JP2002051790A - Arginine repressor-defected strain of coryne form bacterium and method for producing l-arginine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specify the biologically arginine-synthesizing receptor of a coryne form bacterium and improve the L-arginine productivity of the coryne form bacterium. SOLUTION: This coryne form bacterium whose biologically arginine- synthesizing receptor has been defected by breaking a gene encoding the repressor and has L-arginine productivity. The method for producing the L- arginine, characterized by culturing the coryne form bacterium having the L- arginine productivity to produce and accumulate the L-arginine in a culture medium and then collecting the L-arginine from the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a coryneform bacterium capable of producing L-arginine and a method for producing L-arginine using the same. L-arginine is an industrially useful amino acid as a component for liver function promoters, amino acid infusions, general amino acid preparations, and the like.

[0002]

2. Description of the Related Art Heretofore, L-arginine has been produced by a fermentation method using a coryneform bacterium as a wild strain; a coryneform bacterium resistant to drugs such as sulfa drugs, 2-thiazolealanine or α-amino-β-hydroxyvaleric acid; In addition to 2-thiazolealanine resistance, L-histidine, L-proline,
-Coryneform bacterium having a requirement for threonine, L-isoleucine, L-methionine or L-tryptophan (Japanese Patent Application Laid-Open No. 54-44096); Coryneform bacterium having resistance to ketomalonic acid, fluoromalonic acid or monofluoroacetic acid (particularly, No. 57-1889); Coryneform bacteria having resistance to argininol (Japanese Patent Application Laid-Open No. 62-24075)
Or a coryneform bacterium having resistance to X-guanidine (X is a derivative of a fatty acid or a fatty chain) (Japanese Unexamined Patent Publication (Kokai) No. 2-1).
No. 86995).

[0003] On the other hand, various techniques for increasing L-arginine producing ability by enhancing L-arginine biosynthetic enzymes by recombinant DNA techniques have been disclosed. For example, a combination of a DNA fragment containing genes of acetylornithine deacetylase, N-acetylglutamic acid-γ-semialdehyde dehydrogenase, N-acetylglutamic kinase, and argininosuccinase derived from a microorganism belonging to the genus Escherichia, and vector DNA A microorganism belonging to the genus Corynebacterium or Brevibacterium harboring the recombinant DNA (Japanese Patent Publication No. 5-23750).
No.) and a method for producing L-arginine.

[0004] In coryneform bacteria, some L-
It has been investigated that the production of arginine biosynthetic enzymes is suppressed by L-arginine. Furthermore, L-
Some arginine biosynthesis enzymes are inhibited by L-arginine, but in mutants of coryneform bacteria with improved L-arginine accumulation, the suppression of these enzymes by L-arginine is released. (Agric. Biol. Chem., 43 (1), 105, 1979).

On the other hand, in Escherichia coli, a repressor of the L-arginine biosynthesis system and a gene encoding the repressor have been identified (Proc. Natl. Acad. Sc).
i. USA (1987), 84 (19), 6697-701), and the binding interaction between repressor proteins and various L-arginine biosynthesis genes has been examined (Proc. Natl.
Acad. Sci. USA (1987), 84 (19), 6697-701, J. Mo
l. Biol. (1992), 226, 367-386).

[0006] However, in coryneform bacteria, a repressor protein of the L-arginine biosynthesis system has not been identified. Regarding the repressor protein gene (argR), its gene sequence and the amino acid sequence assumed to be encoded by it are registered in the gene database GenBank (AF049897). It is thought that it was named argR based on homology with the presser.

[0007]

As described above, the repressor protein of L-arginine biosynthesis system of coryneform bacterium and its gene have been estimated, but the repressor protein itself has not been identified. It is not disclosed at all. An object of the present invention is to identify a repressor of L-arginine biosynthesis of a coryneform bacterium and improve the L-arginine-producing ability of the coryneform bacterium.

[0008]

Means for Solving the Problems The present inventors have studied a gene (GenB) registered as argR in the gene database.
ank accession AF049897) was isolated from a coryneform bacterium, and amplification of the gene in the coryneform bacterium led to a decrease in L-arginine-producing ability, whereas disruption of the gene improved L-arginine-producing ability. As with Escherichia coli, it was confirmed that L-arginine biosynthesis was suppressed by the repressor and that the gene registered as argR encodes the repressor. Thus, the present invention has been completed. That is, the present invention is as follows.

(1) A coryneform bacterium in which an arginine repressor does not function normally and has an L-arginine-producing ability. (2) The coryneform bacterium is characterized in that the gene encoding the arginine repressor on the chromosome is disrupted,
The coryneform bacterium according to (1), wherein the arginine repressor does not function normally. (3) The coryneform bacterium according to (2), wherein the arginine repressor has an amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having homology to the amino acid sequence. (4) culturing the coryneform bacterium according to any of (1) to (3) in a medium to produce and accumulate L-arginine in the medium;
A method for producing L-arginine, wherein the method is collected from the medium.

In the present invention, the term "arginine repressor" refers to a protein having an action of suppressing L-arginine biosynthesis, and in a coryneform bacterium, when the expression level of the gene encoding the protein is increased, the L-arginine producing ability is reduced. Is decreased, and when the expression level is reduced or disappears, L-
Arginine production ability is improved. Hereinafter, the gene encoding the arginine repressor is also referred to as the argR gene. In the present invention, “L-arginine-producing ability” refers to the ability to accumulate L-arginine in a medium when the microorganism of the present invention is cultured in the medium.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism of the present invention is a coryneform bacterium in which an arginine repressor does not function normally and has an L-arginine-producing ability. Coryneform bacteria of the present invention,
The arginine repressor may have an L-arginine-producing ability by not functioning properly,
Coryneform bacteria having L-arginine-producing ability may be bred so that the arginine repressor does not function normally. After breeding coryneform bacteria so that the arginine repressor does not function properly, L-
Arginine-producing ability may be imparted.

The coryneform bacterium includes a bacterium which has been conventionally classified into the genus Brevibacterium, but is now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 25).
5 (1981)), and also includes bacteria of the genus Brevibacterium which is closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.

Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lilium
Glutamicum) Corynebacterium meracecora Corynebacterium thermoaminogenes Corynebacterium herculis Brevibacterium dibaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium)
(Glutamicum) Brevibacterium immariophyllum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharisticacum Brevibacterium thiogenitalis Brevibacterium album Brevibacterium・ Selinum Microbacterium ・ Ammonia Filum

The coryneform bacterium having L-arginine-producing ability is not particularly limited as long as it has L-arginine-producing ability.
Sulfa drug, 2-thiazolealanine or α-amino-
Coryneform bacteria having resistance to drugs such as β-hydroxyvaleric acid; in addition to 2-thiazolealanine resistance, L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine or L-tryptophan requirement Coryneform bacterium having (JP-A-54-44096);
Coryneform bacteria having resistance to ketomalonic acid, fluoromalonic acid or monofluoroacetic acid (JP-A-57-1889)
No.); Coryneform bacteria having resistance to argininol (Japanese Patent Application Laid-Open No. 62-24075); Coryneform bacteria having resistance to X-guanidine (X is a fatty acid or a derivative of a fatty chain); Is mentioned.

Specifically, the following strains can be exemplified. Brevibacterium flavum AJ11169 (FERM P-4161) Brevibacterium lactofermentum AJ12092 (F
ERM P-7273) Brevibacterium flavum AJ11336 (FERM P-4939) Brevibacterium flavum AJ11345 (FERM P-4948) Brevibacterium lactofermentum AJ12430 (F
ERM BP-2228)

The AJ11169 strain and AJ12092 strain are disclosed in
No. 4096, a 2-thiazolealanine-resistant strain, AJ11
The 336 strain is a strain having the argininol resistance and sulfadiazine resistance described in JP-B-62-24075, AJ1134.
The five strains are argininol-resistant, 2-thiazolealanine-resistant, sulfaguanidine-resistant, and histidine-requiring strains described in JP-B-62-24075, and AJ1243
The 0 strain is a strain having octylguanidine resistance and 2-thiazolealanine resistance described in JP-A-2-18695. AJ11169 was established on August 3, 1977 by the National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology)
F at 305-8566 1-3-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan
Deposited under accession number ERM P-4161, transferred to an international deposit under the Budapest Treaty on September 27, 1999, and transferred to FERM BP-68
Deposited with a deposit number of 92. AJ12092 was launched in 1983
FERM P-7 on April 29
Deposited under accession number 273, transferred to an international deposit under the Budapest Treaty on October 1, 1999, and deposited under accession number FERM BP-6906. AJ11336 was deposited on April 25, 1979 with the Research Institute of Biotechnology and Industrial Technology, under the accession number FERM P-4939.On September 27, 1999, it was transferred to an international deposit under the Budapest Treaty. Deposited with -6893 accession number. AJ11345 was deposited on April 25, 1979 with the Institute of Biotechnology and Industrial Technology, under the accession number FERM P-4948, transferred to an international deposit under the Budapest Treaty on September 27, 1999, and transferred to FERM BP. Deposited with -6894 accession number. AJ12430 is the accession number of FERM BP-2228 to the National Institute of Advanced Industrial Science and Technology on November 26, 1988,
Deposited internationally under the Budapest Treaty.

The coryneform bacterium in which the repressor does not function properly is modified such that the argR gene has a reduced or absent activity of the arginine repressor, which is the gene product, or a reduced or absent transcription of the argR gene. It is obtained by doing. Such a coryneform bacterium is, for example, a homologous recombination method (Experiment
s in Molecular Genetics, Cold Spring Harbor Labora
tory press (1972); Matsuyama, S. and Mizushima,
S., J. Bacteriol., 162, 1196 (1985)), by replacing the argR gene on the chromosome with an argR gene that does not function normally (hereinafter sometimes referred to as a “disrupted argR gene”). be able to.

When a plasmid or the like having a sequence homologous to a sequence on a chromosome is introduced into a bacterial cell, recombination occurs at a site of the homologous sequence at a certain frequency, and the entire introduced plasmid is transferred to the chromosome. Incorporated in After this, if recombination occurs further at the location of the homologous sequence on the chromosome, the plasmid will fall off the chromosome again, but at this time the gene destroyed by the recombination position will be fixed on the chromosome, The original normal gene may fall off the chromosome along with the plasmid. By selecting such a strain, a strain in which a normal argR gene on the chromosome is replaced with a disrupted argR gene can be obtained.

Such a gene disruption technique by homologous recombination has already been established, and a method using linear DNA, a method using a temperature-sensitive plasmid, and the like can be used. Also,
The argR gene can also be disrupted by using a plasmid that contains an argR gene into which a marker gene for drug resistance or the like is inserted and that cannot replicate in the target coryneform bacterial cell. That is, a transformant that has been transformed with the above-described plasmid and has acquired drug resistance has the marker gene incorporated in the chromosomal DNA. Since this marker gene is likely to be integrated by homologous recombination between the argR gene sequence at both ends and the argR gene on the chromosome, a gene-disrupted strain can be selected efficiently.

The disrupted argR gene used for gene disruption is
Specifically, deletion of a certain region of the argR gene by restriction enzyme digestion and recombination, insertion of another DNA fragment (such as a marker gene) into the argR gene, or site-directed mutagenesis (Kr
amer, W. and Frits, HJ, Methods in Enzymology, 1
54, 350 (1987)) and treatment with chemical agents such as sodium hyposulfite and hydroxylamine (Shortle, D. and Nath
ans, D., Proc. Natl. Acad. Sci. USA, 75, 270 (19
78)), by causing substitution, deletion, insertion, addition or inversion of one or more bases in the base sequence such as the coding region or promoter region of the argR gene, Can be obtained by reducing or eliminating the activity of argR gene or by decreasing or eliminating the transcription of the argR gene. Among these embodiments, a method of deleting a certain region of the argR gene by restriction enzyme digestion and recombination, or a method of inserting another DNA fragment into the argR gene is preferable from the viewpoint of reliability and stability. In addition, using a plasmid containing the argR gene and its peripheral region as a template, PCR (polymerase
(Chain reaction) to amplify a part of the argR gene except for the inside or the whole, and circularize the resulting amplification product, whereby a plasmid for disrupting the argR gene can be prepared. In the examples described later, this method
The argR gene was disrupted.

The argR gene is located on the chromosome DN of coryneform bacteria.
A can be obtained from A by a PCR method using an oligonucleotide prepared based on a known nucleotide sequence of the argR gene as a primer. Further, the argR gene can be obtained from a chromosomal DNA library of a coryneform bacterium by a hybridization method using an oligonucleotide prepared based on the base sequence of the known argR gene as a probe. In the present invention, since the argR gene is used for producing a disrupted argR gene, it does not necessarily need to include the full length, but may have a length necessary to cause gene disruption.

The origin of the argR gene is not particularly limited as long as it has homology to homologous recombination with the argR gene of the coryneform bacterium. Specific examples of the argR gene of the coryneform bacterium include the argR gene of Brevibacterium flavum having the nucleotide sequence shown in SEQ ID NO: 17, and the argR gene of Corynebacterium glutamicum (GenBank
accession AF049897). These argR genes are highly homologous, and it is considered that the argR gene of a coryneform bacterium having a different species or genus from a coryneform bacterium that disrupts the argR gene can be used for gene disruption. In the present invention, the amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having homology with the amino acid sequence is an argR gene encoding the amino acid sequence represented by SEQ ID NO: 18 (for example, an argR gene having a base sequence represented by SEQ ID NO: 17) ) And the amino acid sequence encoded by the argR gene having homology to cause homologous recombination.

The primer used for PCR may be any as long as it can amplify the argR gene, and specific examples include oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 19 and 20.

[0024] Examples of the marker gene include drug resistance genes such as a kanamycin resistance gene. A kanamycin resistance gene is a known plasmid containing the kanamycin resistance gene of Streptococcus faecalis,
For example, pDG783 (Anne-Marie Guerout-Fleury et al., G
ene, 167, 335-337 (1995)).

When a drug resistance gene is used as a marker gene, the gene is inserted into an appropriate site of the argR gene in a plasmid, and a microorganism is transformed with the obtained plasmid to select a transformant having become drug resistant. Then ar
A gR gene disrupted strain is obtained. Disruption of the argR gene on the chromosome can be confirmed by analyzing the argR gene or the marker gene on the chromosome by Southern blotting or PCR. Confirmation that the kanamycin resistance gene has been integrated into the chromosomal DNA is performed by PCR using a primer capable of amplifying the kanamycin resistance gene (for example, an oligonucleotide having the nucleotide sequence shown in SEQ ID NOS: 1 and 2). Can be.

The arginine repressor obtained as described above does not function normally, and a coryneform bacterium having L-arginine-producing ability is cultured in a medium, and L-arginine is produced and accumulated in the culture. By collecting L-arginine from the culture, L-arginine can be efficiently produced.

The medium used may be a well-known medium conventionally used for fermentative production of amino acids using microorganisms. That is, it is a normal medium containing a carbon source, a nitrogen source, inorganic ions and other organic components as required.

As the carbon source, sugars such as glucose, sucrose, lactose, galactose, hydrolysates of fructose and starch, alcohols such as glycerol and sorbitol, and organic acids such as fumaric acid, citric acid and succinic acid are used. be able to.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

As organic trace nutrients, vitamin B ₁ ,
Desirable substances such as L-homoserine or yeast extract are desirably contained in appropriate amounts. In addition to these, if necessary, potassium phosphate, magnesium sulfate, iron ions,
A small amount of manganese ions or the like is added.

The cultivation is preferably carried out under aerobic conditions for 1 to 7 days, the culturing temperature is 24 ° C. to 37 ° C., and the pH during culturing is preferably 5 to 9. In addition, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used for pH adjustment. The collection of L-arginine from the fermentation broth can usually be carried out by combining the ion exchange resin method and other known methods.

[0032]

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

[0033]

Example 1 Construction of Shuttle Vector and Temperature-Sensitive Vector for Escherichia coli and Coryneform Bacteria First, a vector for introducing an argR gene into a coryneform bacterium, and an argR-deleted strain of a coryneform bacterium A temperature sensitive vector was constructed. <1> Construction of vector having drug resistance gene of Streptococcus faecalis The kanamycin resistance gene of Streptococcus faecalis was amplified by PCR from a known plasmid containing the gene. The nucleotide sequence of the kanamycin resistance gene of Streptococcus faecalis has already been elucidated (Trieu-Cuot, P. and Courvalin, P .: Gene 23 (3), 331).
-341 (1983)). Based on this sequence, primers represented by SEQ ID NOs: 1 and 2 were synthesized, and pDG783 (Anne-Marie Guerout)
-PCR was performed using Fleury et al., Gene, 167, 335-337 (1995)) as a template to amplify a DNA fragment containing the kanamycin resistance gene and its promoter.

After purifying the above DNA fragment with SUPREC02 manufactured by Takara Shuzo, it was completely digested with restriction enzymes HindIII and HincII and blunt-ended. Blunting Kit manufactured by Takara Shuzo Co., Ltd.
Performed by This DNA fragment and SEQ ID NOs: 3 and 4
PHSG399 (S. Takeshita et al.)
: Gene 61, 63-74 (1987)) was used as a template, and the amplified product obtained by PCR was purified and blunt-ended, and mixed and ligated. The ligation reaction was performed by Takara Shuzo DNA ligat.
Performed with ion kit ver2. The ligated DNA was used to transform competent cells of Escherichia coli JM109 (Takara Shuzo), IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml, X-Gal (5-bromo -4-chloro
-3-indolyl-β-D-galactoside) 40 μg / ml and kanamycin 25 μg / ml L medium (bactotryptone 10 g / ml)
L, Bacto yeast extract 5 g / L, NaCl 5 g / L, agar 15 g / L, pH 7.2), culture overnight, pick up the blue colonies that appeared, separate single colonies, and isolate the transformed strain. Obtained.

Plasmids were prepared from the transformed strains using the alkaline method (Biotechnology Experiments, edited by The Society of Biotechnology, Japan, p. 105, Baifukan, 1992), and a restriction enzyme map was prepared.
The one equivalent to the restriction enzyme map shown in the above was named pK1. This plasmid is stably maintained in Escherichia coli and confers kanamycin resistance on the host. Also,
Since it contains the lazZ 'gene, it is suitable for cloning vectors.

<2> Construction of shuttle vector pSFK6 As a material for obtaining a temperature-sensitive replication control region, a plasmid vector capable of autonomous replication in both Escherichia coli and coryneform bacteria was prepared. Plasmid pAM330 extracted from Brevibacterium lactofermentum ATCC13869 (see JP-A-58-67699)
After complete digestion with HindIII, the ends were blunt-ended, and this pK1 digested completely with the restriction enzyme BsaAI was ligated. Brevibacterium lactofermentum ATCC13869 was transformed using the ligated DNA. The electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) was used for the transformation. Transformant selection includes kanamycin 25 μg / ml
M-CM2B plate (polypeptone 10 g / L, yeast extract 10 g /
L, NaCl 5 g / L, biotin 10 μg / L, agar 15 g / L, pH 7.2). After culturing for two nights, the colonies were picked and separated into single colonies to obtain transformants. Plasmid from transformant
DNA was prepared, a restriction map was prepared, and a restriction map identical to the restriction map shown in FIG.
It was named. This plasmid is capable of autonomous replication in Escherichia coli and coryneform bacteria and confer kanamycin resistance to the host.

<3> Obtaining a plasmid having a temperature-sensitive replication control region pSFK6 was treated with hydroxylamine in vitro. Hydroxylamine treatment can be performed by a known method (GO Humphery
s et al., Molec. Gen. Genet., 145, 101-108 (1976), etc.). The DNA after the treatment was recovered and transformed into Brevibacterium lactofermentum ATCC13869 strain. Transformants were CM2B containing kanamycin 25 μg / ml.
Selected at low temperature (25 ° C.) on plate. Replicate the transformants that appeared on the same selection plate, and heat to 34 ° C.
And cultured. One strain that could not grow on a selection plate containing kanamycin at high temperature was obtained. The plasmid was recovered from this strain and named p48K.

<4> Determination of Base Sequence of Temperature-Sensitive Replication Control Region With respect to plasmid pSFK6 having a wild-type replication control region and plasmid p48K having a temperature-sensitive replication control region, the nucleotide sequences of the respective replication control regions It was determined. The nucleotide sequence was determined using the ABI DNA Sequencing Kit.
Determined by BI company's fully automatic sequencer ABI310. As a result, it was found that there were six base substitutions between the wild-type replication control region and the temperature-sensitive mutant replication control region.
The nucleotide sequence of the replication control region (all the pAM330-derived sequence) that functions in coryneform bacteria contained in pSFK6 is shown in SEQ ID NO: 5.
SEQ ID NO: 7 shows the nucleotide sequence of the temperature-sensitive replication control region contained in p48K that functions in coryneform bacteria. In addition, amino acid sequences that can be encoded by ORFs present in these nucleotide sequences are shown in SEQ ID NOS: 6 and 8. In the temperature-sensitive replication control region, C at position 1255
The 1534th C to T, the 1866th G to A, the 2058th to
G is mutated to A, C at position 2187 is mutated to T, and G at position 3193 is mutated to A. Of these, only the mutation point at position 1534 has an amino acid mutation, which causes substitution of proline to serine.

<5> Construction of Shuttle Vector Having Temperature Sensitive Mutation
It was introduced into FK6 (see Fig. 3). Mutation can be introduced by a known method (Mikaelian, I., Sergeant, A. Nucleic Acids Re
s., 20, 376 (1992)). The specific method is shown below. In order to introduce a C → T mutation at position 1534, primers shown in SEQ ID NOS: 9 and 10 (primers 9,
PCR was performed using the combination of 10) and the combination of the primers (primers 11 and 12) shown in SEQ ID NOS: 11 and 12 with pAM330 as a template. The obtained amplification products were purified by electrophoresis on an agarose gel and then recovered from the gel. DNA fragments were recovered from the gel using EASYTRAPVer.2 manufactured by Takara Shuzo. The purified DNA was mixed so as to have a molar ratio of 1: 1 and a PCR reaction was carried out using this as a template and the primers (primers 13, 14) shown in SEQ ID NOs: 13 and 14. The amplification product was completely digested with the restriction enzyme MluI, and after electrophoresis on an agarose gel, a DNA fragment of about 3.2 Kb was recovered. pSFK6
Similarly, the DNA fragment was completely digested with the restriction enzyme MluI, electrophoresed on an agarose gel, and a DNA fragment of about 3.8 Kb was recovered. The obtained DNA fragments are mixed and ligated, and Escherichia coli JM1
Transform using 09 competent cells (Takara Shuzo Co., Ltd.), apply to L medium containing kanamycin 25 μg / ml,
After overnight culture, appeared colonies were picked and separated into single colonies to obtain transformed strains. A plasmid was prepared from the transformant using the alkaline method, and the nucleotide sequence was determined. It was confirmed that C at position 1534 in the sequence shown in SEQ ID NO: 5 was mutated to T. This plasmid was named pSFKT2 (FIG. 3).

[0040]

Example 2 Cloning of argR gene and amplification effect in coryneform bacterium Brevibacterium flavum wild strain 2247 (AJ14067)
SEQ ID NO: 15 (SEQ ID NO: 17)
SEQ ID NOs: 1717 to 1741) and SEQ ID NO: 16
PCR was performed using oligonucleotides (primers 15, 16) having the nucleotide sequence shown in (sequence complementary to the sequence of nucleotide numbers 2386 to 2362 of SEQ ID NO: 17) as primers. PCR was performed using Pyrobest DNA polymerae (Takara Shuzo) for 30 cycles at 98 ° C. for 10 seconds, 58 ° C. for 1 minute, and 72 ° C. for 3 minutes. The resulting amplified fragment was inserted into the SmaI site of the shuttle vector pSFK6 obtained in Example 1, and a plasmid capable of autonomously replicating in coryneform bacteria.
pWR was obtained.

Coryneform Bacteria in L-Arginine Producing Bacteria
To examine the effect of amplifying the argR gene, pWR was introduced into L-arginine-producing bacterium Brevibacterium flavum strain AJ113455 (FERMBP-6894). The plasmid was introduced by the electric pulse method (Japanese Patent Laid-Open No. 2-207791). Transformants were selected as kanamycin-resistant strains on a CM2G agar medium containing 25 μg / ml of kanamycin (5 g of glucose, 5 g of NaCl, and 15 g of agar in 1 L of pure water; pH 7.2) to obtain AJ11345 / pWR. As a control, pSFK6 was similarly introduced into AJ113455 strain to obtain a transformant AJ11345 / pSFK6.

Each of the above strains was wetted on an agar medium containing 0.5 g / dl of glucose, 1 g / dl of polypeptone, 1 g / dl of yeast extract, and 0.5 g / dl of NaCl, and cultured at 31.5 ° C. for 20 hours. The obtained bacterial cell 1ase was subjected to glucose 4 g / dl, ammonium sulfate 6.5 g / dl, KH ₂ PO ₄ 0.1 g / dl, MgSO ₄ 0.04 g / dl, FeSO ₄
0.001 g / dl, MnSO ₄ 0.001 g / dl, vitamin B ₁₅ μg / dl,
Biotin 5μg / dl, soybean hydrolyzate (N amount 45mg / d
The cells were inoculated into the medium containing l) and cultured in a flask at 31.5 ° C. for 50 hours with shaking. Table 1 shows the results of measuring the amount of L-arginine accumulation (concentration (g / dl)) in each culture solution. As a result, the argR amplified strain hardly accumulated L-arginine. This indicated that the argR gene product functions as an arginine repressor.

[0043]

[Table 1] Table 1 蓄積 Accumulated amount of strain L-alkydnin (g / dl) ──────────── ───────── AJ11345 / pSFK6 1.3 AJ11345 / pWR 0.2 ─────────────────────

The result of determining the nucleotide sequence of the insert fragment cloned into pWR is shown in SEQ ID NO: 17. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NO: 18.

[0045]

[Example 3] Construction of argR-disrupted strain of coryneform bacterium and effect of deletion of arginine repressor <1> Preparation of plasmid for argR disruption Brevibacterium flavum wild strain 2247 strain (AJ1406)
Using the chromosomal DNA of 7) as a template, SEQ ID NO: 19 (SEQ ID NO: 1)
PCR using the oligonucleotides (primers 19 and 20) having the nucleotide sequences shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 20 (sequence complementary to the nucleotide numbers 4230 to 4211 in SEQ ID NO: 17) as primers Was done. P
CR is 98% using Pyrobest DNA polymerae (Takara Shuzo).
The cycle was performed at 30 ° C. for 10 seconds, 58 ° C. for 1 minute, and 72 ° C. for 3 minutes for 30 cycles. The obtained amplified fragment was inserted into the SmaI site in the multiple cloning site of the cloning vector pHSG399.

In order to delete all ORFs which seem to encode an arginine repressor from the inserted DNA fragment, SEQ ID NO: 21 (base number 2372 of SEQ ID NO: 17) was deleted.
Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 2395 to 2395) and SEQ ID NO: 22 (sequences complementary to the nucleotides 1851 to 1827 in SEQ ID NO: 17)
PHSG39 with amplified fragment inserted using 22) as a primer
PCR was performed using 9 as a template. The pssER was constructed by self-ligating the PCR product.

Next, a fragment obtained by digesting pssER with the restriction enzymes SmaI and SalI and a fragment obtained by digesting the temperature-sensitive plasmid pSFKT2 obtained in Example 1 with SmaI and SalI were ligated to obtain a coryneform bacterium. The plasmid pssERT for argR disruption, whose autonomous replication ability became temperature sensitive, was obtained.

<2> Acquisition of an Arginine Repressor-Deficient Strain of Coryneform Bacteria by Homologous Recombination The plasmid pssERT obtained as described above was used to transform Brevibacterium lactofermentum AJ13029 strain (FERM BP-5).
189). The plasmid was introduced by the electric pulse method (Japanese Patent Laid-Open No. 2-207791). Since this plasmid has a temperature-sensitive autonomous replication ability in Brevibacterium lactofermentum, only strains in which this plasmid has been integrated into the chromosome by homologous recombination can be used at 34 ° C, the non-permissive temperature for plasmid replication. It can be selected as a kanamycin resistant strain. The strain in which the argR disruption plasmid was integrated into the chromosome was a CM2G medium plate containing 25 μg / ml kanamycin (10 g of polypeptone, 10 g of yeast extract, 5 g of glucose,
Include 5g of Cl and 15g of agar in 1L of pure water. pH 7.2) and selected as a kanamycin resistant strain. At this stage, the chromosome-derived normal argR gene and the plasmid-derived ORF-deleted argR gene are present in tandem on the chromosome across the plasmid portion.

Next, by homologous recombination of the recombinant strain again and selecting a strain which became kanamycin sensitive at a non-permissive temperature of 34 ° C. for plasmid replication, one of the argR genes was dropped together with the plasmid portion. The strain was selected.
Among these strains, there are strains in which the normal argR gene is left on the chromosome, and strains in which the disrupted argR gene is left. From these, a strain retaining only the disrupted argR gene was selected. Whether the argR gene on the chromosome is in the disrupted form was determined by preparing a chromosome of a kanamycin-sensitive strain at 34 ° C. and using this as a template, SEQ ID NO: 19 and SEQ ID NO: 2
PCR was performed using oligonucleotides (primers 19 and 20) having the nucleotide sequence shown in SEQ ID NO: 0 as primers, and the PCR product was confirmed to be approximately 600 bp shorter than that obtained by similarly performing PCR using the chromosome derived from the parent strain as a template. can do.

The argR-disrupted strain selected as described above
Perform direct sequencing of the PCR product, and a
Confirm that the rgR gene has been disrupted and confirm that AJ13029ΔR
Got the strain.

<3> Production of L-arginine by argR-disrupted strain AJ13029ΔR strain was prepared by adding 0.5 g / dl of glucose and 1 g of polypeptone.
/ dl, 1 g / dl of yeast extract, and 0.5 g / dl of NaCl, and cultured at 31.5 ° C for 20 hours. Obtained bacterial cells 1
Ace, glucose 3 g / dl, ammonium sulfate 6.5 g / d
l, KH ₂ PO ₄ 0.1g / dl, MgSO ₄ 0.04g / dl, FeSO ₄ 0.001g / d
l, MnSO ₄ 0.001 g / dl, vitamin B ₁ 300 μg / dl, biotin
200 μg / dl, containing soybean hydrolyzate (165 mg / dl as N content), inoculated in a medium adjusted to pH 7.0 with NaOH, and incubated at 31.5 ° C. for 24 hours
Time seed culture was performed.

[0052] 1 ml of the above seed culture was added to glucose 4 g / d.
l, ammonium sulfate 6.5g / dl, KH_TwoPO _Four 0.5g / dl, MgSO_Four 
0.04g / dl, FeSO_Four 0.001g / dl, MnSO_Four 0.01g / dl, Vitamin
B₁ 5μg / dl, biotin 5μg / dl, soybean hydrolyzate (N content
45mg / dl), pH adjusted to 7.0 with KOH
And shake culture at 31.5 ° C for 50 hours in a flask.
Was. L-arginine accumulation in the culture of each strain (concentration
(mg / dl)) is shown in Table 2. As a result, arg
R-disrupted strains produce significant amounts of L-arginine compared to the parent strain
did.

[0053]

[Table 2] Table 2-Strain L-Alkydinin accumulation (mg / dl)- ───────── AJ13029 20 AJ13029ΔR 120 ─────────────────────

[0054]

[Description of Sequence Listing] SEQ ID NO: 1: Primer for amplifying kanamycin resistance gene of Streptococcus faecalis SEQ ID NO: 2: Primer for amplifying kanamycin resistance gene of Streptococcus faecalis SEQ ID NO: 3: Primer for partial amplification of vector of pHSG399 SEQ ID NO: 4: pHSG399 SEQ ID NO: 5: Nucleotide sequence of replication control region of pSFK6 SEQ ID NO: 6: Amino acid sequence that can be encoded by ORF in pSFK6 SEQ ID NO: 7: Nucleotide sequence of replication control region of p48K SEQ ID NO: 8 Amino acid sequence that can be encoded by ORF in p48K SEQ ID NO: 9: First PCR primer for introducing 1534th C → T mutation in pSFK6 SEQ ID NO: 10: 1534th C → T mutation in pSFK6 First PCR primer for introduction SEQ ID NO: 11: Introduction of C → T mutation at position 1534 into pSFK6 1st PCR primer for introducing a 1534th C → T mutation into pSFK6 SEQ ID NO: 12: 1534th C → T mutation into pSFK6 No. 14: Primer for the second PCR to introduce the 1534th C → T mutation into pSFK6 SEQ ID NO: 14: Primer for argR gene amplification SEQ ID NO: 16: For argR gene amplification Primer SEQ ID NO: 17: Base sequence of DNA fragment containing argR gene SEQ ID NO: 18: Amino acid sequence that can be encoded by the DNA fragment SEQ ID NO: 19: Primer for amplifying argR gene SEQ ID NO: 20: Primer for amplifying argR gene SEQ ID NO: 21: argR ArgR gene of plasmid containing gene
Primer for amplifying parts other than ORF SEQ ID NO: 22: argR gene of plasmid containing argR gene
Primers for amplifying parts other than ORF

[0055]

According to the present invention, the L-arginine-producing ability of a coryneform bacterium having L-arginine-producing ability can be improved.

[0056]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. (Ajinomoto Co., Inc.) <120> Method for producing arginine repressor-deficient strain of coryneform bacterium and L-arginine <130> P-8772 <140> 141> 2001-04-13 <150> JP2000-129167 <151> 2000-04-28 <160> 22 <170> PatentIn Ver. 2.0

<210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 1 cccgttaact gcttgaaacc caggacaata ac 32

<210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 2 cccgttaaca tgtacttcag aaaagattag 30

<210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 3 gatatctacg tgccgatcaa cgtctc 26

<210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 4 aggccttttt ttaaggcagt tattg 25

<210> 5 <211> 4447 <212> DNA <213> Brevibacterium lactofermentum <220> <221> CDS <222> (1318) .. (2598) <400> 5 aagcttgtct acgtctgatg ctttgaatcg gacggacttg ccgatcttgt atgcggtgat 60 tt tttgcccact ttttaatggt ggccggggtg agagctacgc gggcggcgac 120 ctgctgcgct gtgatccaat attcggggtc gttcactggt tcccctttct gatttctggc 180 atagaagaac ccccgtgaac tgtgtggttc cgggggttgc tgatttttgc gagacttctc 240 gcgcaattcc ctagcttagg tgaaaacacc atgaaacact agggaaacac ccatgaaaca 300 cccattaggg cagtagggcg gcttcttcgt ctagggcttg catttgggcg gtgatctggt 360 ctttagcgtg tgaaagtgtg tcgtaggtgg cgtgctcaat gcactcgaac gtcacgtcat 420 ttaccgggtc acggtgggca aagagaacta gtgggttaga cattgttttc ctcgttgtcg 480 gtggtggtga gcttttctag ccgctcggta aacgcggcga tcatgaactc ttggaggttt 540 tcaccgttct gcatgcctgc gcgcttcatg tcctcacgta gtgccaaagg aacgcgtgcg 600 gtgaccacga cgggcttagc ctttgcctgc gcttctagtg cttcgatggt ggcttgtgcc 660 tgcgcttgct gcgcctgtag tgcctgttga gcttcttgta gttgctgttc tagctgtgcc 720 ttggttgcca tgctttaaga ctctagtagc tttcctgcga tatgtcatgc gcatgcgtag 780 caaacattgt cctgcaactc attcattatg tgcagtgctc ctgttactag tcgtacatac 840 tcatatttac ctagtctgca tgcagtgcat gcacatgcag tcatgtcgtg ctaatgtgta 900 aaacatgtac atgcagattg ctgggggtgc agggggcgga gccaccctgt ccatgcgggg 960 tgtggggctt gccccgccgg tacagacagt gagcaccggg gcacctagtc gcggataccc 1020 cccctaggta tcggacacgt aaccctccca tgtcgatgca aatctttaac attgagtacg 1080 ggtaagctgg cacgcatagc caagctaggc ggccaccaaa caccactaaa aattaatagt 1140 ccctagacaa gacaaacccc cgtgcgagct accaactcat atgcacgggg gccacataac 1200 ccgaaggggt ttcaattgac aaccatagca ctagctaaga caacgggcac aacacccgca 1260 caaactcgca ctgcgcaacc ccgcacaaca tcgggtctag Lyc Val Ast Gag Ser Gag Ser Ast Gag Ser Gag Ast Gag Ser Gag Ast Gag Ser g 1 5 10 15 cgc gct agg gcg tgg cgt agg caa aac gtc atg tac aag atc acc aat 1413 Arg Ala Arg Ala Trp Arg Arg Gln Asn Val Met Tyr Lys Ile Thr Asn 20 25 30 agt aag gct ctg gcg ggg tgc cat agg tgg cgc agg gac gaa gct gtt 1461 Ser Lys Ala Leu Ala Gly Cys His Arg Trp Arg Arg Asp Glu Ala Val 35 40 45 gcg gtg tcc tgg tcg tct aac ggt gct tcg cag ttt gag ggt ctg caa 1509 Ala Val Ser Trp Ser Ser Asn Gly Ala Ser Gln Phe Glu Gly Leu Gln 50 55 60 aac tct cac tct cgc tgg ggg tca cct ctg gct gaa ttg gaa gtc atg 1557 Asn Ser His Ser Arg Trp Gly Ser Pro Leu Ala Glu Leu Glu Val Met 65 70 75 80 ggc gaa cgc cgc att gag ctg gct att gct act aag aat cac ttg gcg 1605 Gly Glu Arg Arg Ile Glu Leu Ala Ile Ala Thr Lys Asn His Leu Ala 85 90 95 gcg ggt ggc gcg ctc atg atg ttt gtg ggc ac gtt 1653 Ala Gly Gly Ala Leu Met Met Phe Val Gly Thr Val Arg His Asn Arg 100 105 110 tca cag tca ttt gcg cag gtt gaa gcg ggt att aag act gcg tac tct 1701 Ser Gln Ser Phe Ala Gln Val Glu Ala Gly Ile Lys Thr Ala Tyr Ser 115 120 125 tcg atg gtg aaa aca tct cag tgg aag aaa gaa cgt gca cgg tac ggg 1749 Ser Met Val Lys Thr Ser Gln Trp Lys Lys Glu Arg Ala Arg Tyr Gly 130 135 140 gtg gag cac acc tat agt gac tat gag gt c aca gac tct tgg gcg aac 1797 Val Glu His Thr Tyr Ser Asp Tyr Glu Val Thr Asp Ser Trp Ala Asn 145 150 155 160 ggt tgg cac ttg cac cgc aac atg ctg ttg ttc ttg gat cgt cca ctg 1845 Gly Trp His Leu His Arg Asn Met Leu Leu Phe Leu Asp Arg Pro Leu 165 170 175 tct gac gat gaa ctc aag gcg ttt gag gat tcc atg ttt tcc cgc tgg 1893 Ser Asp Asp Glu Leu Lys Ala Phe Glu Asp Ser Met Phe Ser Arg Trp 180 185 190 tct gct ggt gtg gtt aag gcc ggt atg gac gcg cca ctg cgt gag cac 1941 Ser Ala Gly Val Val Lys Ala Gly Met Asp Ala Pro Leu Arg Glu His 195 200 205 ggg gtc aaa ctt gat cag gtg tct acc tgg ggt gga gac gct gcg aaa 1989 Gly Val Lys Leu Asp Gln Val Ser Thr Trp Gly Gly Asp Ala Ala Lys 210 215 220 atg gca acc tac ctc gct aag ggc atg tct cag gaa ctg act ggc tcc 2037 Met Ala Thr Tyr Leu Ala Lys Gly Met Ser Gln Glu Leu Thr Gly Ser 225 230 235 240 gct act aaa acc gcg tct aag ggg tcg tac acg ccg ttt cag atg ttg 2085 Ala Thr Lys Thr Ala Ser Lys Gly Ser Tyr Thr Pro Phe Gln Met Leu 245 250 255 gat atg ttg gcc gat caa agc gac gcc ggc gag gat atg gac gct gtt 2133 Asp Met Leu Ala Asp Gln Ser Asp Ala Gly Glu Asp Met Asp Ala Val 260 265 270 ttg gtg gct cgg tgg cgt gag tat gag gtt ggt tct aaa aac ctg Val Ala Arg Trp Arg Glu Tyr Glu Val Gly Ser Lys Asn Leu Arg 275 280 285 tcg tcc tgg tca cgt ggg gct aag cgt gct ttg ggc att gat tac ata 2229 Ser Ser Trp Ser Arg Gly Ala Lys Arg Ala Leu Gly Ile Asp Tyr Ile 290 295 300 gac gct gat gta cgt cgt gaa atg gaa gaa gaa ctg tac aag ctc gcc 2277 Asp Ala Asp Val Arg Arg Glu Met Glu Glu Glu Leu Tyr Lys Leu Ala 305 310 315 320 ggt ctg gaa gca ccg tca acc cgc gtt gct gtt gct 2325 Gly Leu Glu Ala Pro Glu Arg Val Glu Ser Thr Arg Val Ala Val Ala 325 330 335 ttg gtg aag ccc gat gat tgg aaa ctg att cag tct gat ttc gcg gtt 2373 Leu Val Lys Pro Asp Asp Trp Lys Leu Ile Gln Ser Asp Phe Ala Val 340 345 350 agg cag tac gtt cta gat tgc gtg gat aag gct aag gac gtg gcc gct 2421 Arg Gln Tyr Val Leu Asp Cys Val Asp Lys Ala Lys Asp Val Ala Ala 355 360 365 gcg caa cgt gtc gct aat gag gtg ctg gca agt ctg ggt gtg gat tcc 2469 Ala Gln Arg Val Ala Asn Glu Val Leu Ala Ser Leu Gly Val Asp Ser 370 375 380 acc ccg tgc atg atc gtt atg gat gat ggt gac gcg gtt ctg 2517 Thr Pro Cys Met Ile Val Met Asp Asp Val Asp Leu Asp Ala Val Leu 385 390 395 400 cct act cat ggg gac gct act aag cgt gat ctg aat gcg gcg gtg ttc 2565 Pro Thr His Gly Asp Ala Thr Lys Arg Asp Leu Asn Ala Ala Val Phe 405 410 415 gcg ggt aat gag cag act att ctt cgc acc cac taaaagcggc ataaaccccg 2618 Ala Gly Asn Glu Gln Thr Ile Leu Arg Thr His 420 425 ttcgatattt tgtgcgatga atttatggtc aatgtcgcgg gggcaaacta tgatgggtct 2678 tgttgttgac aatggctgat ttcatcagga atggaactgt catgctgtta tgtgcctggc 2738 tcctaatcaa agctggggac aatgggttgc cccgttgatc tgatctagtt cggattggcg 2798 gggcttcact gtatctgggg gtggcatcgt gaatagattg cacaccgtag tgggcagtgt 2858 gcacaccata gtgggcatga gtaataccta cgcgcgcgtg ggctagggct taacgcgcgt 2918 tttgccgtgc tgcggggcat acgttagcgc atacgctttt ttctgtgaaa cctttttgtg 2978 ttgttgtt tc gtgttggttt cctttctgtt ggcggggcaa cttaacgcct gcgggggtgg 3038 ttgttgacgt taacgggggt agtttttatt cccctagtgg tttttcagta cgacaatcga 3098 gaaagacctg tttcagccag ttcgggtcat gttcgtcggt atggccacgt gcatagcgac 3158 cagttttcga gttcactggg atttttggtg catcgaacaa gatgtaggac aatgcggttt 3218 ctaggtctac tttttgcttt atgccgtaca agccccgtgg gtattcagcg attgattcca 3278 aggcggcttc ccagtcctgt tttgtgaagg actggcttag ttctaggtct gtgtctgggt 3338 agtactgctt gtttgtgtaa gcgccgttgg tgctcattga tgattccttt gaagtgtttg 3398 gagttcggct agtagtgcgg cgtatggtgc tgctttttgc tcgtgatagc tcgccttggc 3458 tatgaggtcg gctaggtagg tttccggggt gcctaggttg cgtaggtcta gcaaatcccg 3518 gtatgtggcc tgtgcgctgc gctggtggtg catacagtcg ttaagctggg cttttacgtc 3578 tgcgatgcgg tggcggttag gcatgttggt gtgcttcttc caagtactca cgggcgggtt 3638 ttgtgtatgc ctggcgtgat gcttctttga gctgttggag ttccgcttgg agtgcgggta 3698 gttcgtccgc gaactgcttg tggtactcgt atttctcttg ttcctgggcg atagcatttg 3758 cgttgaattg cagggcggtg agttcgtcca cgcgtcgttt tgctgcgttg gtcatggtgg 3818 cgtgccattt gcg gttgtgg acgcggggtt caaggttgcg cacggctgct tcggctaggt 3878 tggtggctgc ttttttcagt gctcgggctt cccgttcctc gtccaacgag agcacctttg 3938 gtttgttggc ttcggctagt ttttgcttct ccgctttgat gagttggtca acttcgtgtt 3998 gggagaggtc gtttttcacg atgcgtcgaa tgtggtcgtt gtgggtgctg agttggtgtg 4058 agaggtagtg gggttctggg atttcggcga gttggtcgag gttggtgtag tgcgggttgc 4118 ggcctggttg gttgggttcg ctggggaggt cgatgtatcc ggttgagtct ccggcgtggt 4178 tgaagtgaat taggcgttgg tagccgtatt cctggttggg gaggtacgac agaatgagga 4238 agtttggtgc ttctcctgca atgagtcgtg cgtgttcgta gttcggtact gggtcgtgct 4298 cggggagaat gttcttttgg gtcatggctt ctctttctgt tgctctgtgtgta gtccgtatgt 4358 gggcatggga aagccccggc aaccctttgg gtcaaccggg gctgcg gtcgt gtcg gtcg gtg cgtcg gtg cgtcgt cgtcgtcgt4g

<210> 6 <211> 427 <212> PRT <213> Brevibacterium lactofermentum <400> 6 Val Asn Thr Ser Lys Glu Pro Gln Val Asn Glu Gly Ser Lys Val Thr 1 5 10 15 Arg Ala Arg Ala Trp Arg Arg Gln Asn Val Met Tyr Lys Ile Thr Asn 20 25 30 Ser Lys Ala Leu Ala Gly Cys His Arg Trp Arg Arg Asp Glu Ala Val 35 40 45 Ala Val Ser Trp Ser Ser Asn Gly Ala Ser Gln Phe Glu Gly Leu Gln 50 55 60 Asn Ser His Ser Arg Trp Gly Ser Pro Leu Ala Glu Leu Glu Val Met 65 70 75 80 Gly Glu Arg Arg Ile Glu Leu Ala Ile Ala Thr Lys Asn His Leu Ala 85 90 95 Ala Gly Gly Ala Leu Met Met Phe Val Gly Thr Val Arg His Asn Arg 100 105 110 Ser Gln Ser Phe Ala Gln Val Glu Ala Gly Ile Lys Thr Ala Tyr Ser 115 120 125 Ser Met Val Lys Thr Ser Gln Trp Lys Lys Glu Arg Ala Arg Tyr Gly 130 135 140 Val Glu His Thr Tyr Ser Asp Tyr Glu Val Thr Asp Ser Trp Ala Asn 145 150 155 160 Gly Trp His Leu His Arg Asn Met Leu Leu Phe Leu Asp Arg Pro Leu 165 170 175 Ser Asp Asp Glu Leu Lys Ala Phe Glu Asp Ser Met Phe Ser Arg Trp 180 185 190 Ser Ala Gly Val Val Lys Ala Gly Met Asp Ala Pro Leu Arg Glu His 195 200 205 Gly Val Lys Leu Asp Gln Val Ser Thr Trp Gly Gly Asp Ala Ala Lys 210 215 220 Met Ala Thr Tyr Leu Ala Lys Gly Met Ser Gln Glu Leu Thr Gly Ser 225 230 235 240 Ala Thr Lys Thr Ala Ser Lys Gly Ser Tyr Thr Pro Phe Gln Met Leu 245 250 255 Asp Met Leu Ala Asp Gln Ser Asp Ala Gly Glu Asp Met Asp Ala Val 260 265 270 Leu Val Ala Arg Trp Arg Glu Tyr Glu Val Gly Ser Lys Asn Leu Arg 275 280 285 Ser Ser Trp Ser Arg Gly Ala Lys Arg Ala Leu Gly Ile Asp Tyr Ile 290 295 300 Asp Ala Asp Val Arg Arg Glu Met Glu Glu Glu Leu Tyr Lys Leu Ala 305 310 315 320 Gly Leu Glu Ala Pro Glu Arg Val Glu Ser Thr Arg Val Ala Val Ala 325 330 335 Leu Val Lys Pro Asp Asp Trp Lys Leu Ile Gln Ser Asp Phe Ala Val 340 345 350 Arg Gln Tyr Val Leu Asp Cys Val Asp Lys Ala Lys Asp Val Ala Ala 355 360 365 Ala Gln Arg Val Ala Asn Glu Val Leu Ala Ser Leu Gly Val Asp Ser 370 375 380 Thr Pro Cys Met Ile Val Met Asp Asp Val Asp Leu Asp Ala Val Leu 385 390 395 400 400 Pro Thr His Gly Asp Ala Thr Lys Arg Asp Leu Asn Ala Ala Val Phe 405 410 415 Ala Gly Asn Glu Gln Thr Ile Leu Arg Thr His 420 425

<210> 7 <211> 4447 <212> DNA <213> Brevibacterium lactofermentum <220> <221> CDS <222> (1318) .. (2598) <400> 7 aagcttgtct acgtctgatg ctttgaatcg gacggacttg ccgatcttgt atgcggtgcat 60g tttgcccact ttttaatggt ggccggggtg agagctacgc gggcggcgac 120 ctgctgcgct gtgatccaat attcggggtc gttcactggt tcccctttct gatttctggc 180 atagaagaac ccccgtgaac tgtgtggttc cgggggttgc tgatttttgc gagacttctc 240 gcgcaattcc ctagcttagg tgaaaacacc atgaaacact agggaaacac ccatgaaaca 300 cccattaggg cagtagggcg gcttcttcgt ctagggcttg catttgggcg gtgatctggt 360 ctttagcgtg tgaaagtgtg tcgtaggtgg cgtgctcaat gcactcgaac gtcacgtcat 420 ttaccgggtc acggtgggca aagagaacta gtgggttaga cattgttttc ctcgttgtcg 480 gtggtggtga gcttttctag ccgctcggta aacgcggcga tcatgaactc ttggaggttt 540 tcaccgttct gcatgcctgc gcgcttcatg tcctcacgta gtgccaaagg aacgcgtgcg 600 gtgaccacga cgggcttagc ctttgcctgc gcttctagtg cttcgatggt ggcttgtgcc 660 tgcgcttgct gcgcctgtag tgcctgttga gcttcttgta gttgctgttc tagctgtgcc 720 ttggttgcca tgctttaaga ctctagtagc tttcctgcga tatgtcatgc gcatgcgtag 780 caaacattgt cctgcaactc attcattatg tgcagtgctc ctgttactag tcgtacatac 840 tcatatttac ctagtctgca tgcagtgcat gcacatgcag tcatgtcgtg ctaatgtgta 900 aaacatgtac atgcagattg ctgggggtgc agggggcgga gccaccctgt ccatgcgggg 960 tgtggggctt gccccgccgg tacagacagt gagcaccggg gcacctagtc gcggataccc 1020 cccctaggta tcggacacgt aaccctccca tgtcgatgca aatctttaac attgagtacg 1080 ggtaagctgg cacgcatagc caagctaggc ggccaccaaa caccactaaa aattaatagt 1140 tcctagacaa gacaaacccc cgtgcgagct accaactcat atgcacgggg gccacataac 1200 ccgaaggggt ttcaattgac aaccatagca ctagctaaga caacgggcac aacatccgca 1260 caaactcgca ctgcgcaacc ccgcacaaca tcgggtctag Lyta Val Ast Gag Ser Gag Ast Gag Ser Gag Ast Gag Ser Gag Ser g 1 5 10 15 cgc gct agg gcg tgg cgt agg caa aac gtc atg tac aag atc acc aat 1413 Arg Ala Arg Ala Trp Arg Arg Gln Asn Val Met Tyr Lys Ile Thr Asn 20 25 30 agt aag gct ctg gcg ggg tgc cat agg tgg cgc agg gac gaa gct gtt 1461 Ser Lys Ala Leu Ala Gly Cys His Arg Trp Arg Arg Asp Glu Ala Val 35 40 45 gcg gtg tcc tgg tcg tct aac ggt gct tcg cag ttt gag ggt ctg caa 1509 Ala Val Ser Trp Ser Ser Asn Gly Ala Ser Gln Phe Glu Gly Leu Gln 50 55 60 aac tct cac tct cgc tgg ggg tca tct ctg gct gaa ttg gaa gtc atg 1557 Asn Ser His Ser Arg Trp Gly Ser Ser Leu Ala Glu Leu Glu Val Met 65 70 75 80 ggc gaa cgc cgc att gag ctg gct att gct act aag aat cac ttg gcg 1605 Gly Glu Arg Arg Ile Glu Leu Ala Ile Ala Thr Lys Asn His Leu Ala 85 90 95 gcg ggt ggc gcg ctc atg atg ttt gtg ggc ac gtt 1653 Ala Gly Gly Ala Leu Met Met Phe Val Gly Thr Val Arg His Asn Arg 100 105 110 tca cag tca ttt gcg cag gtt gaa gcg ggt att aag act gcg tac tct 1701 Ser Gln Ser Phe Ala Gln Val Glu Ala Gly Ile Lys Thr Ala Tyr Ser 115 120 125 tcg atg gtg aaa aca tct cag tgg aag aaa gaa cgt gca cgg tac ggg 1749 Ser Met Val Lys Thr Ser Gln Trp Lys Lys Glu Arg Ala Arg Tyr Gly 130 135 140 gtg gag cac acc tat agt gac tat gag gt c aca gac tct tgg gcg aac 1797 Val Glu His Thr Tyr Ser Asp Tyr Glu Val Thr Asp Ser Trp Ala Asn 145 150 155 160 ggt tgg cac ttg cac cgc aac atg ctg ttg ttc ttg gat cgt cca ctg 1845 Gly Trp His Leu His Arg Asn Met Leu Leu Phe Leu Asp Arg Pro Leu 165 170 175 tct gac gat gaa ctc aag gca ttt gag gat tcc atg ttt tcc cgc tgg 1893 Ser Asp Asp Glu Leu Lys Ala Phe Glu Asp Ser Met Phe Ser Arg Trp 180 185 190 tct gct ggt gtg gtt aag gcc ggt atg gac gcg cca ctg cgt gag cac 1941 Ser Ala Gly Val Val Lys Ala Gly Met Asp Ala Pro Leu Arg Glu His 195 200 205 ggg gtc aaa ctt gat cag gtg tct acc tgg ggt gga gac gct gcg aaa 1989 Gly Val Lys Leu Asp Gln Val Ser Thr Trp Gly Gly Asp Ala Ala Lys 210 215 220 atg gca acc tac ctc gct aag ggc atg tct cag gaa ctg act ggc tcc 2037 Met Ala Thr Tyr Leu Ala Lys Gly Met Ser Gln Glu Leu Thr Gly Ser 225 230 235 240 gct act aaa acc gcg tct aaa ggg tcg tac acg ccg ttt cag atg ttg 2085 Ala Thr Lys Thr Ala Ser Lys Gly Ser Tyr Thr Pro Phe Gln Met Leu 245 250 255 gat atg ttg gcc gat caa agc gac gcc ggc gag gat atg gac gct gtt 2133 Asp Met Leu Ala Asp Gln Ser Asp Ala Gly Glu Asp Met Asp Ala Val 260 265 270 ttg gtg gct cgg tgg cgt gag tat gag gtt ggt tct aaa aac ctg Val Ala Arg Trp Arg Glu Tyr Glu Val Gly Ser Lys Asn Leu Arg 275 280 285 tcg tct tgg tca cgt ggg gct aag cgt gct ttg ggc att gat tac ata 2229 Ser Ser Trp Ser Arg Gly Ala Lys Arg Ala Leu Gly Ile Asp Tyr Ile 290 295 300 gac gct gat gta cgt cgt gaa atg gaa gaa gaa ctg tac aag ctc gcc 2277 Asp Ala Asp Val Arg Arg Glu Met Glu Glu Glu Leu Tyr Lys Leu Ala 305 310 315 320 ggt ctg gaa gca ccg tca acc cgc gtt gct gtt gct 2325 Gly Leu Glu Ala Pro Glu Arg Val Glu Ser Thr Arg Val Ala Val Ala 325 330 335 ttg gtg aag ccc gat gat tgg aaa ctg att cag tct gat ttc gcg gtt 2373 Leu Val Lys Pro Asp Asp Trp Lys Leu Ile Gln Ser Asp Phe Ala Val 340 345 350 agg cag tac gtt cta gat tgc gtg gat aag gct aag gac gtg gcc gct 2421 Arg Gln Tyr Val Leu Asp Cys Val Asp Lys Ala Lys Asp Val Ala Ala 355 360 365 gcg caa cgt gtc gct aat gag gtg ctg gca agt ctg ggt gtg gat tcc 2469 Ala Gln Arg Val Ala Asn Glu Val Leu Ala Ser Leu Gly Val Asp Ser 370 375 380 acc ccg tgc atg atc gtt atg gat gat ggt gac gcg gtt ctg 2517 Thr Pro Cys Met Ile Val Met Asp Asp Val Asp Leu Asp Ala Val Leu 385 390 395 400 cct act cat ggg gac gct act aag cgt gat ctg aat gcg gcg gtg ttc 2565 Pro Thr His Gly Asp Ala Thr Lys Arg Asp Leu Asn Ala Ala Val Phe 405 410 415 gcg ggt aat gag cag act att ctt cgc acc cac taaaagcggc ataaaccccg 2618 Ala Gly Asn Glu Gln Thr Ile Leu Arg Thr His 420 425 ttcgatattt tgtgcgatga atttatggtc aatgtcgcgg gggcaaacta tgatgggtct 2678 tgttgttgac aatggctgat ttcatcagga atggaactgt catgctgtta tgtgcctggc 2738 tcctaatcaa agctggggac aatgggttgc cccgttgatc tgatctagtt cggattggcg 2798 gggcttcact gtatctgggg gtggcatcgt gaatagattg cacaccgtag tgggcagtgt 2858 gcacaccata gtgggcatga gtaataccta cgcgcgcgtg ggctagggct taacgcgcgt 2918 tttgccgtgc tgcggggcat acgttagcgc atacgctttt ttctgtgaaa cctttttgtg 2978 ttgttgtt tc gtgttggttt cctttctgtt ggcggggcaa cttaacgcct gcgggggtgg 3038 ttgttgacgt taacgggggt agtttttatt cccctagtgg tttttcagta cgacaatcga 3098 gaaagacctg tttcagccag ttcgggtcat gttcgtcggt atggccacgt gcatagcgac 3158 cagttttcga gttcactggg atttttggtg catcaaacaa gatgtaggac aatgcggttt 3218 ctaggtctac tttttgcttt atgccgtaca agccccgtgg gtattcagcg attgattcca 3278 aggcggcttc ccagtcctgt tttgtgaagg actggcttag ttctaggtct gtgtctgggt 3338 agtactgctt gtttgtgtaa gcgccgttgg tgctcattga tgattccttt gaagtgtttg 3398 gagttcggct agtagtgcgg cgtatggtgc tgctttttgc tcgtgatagc tcgccttggc 3458 tatgaggtcg gctaggtagg tttccggggt gcctaggttg cgtaggtcta gcaaatcccg 3518 gtatgtggcc tgtgcgctgc gctggtggtg catacagtcg ttaagctggg cttttacgtc 3578 tgcgatgcgg tggcggttag gcatgttggt gtgcttcttc caagtactca cgggcgggtt 3638 ttgtgtatgc ctggcgtgat gcttctttga gctgttggag ttccgcttgg agtgcgggta 3698 gttcgtccgc gaactgcttg tggtactcgt atttctcttg ttcctgggcg atagcatttg 3758 cgttgaattg cagggcggtg agttcgtcca cgcgtcgttt tgctgcgttg gtcatggtgg 3818 cgtgccattt gcg gttgtgg acgcggggtt caaggttgcg cacggctgct tcggctaggt 3878 tggtggctgc ttttttcagt gctcgggctt cccgttcctc gtccaacgag agcacctttg 3938 gtttgttggc ttcggctagt ttttgcttct ccgctttgat gagttggtca acttcgtgtt 3998 gggagaggtc gtttttcacg atgcgtcgaa tgtggtcgtt gtgggtgctg agttggtgtg 4058 agaggtagtg gggttctggg atttcggcga gttggtcgag gttggtgtag tgcgggttgc 4118 ggcctggttg gttgggttcg ctggggaggt cgatgtatcc ggttgagtct ccggcgtggt 4178 tgaagtgaat taggcgttgg tagccgtatt cctggttggg gaggtacgac agaatgagga 4238 agtttggtgc ttctcctgca atgagtcgtg cgtgttcgta gttcggtact gggtcgtgct 4298 cggggagaat gttcttttgg gtcatggctt ctctttctgt tgctctgtgtgta gtccgtatgt 4358 gggcatggga aagccccggc aaccctttgg gtcaaccggg gctgcg gtcgt gtcg gtcg gtg cgtcg gtg cgtcgt cgtcgtcgt4g

<210> 8 <211> 427 <212> PRT <213> Brevibacterium lactofermentum <400> 8 Val Asn Thr Ser Lys Glu Pro Gln Val Asn Glu Gly Ser Lys Val Thr 1 5 10 15 Arg Ala Arg Ala Trp Arg Arg Gln Asn Val Met Tyr Lys Ile Thr Asn 20 25 30 Ser Lys Ala Leu Ala Gly Cys His Arg Trp Arg Arg Asp Glu Ala Val 35 40 45 Ala Val Ser Trp Ser Ser Asn Gly Ala Ser Gln Phe Glu Gly Leu Gln 50 55 60 Asn Ser His Ser Arg Trp Gly Ser Ser Leu Ala Glu Leu Glu Val Met 65 70 75 80 Gly Glu Arg Arg Ile Glu Leu Ala Ile Ala Thr Lys Asn His Leu Ala 85 90 95 Ala Gly Gly Ala Leu Met Met Phe Val Gly Thr Val Arg His Asn Arg 100 105 110 Ser Gln Ser Phe Ala Gln Val Glu Ala Gly Ile Lys Thr Ala Tyr Ser 115 120 125 Ser Met Val Lys Thr Ser Gln Trp Lys Lys Glu Arg Ala Arg Tyr Gly 130 135 140 Val Glu His Thr Tyr Ser Asp Tyr Glu Val Thr Asp Ser Trp Ala Asn 145 150 155 160 Gly Trp His Leu His Arg Asn Met Leu Leu Phe Leu Asp Arg Pro Leu 165 170 175 Ser Asp Asp Glu Leu Lys Ala Phe Glu Asp Ser Met Phe Ser Arg Trp 180 185 190 Ser Ala Gly Val Val Lys Ala Gly Met Asp Ala Pro Leu Arg Glu His 195 200 205 Gly Val Lys Leu Asp Gln Val Ser Thr Trp Gly Gly Asp Ala Ala Lys 210 215 220 Met Ala Thr Tyr Leu Ala Lys Gly Met Ser Gln Glu Leu Thr Gly Ser 225 230 235 240 Ala Thr Lys Thr Ala Ser Lys Gly Ser Tyr Thr Pro Phe Gln Met Leu 245 250 255 Asp Met Leu Ala Asp Gln Ser Asp Ala Gly Glu Asp Met Asp Ala Val 260 265 270 Leu Val Ala Arg Trp Arg Glu Tyr Glu Val Gly Ser Lys Asn Leu Arg 275 280 285 Ser Ser Trp Ser Arg Gly Ala Lys Arg Ala Leu Gly Ile Asp Tyr Ile 290 295 300 Asp Ala Asp Val Arg Arg Glu Met Glu Glu Glu Leu Tyr Lys Leu Ala 305 310 315 320 Gly Leu Glu Ala Pro Glu Arg Val Glu Ser Thr Arg Val Ala Val Ala 325 330 335 Leu Val Lys Pro Asp Asp Trp Lys Leu Ile Gln Ser Asp Phe Ala Val 340 345 350 Arg Gln Tyr Val Leu Asp Cys Val Asp Lys Ala Lys Asp Val Ala Ala 355 360 365 Ala Gln Arg Val Ala Asn Glu Val Leu Ala Ser Leu Gly Val Asp Ser 370 375 380 Thr Pro Cys Met Ile Val Met Asp Asp Val Asp Leu Asp Ala Val Leu 385 390 395 400 400 Pro Thr His Gly Asp Ala Thr Lys Arg Asp Leu Asn Ala Ala Val Phe 405 410 415 Ala Gly Asn Glu Gln Thr Ile Leu Arg Thr His 420 425

<210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 9 aaacccgggc tacgtctgat gctttgaatc 30

<210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 10 tttgatcccc cgttaacgtc aacaacc 27

<210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 11 ttttcccggg agcttgccac accccgag 28

<210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 12 gggggtcatc tctggctgaa ttgg 24

<210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 13 gaggttttca ccgttctgca tgcc 24

<210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for introducing mutation to pAM330 <400> 14 aactcaccgc cctgcaattc aac 23

<210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 15 gcctaccgcg gcaaagaagt ggcag 25

<210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 16 gccttgaact aggggcgctt taagt 25

<210> 17 <211> 4235 <212> DNA <213> Brevibacterium flavum <220> <221> CDS <222> (1852) .. (2364) <400> 17 aaacccgggt tttcttctgc aactcgggcg ccgaagcaaa cgaggctgct ttcaagattg 60 cacgctg tggtcgttcc cggattctgg ctgcagttca tggtttccac ggccgcacca 120 tgggttccct cgcgctgact ggccagccag acaagcgtga agcgttcctg ccaatgccaa 180 gcggtgtgga gttctaccct tacggcgaca ccgattactt gcgcaaaatg gtagaaacca 240 acccaacgga tgtggctgct atcttcctcg agccaatcca gggtgaaacg ggcgttgttc 300 cagcacctga aggattcctc aaggcagtgc gcgagctgtg cgatgagtac ggcatcttga 360 tgatcaccga tgaagtccag actggcgttg gccgtaccgg cgatttcttt gcacatcagc 420 acgatggcgt tgttcccgat gtggtgacca tggccaaggg acttggcggc ggtcttccca 480 tcggtgcttg tttggccact ggccgtgcag ctgaattgat gaccccaggc aagcacggca 540 ccactttcgg tggcaaccca gttgcttgtg cagctgccaa ggcagtgctg tctgttgtcg 600 atgacgcttt ctgcgcagaa gttacccgca agggcgagct gttcaaggta cttcttgcca 660 aggttgacgg cgttgtagac gtccgtggca ggggcttgat gttgggcgtg gtgctggagc 720 gcgacgtcgc aaagcaagct gttcttgatg gtttt aagca cggcgttatt ttgaatgcac 780 cggcggacaa cattatccgt ttgaccccgc cgctggtgat caccgacgaa gaaatcgcag 840 acgcagtcaa ggctattgcc gagacaatcg cataaaggac ttaaacttat gacttcacaa 900 ccacaggttc gccatttcct ggctgatgat gatctcaccc ctgcagagca ggcagaggtt 960 ttgaccctag ccgcaaagct caaggcagcg ccgttttcgg agcgtccact cgagggacca 1020 aagtccgttg cagttctttt tgataagact tcaactcgta ctcgcttctc cttcgacgcg 1080 ggcatcgctc atttgggtgg acatgccatc gtcgtggatt ccggcagctc acagatgggt 1140 aagggcgaga ccctgcagga caccgcagct gtattgtccc gctacgtgga agcaattgtg 1200 tggcgcacct acgcacacag caatttccac gccatggcgg agacgtccac tgtgccgctg 1260 gtgaactcct tgtccgatga tctgcaccca tgccagattc tggctgatct gcagaccatc 1320 gtggaaaacc tcagccctga agaaggccca gcaggcctta agggtaagaa ggctgtgtac 1380 ctgggcgatg gcgacaacaa catggccaac tcctacatga ttggctttgc caccgcgggc 1440 atggatattt ccatcatcgc tcctgaaggg ttccagcctc gtgcggaatt cgtggagcgc 1500 gcggaaaagc gtggccagga aaccggcgcg aaggttgttg tcaccgacag cctcgacgag 1560 gttgccggcg ccgatgttgt catcaccgat acctgggtat ccat gggtat ggaaaacgac 1620 ggcatcgatc gcaccacacc tttcgttcct taccaggtca acgatgaggt catggcgaaa 1680 gctaacgacg gcgccatctt cctgcactgc cttcctgcct accgcggcaa agaagtggca 1740 gcctccgtga ttgatggacc agcgtccaaa gttttcgatg aagcagaaaa ccgcctccac 1800 gctcagaaag cactgctggt gtggctgctg gccaaccagc cgaggtaaga c atg tct 1857 Met Ser 1 ctt ggc tca acc ccg tca aca ccg gaa aac tta aat ccc gtg act cgc 1905 Leu Gly Ser Thr Pro Ser Thr Pro Glu Asn Leu Asn Pro Val Thr Arg 5 10 15 act gca cgc caa gct ctc att ttg cag att ttg gac aaa caa aaa gtc 1953 Thr Ala Arg Gln Ala Leu Ile Leu Gln Ile Leu Asp Lys Gln Lys Val 20 25 30 acc agc cag gta caa ctg tct gaa ttg ctg ctg gat gaa ggc atc gat 2001 Thr Ser Gln Val Gln Leu Ser Glu Leu Leu Leu Asp Glu Gly Ile Asp 35 40 45 50 atc acc cag gcc acc ttg tcc cgg gat ctc gat gaa ctc ggt gca cgc 2049 Ile Thr Gln Ala Thr Leu Ser Arg Asp Leu Asp Glu Leu Gly Ala Arg 55 60 65 aag gtt cgc ccc gat ggg gga cgc gcc tac tac gcg gtc ggc cta g Arg Pro Asp Gly Gly Arg Ala Tyr Tyr A la Val Gly Pro Val 70 75 80 gat agc atc gcc cgc gaa gat ctc cgg ggt ccg tcg gag aag ctg cgc 2145 Asp Ser Ile Ala Arg Glu Asp Leu Arg Gly Pro Ser Glu Lys Leu Arg 85 90 95 cgc atg ctt gat gaag ctg gtt tct aca gat cat tcc ggc aac atc 2193 Arg Met Leu Asp Glu Leu Leu Val Ser Thr Asp His Ser Gly Asn Ile 100 105 110 gcg atg ctg cgc acc ccg ccg gga gct gcc cag tac ctg gca agt ttc Leu Met Leu Arg Thr Pro Pro Gly Ala Ala Gln Tyr Leu Ala Ser Phe 115 120 125 130 atc gat agg gtg ggg ctg aaa gaa gtc gtt ggc acc atc gct ggc gat 2289 Ile Asp Arg Val Gly Leu Lys Glu Val Val Gly Thr Ile Ala Gly Asp 135 140 145 gac acc gtt ttt gtt ctc gcc cgt gat ccg ctc aca ggt aaa gaa cta 2337 Asp Thr Val Phe Val Leu Ala Arg Asp Pro Leu Thr Gly Lys Glu Leu 150 155 160 ggt gaa tta ctc agc ggg cgc acc act cataagcag 2384 Gly Glu Leu Leu Ser Gly Arg Thr Thr 165 170 gcttgttaat cgcttgttaa tgcaggcagg taaggtataa cccgagtgtt ttttcgagga 2444 ataccaaccc tttcaacaca ataattttct ttaaacatcc ttgctgtcca ccacggctgg 25 04 caaggaactt aaaatgaagg agcacacctc atgactaacc gcatcgttct tgcatactcc 2564 ggcggtctgg acaccactgt ggcaattcca tacctgaaga agatgattga tggtgaagtc 2624 atcgcagttt ctctcgacct gggccagggt ggagagaaca tggacaacgt tcgccagcgt 2684 gcattggatg ccggtgcagc tgagtccatc gttgttgatg caaaggatga gttcgctgag 2744 gagtactgcc tgccaaccat caaggcaaac ggcatgtaca tgaagcagta cccactggtt 2804 tctgcaatct cccgcccact gatcgtcaag cacctcgttg aggctggcaa gcagttcaac 2864 ggtacccacg ttgcacacgg ctgcactggt aagggcaacg accaggttcg tttcgaggtc 2924 ggcttcatgg acaccgatcc aaacctggag atcattgcac ctgctcgtga cttcgcatgg 2984 acccgcgaca aggctatcgc cttcgccgag gagaacaacg ttccaatcga gcagtccgtg 3044 aagtccccat tctccatcga ccagaacgtc tggggccgcg ctattgagac cggttacctg 3104 gaagatctgt ggaatgctcc aaccaaggac atctacgcat acaccgagga tccagctctg 3164 ggtaacgctc cagatgaggt catcatctcc ttcgagggtg gcaagccagt ctccatcgat 3224 ggccgtccag tctccgtact gcaggctatt gaagagctga accgtcgtgc aggcgcacag 3284 ggcgttggcc gccttgacat ggttgaggac cgtctcgtgg gcatcaagtc ccgcgaaatc 3344 tac gaagcac caggcgcaat cgcactgatt aaggctcacg aggctttgga agatgtcacc 3404 atcgagcgcg aactggctcg ctacaagcgt ggcgttgacg cacgttgggc tgaggaagta 3464 tacgacggcc tgtggttcgg acctctgaag cgctccctgg acgcgttcat tgattccacc 3524 caggagcacg tcaccggcga tatccgcatg gttctgcacg caggttccat caccatcaat 3584 ggtcgtcgtt ccagccactc cctgtacgac ttcaacctgg ctacctacga caccggcgac 3644 accttcgacc agaccctggc taagggcttt gtccagctgc acggtctgtc ctccaagatc 3704 gctaacaagc gcgatcgcga agctggcaac aactaagcca ccttttcaag catccagact 3764 agaacttcaa gtatttagaa agtagaagaa caccacatgg aacagcacgg aaccaatgaa 3824 ggtgcgctgt ggggcggccg cttctccggt ggaccctccg aggccatgtt cgccttgagt 3884 gtctccactc atttcgactg ggttttggcc ccttatgatg tgttggcctc caaggcacac 3944 gccaaggttt tgcaccaagc agagctactt tctgatgaag atctagccac catgctggct 4004 ggtcttgatc agctgggcaa ggatgtcgcc gacggaacct tcggtccgct gccttctgat 4064 gaggatgtgc acggcgcgat ggaacgcggt ctgattgacc gcgttggtcc tgaggtgggc 4124 ggccgtctgc gcgctggtcg ttcccgcaac gaccaggtgg caaccctgtt ccgcatgtgg 4184 gtccgcgac g cagtgcgcga catcgcgctg ggaacaaccg agcttgtcga c 4235

<210> 18 <211> 171 <212> PRT <213> Brevibacterium flavum <400> 18 Met Ser Leu Gly Ser Thr Pro Ser Thr Pro Glu Asn Leu Asn Pro Val 1 5 10 15 Thr Arg Thr Ala Arg Gln Ala Leu Ile Leu Gln Ile Leu Asp Lys Gln 20 25 30 Lys Val Thr Ser Gln Val Gln Leu Ser Glu Leu Leu Leu Asp Glu Gly 35 40 45 Ile Asp Ile Thr Gln Ala Thr Leu Ser Arg Asp Leu Asp Glu Leu Gly 50 55 60 Ala Arg Lys Val Arg Pro Asp Gly Gly Arg Ala Tyr Tyr Ala Val Gly 65 70 75 80 Pro Val Asp Ser Ile Ala Arg Glu Asp Leu Arg Gly Pro Ser Glu Lys 85 90 95 Leu Arg Arg Met Leu Asp Glu Leu Leu Val Ser Thr Asp His Ser Gly 100 105 110 Asn Ile Ala Met Leu Arg Thr Pro Pro Gly Ala Ala Gln Tyr Leu Ala 115 120 125 Ser Phe Ile Asp Arg Val Gly Leu Lys Glu Val Val Gly Thr Ile Ala 130 135 140 Gly Asp Asp Thr Val Phe Val Leu Ala Arg Asp Pro Leu Thr Gly Lys 145 150 155 160 Glu Leu Gly Glu Leu Leu Ser Gly Arg Thr Thr 165 170

<210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 19 cccgggtttt cttctgcaac tcggg 25

<210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 20 gtcgacaagc tcggttgttc ccagc 25

<210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 21 cccctagttc aaggcttgtt aatc 24

<210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 22 gtcttacctc ggctggttgg ccagc 25

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction process of plasmid pK1.

FIG. 2 is a diagram showing a construction process of a plasmid pSFK6.

FIG. 3 is a diagram showing a construction process of a plasmid pSFKT2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:15) (C12P 13/10 B (C12N 15/09 C12R 1:15) C12R 1:15) C12N 15/00 ZNAA (C12P 13/10 A C12R 1:15) C12R 1:15) (72) Inventor Yukiko Mori 1-1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. Fermentation Technology Research Laboratories (72) Inventor Hisao Ito 1-1 Ajinomoto Co., Ltd. Fermentation Technology Laboratory, Suzuki-cho, Kawasaki-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Osamu Kurahashi 1-1 Fermentation Technology Research Laboratories, Ajinomoto Co., Ltd. Ajinomoto Co., Ltd. F-term (reference) 4B024 AA01 BA71 CA01 DA10 GA11 4B064 AE24 CA02 CA19 CC24 DA01 4B065 AA22X AA22Y AA24X AA24Y AB01 AC14 BA02 CA17 CA44

Claims (4)

[Claims] 1. A coryneform bacterium in which an arginine repressor does not function normally and has an L-arginine-producing ability. 2. The coryneform bacterium according to claim 1, wherein the arginine repressor does not function normally because the gene encoding the arginine repressor on the chromosome is disrupted. 3. The coryneform bacterium according to claim 2, wherein the arginine repressor has an amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having homology to the amino acid sequence. 4. The method according to claim 1, wherein the coryneform bacterium according to claim 1 is cultured in a medium, L-arginine is produced and accumulated in the medium, and the L-arginine is collected from the medium. A method for producing L-arginine.