JP3767066B2 - New gene - Google Patents

Description

【００３９】

【００４０】

【００４１】

【００４２】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel gene of coryneform bacteria, and particularly to a gene similar to a surfactant resistance gene.
[0002]
[Prior art]
Conventionally, L-lysine and L-glutamic acid are industrially produced by fermentation using coryneform bacteria belonging to the genus Brevibacterium or Corynebacterium having these amino acid-producing abilities. In this method, it is known that coryneform bacteria require biotin for growth, but L-glutamic acid does not accumulate if an excessive amount of biotin is present in the medium. Therefore, in the conventional method for producing L-glutamic acid, when culture is carried out in a medium with a limited biotin concentration or when a medium containing a sufficient amount of biotin is used, an interface as a biotin action inhibitory substance is initially or during the culture. Either a method of culturing an active agent or a lactam antibiotic in a medium is employed. However, in particular, when a raw material containing an excessive amount of biotin such as molasses is used as a carbon source of the medium, a biotin action inhibitory substance that needs to be added to the medium causes high production costs. It was.
[0003]
On the other hand, the present inventors have identified the presence of a gene ( dts R gene) derived from a Corynebacterium bacterium and encoding a protein (DTSR protein) that imparts resistance to a surfactant to the bacterium, The coryneform L-glutamic acid-producing bacterium in which the gene is disrupted produces a significant amount of L-glutamic acid even in the presence of an amount of biotin in which the wild strain hardly generates L-glutamic acid, and L-lysine production It has been found that a coryneform L-glutamic acid-producing bacterium having an ability is capable of producing a significant amount of L-lysine when the dts R gene is amplified (WO95 / 23224 international publication pamphlet).
[0004]
Further, the present inventors can stably produce L-glutamic acid by fermentation in the presence of biotin by imparting temperature sensitivity to a biotin action-inhibiting substance to coryneform L-glutamic acid-producing bacteria. It has been found that by giving L-lysine productivity to a temperature-sensitive strain for such a biotin action inhibitory substance, L-lysine and L-glutamic acid can be stably produced by fermentation simultaneously in the presence of biotin. (WO96 / 06180 international publication pamphlet).
[0005]
Examples of such a gene involved in temperature sensitivity to a biotin action inhibitory substance include the above dts R gene, and the amino acid sequence of the DTSR protein encoded by this gene is Proc.Nati.Acad.Sci.USA. vol.83 (1986) 8049-8053; Proc.Nati.Acad.Sci.USA vol.83 (1986) 4864-4868; Gene vol. 122 (1992) 199-202, Propionylcoe (CoA) carboxylase (PCC) ) It has been found to be homologous to the proteins described as protein β subunits. Propionyl coe carboxylate is an enzyme that catalyzes one of the metabolic pathways that convert α-ketoglutarate through 2-hydroxyglutarate, propionyl coe, D-methyl malonyl coe, and L-methyl malonyl coe to succinyl coe. Yes, the metabolic pathway appears to bypass the reaction catalyzed by alpha ketoglutarate dehydrogenase in the TCA cycle. Propionyl coace carboxylase is an enzyme using biotin as a coenzyme.
[0006]
From these, it is suggested that the reaction catalyzed by propionylcoe carboxylase, and further, the metabolic pathway including this reaction or a part thereof is involved in surfactant resistance. Therefore, genes involved in surfactant resistance include, in addition to the dts R gene, a gene encoding propionylcoe carboxylase α subunit, another enzyme that catalyzes each reaction of the above metabolic pathway, or a subunit thereof. It was considered likely to be included. In addition, the present inventors have found that DTSR protein-deficient strains require oleic acid for culture, and the structure of acetylcoe carboxy race using biotin as a coenzyme is similar to that of propionyl coe carboxy race. Furthermore, it was considered that an enzyme that catalyzes each reaction in the fatty acid metabolic pathway or a gene encoding a subunit thereof may be involved in surfactant resistance.
[0007]
[Problems to be solved by the invention]
This invention is made | formed from the said viewpoint, and makes it a subject to provide the gene concerned in the tolerance with respect to biotin action suppression substances, such as surfactant of a coryneform bacterium.
[0008]
[Means for Solving the Problems]
The present inventor found that when the dts R gene was cloned, another ORF was present in the middle immediately downstream of the ORF (open reading frame) encoding the DTSR protein. And it succeeded in obtaining the clone containing this ORF full length, and discovered that the protein which this ORF codes has a homology with DTSR protein.
[0009]
That is, the present invention is a DNA encoding the protein shown in the following (A) or (B).
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.
(B) A protein having an amino acid sequence containing a substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing.
[0010]
Specific examples of the DNA include DNA shown in the following (a) or (b).
(A) DNA containing a base sequence consisting of base numbers 2331 to 3941 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) A DNA that hybridizes under stringent conditions with a base sequence consisting of base numbers 2331 to 3941 among the base sequences set forth in SEQ ID NO: 1 in the Sequence Listing.
[0011]
Hereinafter, any of the above DNAs may be referred to as “the gene of the present invention” or “ dts R2 gene”, and the protein encoded by these DNAs may be referred to as “DTSR2 protein”.
[0012]
The coryneform bacterium referred to in the present invention is a group of microorganisms defined in Bargey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974), An aerobic, Gram-positive, non-acidic, and spore-forming gonococcus, previously classified as a genus Brevibacterium but now integrated as a Corynebacterium bacterium (Int. J Syst. Bacteriol., 41, 255 (1981)), and Brevibacterium and Microbatterium bacteria closely related to Corynebacterium.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The gene of the present invention is prepared by linking various fragments of chromosomal DNA of a wild-type coryneform bacterium with a vector that functions in the coryneform bacterium to produce various recombinant DNAs. Transform by introducing into a sensitive mutant, select the strain that lost the surfactant sensitivity from the transformed strain, collect the recombinant DNA from the transformed strain that lost the surfactant sensitivity, By analyzing the structure of the chromosomal DNA fragment of the wild-type coryneform bacterium linked to the vector, it was obtained as an ORF immediately downstream of the dts R gene. Thus, the gene of the present invention can be obtained in the same manner as the method for obtaining the dts R gene described in WO95 / 23224 international publication pamphlet. Hereinafter, a method for obtaining the gene of the present invention using a surfactant-sensitive mutant of coryneform bacteria will be described.
[0014]
A surfactant-sensitive mutant strain refers to a mutant strain of coryneform bacteria that grows poorly in a medium containing a surfactant at a concentration that does not affect the growth of wild-type coryneform bacteria. For example, when the surfactant is polyoxyethylene sorbitan monopalmitate, a surfactant-sensitive mutant of coryneform bacterium grows when the above surfactant is added to the medium at a concentration of 0.1 to 1 mg / dl. Worse compared to wild strains. On the other hand, the wild-type coryneform bacterium does not show a significant change in growth even in a medium supplemented with the above-described surfactant at a concentration of 0.1 to 1 mg / dl. Specific examples of such surfactant-sensitive mutants include Corynebacterium glutamicum AJ11060. This stock has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, and has been assigned the deposit number FERM P-3678.
[0015]
Chromosomal DNA is recovered from the surfactant-sensitive mutant of the coryneform bacterium as described above, for example, according to the method of Saito et al. (H. Saito and K. Miura Biochem. Biophys. Acta 72, 619 (1963)). The recovered chromosomal DNA is cleaved using a restriction enzyme and ligated to a vector that functions in coryneform bacteria to obtain various recombinant DNAs.
[0016]
In order to introduce these various recombinant DNAs into a surfactant-sensitive mutant of a coryneform bacterium, transformation methods that have been reported so far may be used. For example, as reported for Escherichia coli K-12, a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. , 53 , 159 (1970)), and a method of introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE). Gene, 1 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily incorporates recombinant DNA. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168 , 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274 , 398 (1978); Hinnen, A. Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). The transformation method used in the examples of the present invention is the electric pulse method (see JP-A-2-207791).
[0017]
Next, the transformed strain was once added with a surfactant-free M-CM2G agar plate (glucose 5 g, polypeptone 10 g, yeast extract 10 g, NaCl 5 g, DL-methionine 0.2 g, agar 15 g and chloramphenicol 4 mg. It is contained in 1 L of pure water and applied to pH 7.2) to form tens of thousands of colonies. The colony is replicated on an M-CM2G plate containing 30 mg / L surfactant (Tween 40), and the ones that show good growth on the surfactant-containing M-CM2G plate are obtained, whereby the surfactant sensitivity is increased. The lost stock can be acquired.
[0018]
Recombinant DNA is prepared from a transformed strain that has lost its detergent sensitivity in the same manner as the chromosomal DNA of the wild-type coryneform bacterium, and the nucleotide sequence of the chromosomal DNA fragment of the wild-type coryneform bacterium linked to the vector To decide.
As an example of the gene of the present invention obtained as described above, the base sequence of a DNA fragment containing the dts R2 gene obtained from Brevibacterium lactofermentum ATCC 13869 is shown in SEQ ID NO: 1 in the sequence listing. In the base sequence shown in SEQ ID NO: 1, the dts R2 gene corresponds to base numbers 2331-3941. In the base sequence shown in SEQ ID NO: 1, base numbers 359 to 1987 are the coding sequence of the dts R gene. Further, the amino acid sequence of the DTSR2 protein encoded by the dts R2 gene is shown in SEQ ID NO: 3, and the amino acid sequence of the DTSR protein encoded by the dts R gene is shown in SEQ ID NO: 2.
[0019]
In the examples of the present invention, the DNA fragment obtained as described above lacked a part of the 3 ′ end of the gene of the present invention. Therefore, a polymerase using an oligonucleotide primer having the base sequence shown in SEQ ID NO: 4 (corresponding to base numbers 3624 to 3653 of SEQ ID NO: 1) and SEQ ID NO: 5 (corresponding to base numbers 3721 to 3750 of SEQ ID NO: 1) The chain reaction method (PCR: polymerase chain reaction; White, TJ et al; see Trends Genet. 5,185 (1989)) is used to amplify the 3 ′ terminal portion of the gene of the present invention from Brevibacterium lactofermentum ATCC 13869 By doing this, this part was acquired and the base sequence was determined.
[0020]
Since the nucleotide sequence of the gene of the present invention has been clarified, the gene of the present invention is selected from a chromosomal DNA library of coryneform bacteria by hybridization using an oligonucleotide prepared based on the nucleotide sequence shown in SEQ ID NO: 1 as a probe. Can be acquired. The gene of the present invention can also be obtained by amplifying the full length of the gene of the present invention from the chromosomal DNA of coryneform bacteria by PCR using an oligonucleotide prepared based on the nucleotide sequence shown in SEQ ID NO: 1 as a primer. Can do. The Escherichia coli JM109 / pDTR6 (private number AJ12967) strain carrying the plasmid pDTR6 containing the dts R gene was transferred to the Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry on February 22, 1994. It was deposited as -14168, transferred to an international deposit based on the Budapest Treaty on February 9, 1995, and assigned the deposit number FERM BP-4994. The d ts R gene and its downstream region contained in pDTR6 prepared from this strain can be used as the hybridization probe.
[0021]
The gene of the present invention includes a DNA having the base sequence encoding the amino acid sequence shown in SEQ ID NO: 3 in addition to the DNA having the base sequence represented by base numbers 2331 to 3941 in the base sequence shown in SEQ ID NO: 1. In addition, in the amino acid sequence shown in SEQ ID NO: 3, even a protein having an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues can interact with coryneform bacteria. As long as it has a function of imparting resistance to an active agent, DNA encoding such a protein is included in the gene of the present invention.
[0022]
In the amino acid sequence shown in SEQ ID NO: 3, a DNA encoding a protein having an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acid residues is, for example, a coryneform bacterium A DNA that hybridizes under stringent conditions with a DNA having at least a part of the base sequence represented by base numbers 2331 to 3941 in the base sequence shown in SEQ ID NO: 1 in the sequence listing is isolated from a wild strain or a mutant strain Can be obtained. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, DNAs with high homology, for example, DNAs having 90% or more homology hybridize and DNA with lower homology than that. Conditions for not hybridizing to each other, or Tm to (Tm-30) ° C., preferably Tm to (Tm-20) ° C. of a hybrid whose temperature is perfectly matched, and 1 × SSC, preferably 0.1 × A condition for hybridizing at a salt concentration corresponding to SSC is mentioned. In the base sequence shown in SEQ ID NO: 1 in the sequence listing, the base sequence represented by base numbers 2331 to 3941 and the coding sequence of the dts R gene do not hybridize under stringent conditions.
[0023]
In addition, the DNA encoding the DTSR2 protein having the mutation as described above has a nucleotide sequence that causes substitution, deletion, insertion, impossibility or inversion of the amino acid residue at a specific site by site-specific mutagenesis. It is obtained by modifying. Moreover, the dts R2 gene having such a mutation can be obtained by a conventionally known mutation treatment. Mutation treatment includes a method of treating DNA containing dts R2 gene in vitro with hydroxylamine or the like, and a microorganism holding DNA containing dts R2 gene by ultraviolet irradiation or N-methyl-N′-nitro-N-nitroso. Examples include treatment with a mutating agent that is usually used for artificial mutation such as guanidine (NTG) or nitrous acid. After the mutation treatment, the DTSR2 protein encoded or produced from the mutation-treated DNA or the mutation-treated microorganism retains the function of imparting resistance to the surfactant to the coryneform bacterium, and the DTSR2 protein By selecting the amino acid sequence that has been mutated, the position where the mutation can be introduced or the position where the mutation has occurred can be determined. The position of the introduced mutation is not particularly limited as long as it does not substantially affect the function of the DTSR2 protein. The number of mutations to be introduced also varies depending on the position and type of amino acid to be mutated in the three-dimensional structure of the protein, and is not particularly limited as long as it does not substantially affect the function of the DTSR2 protein. The number is preferably 1 to 10.
[0024]
The DSTR protein encoded by the dts R2 gene is highly homologous to the DTSR protein encoded by the dts R gene, and is presumed to have the same function. Therefore, it is expected that the dts R2 gene can be used for breeding coryneform bacteria used for the production of L-glutamic acid, L-lysine and the like, like the dts R gene.
[0025]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0026]
[Example 1]
Preparation of Chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 (Wild strain of Corynebacterium) Brevibacterium lactofermentum ATCC 13869 was prepared from TY medium (Bacto-trypton (Difco) 1%, Bacto-yeasttextract ( Difco) 0.5%, NaCl 0.5% (pH 7.2)) 100 ml was inoculated and cultured at a temperature of 31.5 ° C. for 8 hours to obtain a culture. After centrifuging this culture for 15 minutes at 3,000 rpm to obtain 0.5 g of wet cells, Saito and Miura's method (Biochem. Biophys. Acta., 72, 619 (1963)) was used. Chromosomal DNA was obtained. Next, 60 μg of this chromosomal DNA and 3 units of restriction enzyme Sau 3AI were mixed with 10 mM Tris-HCl buffer solution (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4)), and the temperature was changed to 30 ° C. for 30 minutes. Reacted for 1 minute. The reaction-terminated solution was subjected to phenol extraction treatment in the usual manner, ethanol precipitation treatment, and 50 μg of a chromosomal DNA fragment of Brevibacterium lactofermentum ATCC 13869 digested with Sau 3AI.
[0027]
[Example 2]
Preparation of Brevibacterium lactofermentum ATCC 13869 gene library using plasmid vector DNA Plasmid vector DNA (pSAC4) capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria and restriction enzyme Bam HI200 unit was mixed with 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM magnesium sulfate (pH 7.4)) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digested solution. Ethanol precipitation was performed. Thereafter, in order to prevent recombination of DNA fragments derived from the plasmid vector, the method of Molecular Cloning 2nd editon (J. Sambrook, EFFritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p1.56 (1989)) is used. The DNA fragment was dephosphorylated by a bacterial alkaline phosphatase treatment, phenol extracted by a conventional method, and further subjected to an ethanol precipitation treatment.
[0028]
1 μg of pSAC4 digested with Bam HI, 1 μg of chromosomal DNA fragment of Brevibacterium lactofermentum ATCC 13869 digested with Sau 3AI obtained in Example 1, and 2 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) Was added to 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM magnesium chloride, 10 mM dithiothreitol and 10 mM ATP, and reacted at a temperature of 16 ° C. for 16 hours to ligate DNA. Subsequently, Escherichia coli DH5 was transformed with the DNA mixture by a conventional method, and this was spread on an L agar medium containing 170 μg / ml of chloramphenicol to obtain about 20,000 colonies to obtain a gene library. .
[0029]
[Example 3]
Transformation of Brevibacterium lactofermentum AJ11060 Recombinant DNA was recovered from the approximately 20,000 colonies described above. The method of recovery was according to the method of Saito and Miura shown above.
[0030]
The recombinant DNA mixture divided into 50 batches was introduced into a mutant AJ11060 with increased sensitivity to a surfactant according to a conventional method of transformation using an electric pulse method (Japanese Patent Laid-Open No. 2-207791). The transformant was inoculated on agar medium supplemented with glucose and statically cultured at 31.5 ° C., and about 20,000 transformants appeared. Next, these transformants were replicated on the same plate containing 30 mg / l of a surfactant, and several strains that were resistant to the surfactant and were able to grow on the plate were obtained.
[0031]
Recombinant DNA was extracted from each of the several strains obtained above, and AJ11060 strain was retransformed using the DNA. Again, a strain that was resistant to the surfactant was obtained. The recombinant DNA retained by one of these strains was named pDTR6, and the recombinant DNA retained by the other strain was named pDTR98. Inhibition of growth in a liquid medium supplemented with 3 g / L surfactant was suppressed in AJ11060 bacteria into which pDTR6 was introduced (see WO95 / 23224 International Publication Pamphlet).
[0032]
[Example 4]
Preparation of DNA A plasmid was prepared from AJ11060 / pDTR98 containing the recombinant DNA obtained above according to a conventional method, and introduced into Escherichia coli JM109. The resulting Escherichia coli JM109 / pDTR98 was pre-cultured at 20 ° C. for 24 hours in 20 ml of a medium consisting of 1% tryptone, 0.5% yeast extract and 0.5% NaCl. After inoculating 1 l of the medium having the same composition and culturing at 37 ° C. for 3 hours, 0.2 g of chloramphenicol was added, and further culturing was carried out at the same temperature for 20 hours to obtain a culture solution. Next, this culture solution was centrifuged at 3,000 rpm for 10 minutes to obtain 2 g of wet cells, and this was suspended in 350 mM Tris-HCl buffer (pH 8.0) containing 25 ml of 25% sucrose, Furthermore, 10 mg of lysozyme (manufactured by Sigma), 8 ml of 0.25M EDTA solution (pH 8.0) and 8 ml of 20% sodium dodecyl sulfate solution were added, respectively, and incubated at a temperature of 60 ° C. for 30 minutes to obtain a lysate. . To this lysate, 13 ml of a 5M NaCl solution was added, treated at a temperature of 4 ° C. for 16 hours, and then centrifuged at 15,000 rpm for 30 minutes. The obtained supernatant was subjected to phenol extraction treatment and ethanol precipitation treatment in the usual manner to precipitate DNA.
[0033]
This precipitate was dried under reduced pressure, dissolved in 6 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, and further added with 6 g of cesium chloride and 0.2 ml of ethidium bromide (19 mg / ml). Then, an equilibrium density gradient centrifugation treatment was performed using an ultracentrifuge for 42 hours at 39,000 rpm, and DNA was isolated. Furthermore, after removing ethidium bromide using n-butanol, about 500 μg of purified recombinant DNA pDTR98 was dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA. Obtained.
[0034]
[Example 5]
Analysis of the base sequence of the cloned DNA Using the recombinant DNA pDTR98 obtained in Example 4, the base sequence of about 3.8 kbp of the cloned DNA fragment (about 4.8 kbp) was determined. The base sequence was determined according to the method of Sanger using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biochemical). As a result, the cloned DNA fragment possessed by pDTR98 contained the entire coding region of the dts R gene, and further contained a region of about 1 kbp containing an ORF encoding a protein having high homology with the DTSR protein. . The base sequence of this cloned DNA fragment corresponds to base numbers 1 to 3869 of SEQ ID NO: 1 in the sequence listing.
[0035]
Since no termination codon was detected in the above ORF, the region downstream of this region was obtained by amplification by PCR using the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 as a template. PCR uses a kit using a DNA polymerase having 3 ′ → 5 ′ exonuclease activity (LA PCR in vitro cloning Kit, manufactured by Takara Shuzo Co., Ltd.), and the primer is SEQ ID NO: 4 (base numbers 3624 to 3653 of SEQ ID NO: 1). And an oligonucleotide having a base sequence shown in SEQ ID NO: 5 (corresponding to base numbers 3721 to 3750 of SEQ ID NO: 1). Specifically, a chromosomal gene of Brevibacterium lactofermentum ATCC 13869 was obtained by a conventional method, and 1 μg of this DNA was completely digested with the restriction enzyme EcoRI. Next, using this DNA, the EcoRI cassette primer included in the kit, and the primer of SEQ ID NO: 4, a cycle consisting of 94 ° C., 30 seconds; 55 ° C., 2 minutes; 72 ° C., 1 minute is repeated 30 times. Repeated. Next, this reaction solution was diluted 100 times, and 1 μl thereof was used as a template, and the PCR reaction was performed again using the primer of SEQ ID NO: 5 and the cassette primer C2 included in the kit. The PCR reaction was performed in the same cycle as described above.
[0036]
The base sequence of the amplified DNA fragment of about 1.2 kbp was determined by the same method as described above. This DNA fragment contained the ORF in an overlapping manner and further contained a termination codon. This gene was named dtsR2 gene, and the protein encoded by this gene was named DTSR2 protein.
[0037]
【The invention's effect】
According to the present invention, a novel gene, dtsR2 gene, is provided. This gene has high homology to the dts R gene, which is a gene involved in temperature sensitivity of coryneform bacteria to a biotin action inhibitor, and is expected to be usable for the production of various amino acids.
[0038]
[Sequence Listing]

[0039]

[0040]

[0041]

[0042]

Claims (2)

DNA encoding the protein shown in (A) or (B) below.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.
(B) in the amino acid sequence set forth in SEQ ID NO: 3, substitution of 1 to 10 amino acids, deletion, insertion, addition, or inversion of possess an amino acid sequence comprising, surfactant coryneform bacterium A protein that confers resistance to . The DNA according to claim 1, which is the DNA shown in (a) or (b) below.
(A) DNA containing a base sequence consisting of base numbers 2331 to 3941 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) a protein that hybridizes under stringent conditions with the nucleotide sequence consisting of nucleotide numbers 2331 to 3941 among the nucleotide sequences set forth in SEQ ID NO: 1 in the sequence listing, and imparts resistance to a surfactant to coryneform bacteria DNA encoding