JP2005040064A - Protein scdh2, gene encoding the same, microorganism having high acetic acid resistance, and method for producing vinegar using the microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To elucidate at genetic level a protein enhancing acetic acid resistance, to obtain microorganisms having high acetic acid resistance and capable of efficiently producing acetic acid in high concentrations, and to provide a method for efficiently producing a vinegar rich in acetic acid using the microorganisms. <P>SOLUTION: The protein SCDH2(short-chain dehydrogenase/reductase-2) is provided. A DNA for a gene encoding the protein SCDH2 is provided. The microorganisms having high acetic acid resistance are obtained by amplifying the copy number of the DNA in the microorganismal cells. The method for producing the vinegar comprises culturing in an alcohol-containing medium microorganisms having the ability to oxidize alcohol among the above-microorganisms and producing and accumulating acetic acid in the medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a protein SCDH2, a gene thereof, a microorganism having a high degree of acetic acid resistance, and a method for producing vinegar using the microorganism, and more specifically, a protein SCDH2 having a function of enhancing acetic acid resistance, and a gene encoding the protein SCDH2 The present invention relates to a microorganism having high acetic acid resistance and capable of efficiently producing a high concentration of acetic acid, and a method for efficiently producing vinegar having a high acetic acid concentration using the microorganism.

In industrial vinegar production, acetic acid fermentation by microorganisms is used. Such microorganisms have alcohol oxidizing ability and are generally called acetic acid bacteria. Among acetic acid bacteria, in particular, acetic acid bacteria belonging to the genus Acetobacter and Glucon acetobacter are widely used for industrial acetic acid fermentation.
In acetic acid fermentation, ethanol in the medium is oxidized by acetic acid bacteria and converted into acetic acid. As a result, acetic acid accumulates in the medium. Accumulated acetic acid is also inhibitory to acetic acid bacteria, and as the acetic acid accumulation amount increases and the acetic acid concentration in the medium increases, the acetic acid bacteria growth ability and fermentation ability gradually decrease.
Therefore, in industrial vinegar production, it is required to develop an acetic acid bacterium that does not decrease its ability to grow or ferment even at a higher acetic acid concentration, that is, an acetic acid bacterium having high acetic acid resistance. As one means, cloning of a protein gene (acetic acid resistant gene) involved in acetic acid resistance and having a function of enhancing acetic acid resistance, breeding and improvement of acetic acid bacteria using the acetic acid resistant gene has been attempted.

Previous knowledge of acetic acid resistance genes includes three genes that form clusters, ie complementary genes that can restore acetic acid-sensitive strains by mutating the acetic acid resistance of acetic acid bacteria belonging to the genus Acetobacter. There is one related to cloning of (aarA, aarB, aarC) (for example, see Non-Patent Document 1). Of the three genes, the aarA gene is a gene that encodes citrate synthase, and the aarC gene is presumed to be an enzyme that encodes an enzyme involved in the utilization of acetic acid. Was unknown (for example, refer nonpatent literature 2).
A transformant obtained by cloning a gene fragment containing these three genes into a multicopy plasmid and transforming it into an Acetobacter aceti subsp. Xylinum IFO3288 strain, The acetic acid resistance was only slightly improved, and the acetic acid resistance ability in actual acetic acid fermentation was unknown (for example, see Patent Document 1).

On the other hand, an example has been disclosed in which improvement of the final acetic acid concentration in acetic acid fermentation has been confirmed by introducing a gene encoding membrane-bound aldehyde dehydrogenase (ALDH) cloned from acetic acid bacteria into acetic acid bacteria. (For example, refer to Patent Document 2).
However, ALDH is an enzyme having a function to oxidize aldehydes and not an enzyme directly related to acetic acid resistance. Therefore, it cannot be determined that the gene encoding ALDH is truly an acetic acid resistant gene.

“Journal of Bacteriology”, 172, 2096-2104, 1990 ” "Journal of Fermentation and Bioengineering, Vol. 76, 270-275, 1993" JP-A-3-21878 JP-A-2-2364

As described above, there has been no report yet of a case where acetic acid resistance of acetic acid bacteria has been elucidated at the gene level and a practical acetic acid bacteria having high acetic acid resistance has been successfully developed.
Therefore, an object of the present invention is to first elucidate a protein that enhances acetic acid resistance at the gene level in order to improve acetic acid resistance of acetic acid bacteria. Furthermore, another object of the present invention is to obtain an acetic acid resistance enhancing gene encoding the protein, and to use this gene to have a high acetic acid resistance and to efficiently produce a high concentration of acetic acid. It is to obtain a microorganism and to provide a method for efficiently producing vinegar having a high acetic acid concentration using the microorganism.

  The inventors of the present invention have made extensive studies to achieve the above object, and in the process, acetic acid bacteria that can grow and ferment even in the presence of acetic acid have specific acetic acid resistance that does not exist in other microorganisms. Hypothesized that there is a gene involved in. And if such a gene is used, the acetic acid tolerance of microorganisms can be imparted and enhanced more than before, and furthermore, such microorganisms can be used to produce vinegar with a high acetic acid concentration that could not be obtained conventionally. I got a new idea that it would be possible to develop efficient manufacturing methods.

As a method for obtaining an acetic acid resistant gene in the prior art, a method of cloning a gene that complements an acetic acid-sensitive mutant of acetic acid bacteria is generally used.
However, the present inventors considered that it is difficult to find an industrially useful acetate-resistant gene by this method, and studied other acquisition methods. As a result, by constructing a chromosomal DNA library of acetic acid bacteria and transforming the chromosomal DNA library into acetic acid bacteria, the acetic acid bacteria that can usually grow only in the presence of about 1% acetic acid are transformed into 2% acetic acid. We have developed a method for obtaining genes by screening for genes that can grow in the presence of.
By this acquisition method, a novel protein SCDH2 gene (acetic acid resistance enhancing gene) having a function of enhancing acetic acid resistance at a practical level from the acetic acid bacteria of the genus Acetobacter and Glucon Acetobacter that are actually used in vinegar production The first successful cloning of DNA encoding

  The resulting coding region of the acetic acid resistance-enhancing gene has a certain degree of homology with a protein called oxidoreductase or short-chain dehydrogenase / reductase family, but the degree of homology is low, so it is specific to acetic acid bacteria. DNA that encodes a novel protein gene that is somewhat similar to a known protein gene called oxidoreductase or short-chain dehydrogenase / reductase family.

  In addition, a microorganism obtained by amplifying the copy number of DNA encoding an acetic acid resistance enhancing gene has a markedly enhanced acetic acid resistance, and thus it has been found that vinegar with a high acetic acid concentration can be efficiently produced, and the present invention has been completed. It came to do.

That is, the present invention described in claim 1 provides the protein SCDH2 shown in the following (A) or (B).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A function comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and enhancing acetate resistance A protein having

The present invention according to claim 2 provides DNA of a gene encoding the protein SCDH2 shown in (A) or (B).
The present invention according to claim 3 provides the DNA of the gene according to claim 2, which is the DNA shown in the following (a) or (b).
(A) DNA comprising a base sequence consisting of base numbers 216 to 974 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 1 in the Sequence Listing, the nucleotide sequence consisting of nucleotide numbers 216 to 974, or the DNA of the nucleotide sequence that can be a probe prepared from at least a part of the nucleotide sequence, and stringent conditions DNA encoding a protein that hybridizes underneath and has a function of enhancing acetic acid resistance.

The present invention according to claim 4 provides a microorganism having high acetate resistance, wherein the copy number of the DNA according to claim 2 or 3 in the microorganism cell is amplified.
The present invention according to claim 5 provides the microorganism according to claim 4, wherein the microorganism is an acetic acid bacterium of the genus Acetobacter or Glucon acetobacter.

  The present invention according to claim 6 is characterized in that the microorganism having the ability to oxidize alcohol among the microorganisms according to claim 4 or 5 is cultured in a medium containing alcohol and acetic acid is produced and accumulated in the medium. A method for producing vinegar is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the novel protein which has a function which enhances an acetic acid tolerance, and DNA of the gene are provided, Furthermore, microorganisms which can produce a higher concentration acetic acid efficiently can be obtained using this DNA. It is possible to provide a method for producing vinegar having a high acetic acid concentration with high efficiency by using the microorganism.

  According to the present invention, a novel protein having a function of enhancing acetic acid resistance and a DNA of the gene are provided, and a microorganism capable of efficiently producing a higher concentration of acetic acid can be obtained using the DNA. Furthermore, it has become possible to provide a method for producing a highly acetic acid vinegar using the microorganism with high efficiency.

Hereinafter, the present invention will be described in detail.
(1) Protein SCDH2 of the present invention
As described in claim 1, the protein SCDH2 of the present invention is a protein shown in the following (A) or (B).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A function comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and enhancing acetate resistance A protein having

Here, (A) the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing has a certain degree of homology with the amino acid sequences of various microorganisms called oxidoreductase or short-chain dehydrogenase / reductase family. However, since the degree of homology is as low as about 50%, it is a protein gene specific to acetic acid bacteria, which is somewhat similar to a protein gene called oxidoreductase or short-chain dehydrogenase / reductase family It was presumed to be DNA encoding a gene.
That is, when a homology search is performed at the amino acid sequence level in DDBJ / EMBL / Genbank and SWISS-PROT / PIR, the known oxidoreductase found in Mesorhizobium loti is 51.6% at the amino acid sequence level. It has 48.8% homology with Sinorhizobium meliloti oxidoreducase and 49.6%, and Bradyrhizobium japonicum short-chain dehydrogenase.

  As described above, the protein SCDH2 of the present invention may be a protein consisting of the amino acid sequence described in SEQ ID NO: 2 in the (A) Sequence Listing, but (B) the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing. A protein comprising an amino acid sequence including substitution, deletion, insertion, addition or inversion of 1 or several, preferably 1 to 5 amino acids, and having a function of enhancing acetic acid resistance, (A) The protein may be substantially the same protein as.

  Such a protein (B) is substituted or deleted by 1 or several, preferably 1 to 5 amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, for example, by site-directed mutagenesis. It can also be obtained by modifying the amino acid sequence to include an insertion, addition or inversion. It can also be obtained by conventionally known mutation treatments, and can be obtained from all acetic acid bacteria, especially from the species, strains, mutants, and variants of the genus Acetobacter and Gluconacetobacter. The details of the acquisition method will be described in detail in (2) the description of the DNA of the present invention described later, including means for confirming that it has a function of enhancing acetic acid resistance.

  Such a protein SCDH2 of the present invention is a microorganism such as an acetic acid bacterium belonging to the genus Acetobacter or Gluconacetobacter, specifically, for example, Acetobacter aceti No. 1023 strain (Acetobacter aceti No. 1023 .1023) (deposited with FERM BP-2287 at Patent Biological Depositary Center), Acetobacter aceti subspecies zylinum IFO3288 strain, Acetobacter aceti IFO3283 strain, Glucon strain Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) strain (patented in Gluconacetobacter europaeus DSM6160), Gluconacetobacter entanii (Deposited as FERM BP-491 in the biological deposit center).

(2) DNA of the present invention
As described in claim 2, the DNA of the present invention is a DNA of a gene encoding the protein SCDH2 shown in (A) or (B) above. Specifically, as described in claim 3, it is a DNA shown in the following (a) or (b).
(A) DNA comprising a base sequence consisting of base numbers 216 to 974 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 1 in the Sequence Listing, the nucleotide sequence consisting of nucleotide numbers 216 to 974, or the DNA of the nucleotide sequence that can be a probe prepared from at least a part of the nucleotide sequence, and stringent conditions DNA encoding a protein that hybridizes underneath and has a function of enhancing acetic acid resistance.
Needless to say, the DNA of the present invention may contain a base sequence regulatory element and a structural portion of the gene.

(A) a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, specifically, (a) among the base sequences described in SEQ ID NO: 1 in the sequence listing, consisting of base numbers 216 to 974 DNA containing the base sequence can be obtained from Gluconacetobacter entanii as follows.
First, chromosomal DNA of Glucon Acetobacter enterii, for example, Acetobacter altoacetigenes MH-24 (deposited as FERM BP-491 at the Patent Organism Depositary) is obtained. Chromosomal DNA can be obtained, for example, by the method disclosed in JP-A-60-9489.

Next, a chromosomal DNA library is prepared in order to isolate the gene that improves the acetic acid resistance of the present invention from the obtained chromosomal DNA.
That is, first, chromosomal DNA is partially decomposed with an appropriate restriction enzyme to obtain a mixture of various chromosomal DNA fragments. A wide variety of enzymes can be used as restriction enzymes, and the degree of cleavage is adjusted by adjusting the cleavage reaction time and the like according to the enzyme used. For example, Sau3AI is digested by acting on chromosomal DNA at a temperature of 30 ° C. or higher, preferably 37 ° C., at an enzyme concentration of 1 to 10 units / ml for various times (1 minute to 2 hours). In the examples described later, Sau3AI was used.

Next, the cleaved chromosomal DNA fragment is ligated to a vector DNA capable of autonomous replication in acetic acid bacteria.
In order to link a chromosomal DNA fragment to vector DNA, it is necessary to cleave and cleave the vector DNA using a restriction enzyme or the like. As a restriction enzyme, a restriction enzyme (for example, BamHI) that generates a terminal base sequence complementary to the restriction enzyme (for example, Sau3AI) used in obtaining the DNA fragment mixture can be used. In the case of using BamHI, the DNA is completely digested by acting on the vector DNA for 1 hour or more under conditions of a temperature of 30 ° C. and an enzyme concentration of 1 to 100 units / ml, and then cleaved.

The cleaved and cleaved vector DNA is mixed with a chromosomal DNA fragment mixture, and T ₄ DNA ligase is allowed to act on the mixture to obtain the desired recombinant DNA (DNA library). The working conditions of T ₄ DNA ligase can be, for example, 1 hour or more, preferably 6 to 24 hours under conditions of a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units / ml.

From the recombinant DNA (DNA library) thus obtained, a gene encoding the protein (A), specifically, the DNA (a) is selected.
The selection can be performed by an acetic acid resistance enhancement function. For example, acetic acid bacteria that cannot normally grow in the presence of acetic acid at a concentration higher than 1% on an agar medium, for example, Acetobacter aceti No. 1023 strain (FERM at the Patent Organism Depositary) BP-2287) is transformed with the recombinant DNA, and then applied to agar medium containing 2% acetic acid and cultured. The resulting colonies are inoculated into a liquid medium and cultured, and the plasmid is recovered from the resulting bacterial cells, whereby the target DNA can be obtained.

Thus, (A) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, specifically, (a) among the base sequences set forth in SEQ ID NO: 1 in the sequence listing, from base numbers 216 to 974 DNA containing the nucleotide sequence can be obtained. Here, among the base sequences described in SEQ ID NO: 1 in the sequence listing, the base sequence consisting of base numbers 216 to 974 is a coding region, and the amino acid sequence described in SEQ ID NO: 2 in the sequence listing corresponds to the coding region. Is.
(A) a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, specifically, (a) among the base sequences described in SEQ ID NO: 1 in the sequence listing, consisting of base numbers 216 to 974 Since the DNA containing the base sequence has about 50% homology with the amino acid sequence of a known oxidoreductase gene found in Mesorhizobium loti, it is a novel protein that resembles the oxidoreductase protein to some extent It is presumed to be a novel gene coding for. This point is as already described in the above section (1) Protein SCDH2 of the present invention.
The fact that the oxidoreductase protein gene is related to acetic acid resistance has never been known so far, and was first discovered by the present inventors.

  Furthermore, a gene encoding protein (A), specifically, DNA (a) is an acetic acid resistant gene (aarA, aarB, aarC) of an acetic acid bacterium already obtained or an ADH gene having a function of enhancing acetic acid resistance It was identified as a gene having a function of enhancing acetic acid resistance, which is different from the above.

Such a gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing, specifically, (a) among the base sequences described in SEQ ID NO: 1 in the Sequence Listing, the base number 216 The production of DNA containing a base sequence consisting of ˜974 is not limited to the above-mentioned method. For example, the genomic DNA of Acetobacter glucosacetobacter enterii is used as a template, and an oligonucleotide synthesized based on the base sequence is used as a primer It can also be obtained by polymerase chain reaction (PCR reaction) used in the above, or by hybridization using an oligonucleotide synthesized based on the base sequence as a probe.
Here, the synthesis of the oligonucleotide used as a primer can be synthesized according to a conventional method using, for example, various commercially available DNA synthesizers. The PCR reaction is performed using a thermal cycler, Gene Amp PCR System 2400, manufactured by Applied Biosystems, Taq DNA polymerase (manufactured by Takara Bio), KOD-Plus- (manufactured by Toyobo), and the like. Can be carried out according to the standard method.

The DNA of the present invention comprises (A) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing, specifically, (a) among the base sequences set forth in SEQ ID NO: 1 in the Sequence Listing. DNA containing a base sequence consisting of Nos. 216 to 974 may be used, but (B) substitution of 1 or several, preferably 1 to 5 amino acids in the amino acid sequence shown in SEQ ID No. 2 of the Sequence Listing, A protein comprising an amino acid sequence containing a deletion, insertion, addition, or inversion, and having a function of enhancing acetic acid resistance, that is, encoding a protein substantially identical to the protein of (A) good.
Such a DNA encoding the protein (B) is substituted by 1 or several, preferably 1 to 5 amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing by, for example, site-directed mutagenesis. It can also be obtained by modifying the base sequence so as to encode a protein consisting of an amino acid sequence containing a deletion, insertion, addition or inversion. The DNA encoding the protein (B) as described above can also be obtained by a known mutation treatment.
In general, it is known that the amino acid sequence of a protein and the base sequence of DNA encoding the same are slightly different between species, strains, mutants, and variants. Therefore, the DNA encoding the protein (B) is: It is possible to obtain DNA encoding the protein (B) as described above from all acetic acid bacteria, in particular from species, strains, mutants, and variants of the genus Acetobacter or Gluconacetobacter.

DNA encoding such a protein (B) was prepared from a base sequence consisting of base numbers 216 to 974 or at least a part of the base sequence in the base sequence described in SEQ ID NO: 1 in the sequence listing (b) It can be obtained as DNA encoding a protein that hybridizes with a DNA having a base sequence that can serve as a probe under stringent conditions and has a function of enhancing acetic acid resistance.
For example, acetic acid bacteria in general, especially Acetobacter or Gluconacetobacter species, strains, mutants, variants, more specifically Acetobacter or Gluconacetobacter acetic acid bacteria, these naturally or artificially It can be a probe prepared from at least a part of the base sequence consisting of base numbers 216 to 974 out of the base sequence described in SEQ ID NO: 1 in the sequence listing from the mutant (mutant strain or variant) obtained by mutation It can be obtained by hybridizing with DNA of a base sequence under stringent conditions.

  The stringent condition referred to here is a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify this condition clearly, for example, DNAs with high homology, for example, DNAs having homology of 70% or more hybridize and DNA with lower homology than that. Examples include conditions where they do not hybridize with each other or normal hybridization washing conditions, for example, conditions in which washing is performed at 60 ° C. at a salt concentration corresponding to 0.1% SDS with 1 × SSC.

  Confirmation of having a function of enhancing acetic acid resistance is, for example, as described in Examples below, Acetobacter aceti No. which can usually grow only up to about 1% acetic acid concentration on an agar medium. The target DNA can be transformed into strain 1023 (deposited as FERM BP-2287 at the Patent Organism Depository Center), cultured in a medium containing 2% acetic acid, and examined for growth.

(3) Microorganism of the present invention As described in claim 4, the microorganism of the present invention has a high acetic acid resistance, wherein the copy number of the DNA according to claim 2 or 3 is amplified in a microbial cell. It is a microorganism.

Examples of such microorganisms include acetic acid bacteria having an ability to oxidize alcohol. Among them, the acetic acid bacteria belonging to the genus Acetobacter or Glucon acetobacter according to claim 5 can increase the production amount and production efficiency of acetic acid. It is preferable in that it can be performed.
The acetic acid bacterium used for the acquisition of the microorganism having high acetic acid resistance is an acetic acid bacterium of the genus Acetobacter or glucoconacetobacter, and the acetic acid bacterium of the genus Acetobacter is, for example, Acetobacter aceti Specifically, for example, Acetobacter aceti No. 1023 strain (Acetobacter aceti No.1023) (deposited as FERM BP-2287 at the Patent Biological Depositary Center), Acetobacter aceti subspecies zylinum IFO3288 (Acetobacter aceti subsp. Xylinum IFO3288) strain, Acetobacter aceti IFO3283 (Acet aceti IFO3283) strain can be used.
Examples of acetic acid bacteria belonging to the genus Gluconacetobacter include Gluconacetobacter europaeus DSM6160 (Gluconacetobacter europaeus DSM6160) strain and Gluconacetobacter entanii, specifically, for example, Acetobacter alto Acetigenes MH-24 (Acetobacter altoacetigenes MH-24) strain (deposited as FERM BP-491 at the Patent Organism Depositary) can be used.

The microorganism having high acetic acid resistance according to the present invention is a microorganism in which the copy number of the DNA according to claim 2 or 3 in the microorganism cell is preferably amplified twice or more, more preferably 3 to 20 times. .
Examples of a method for amplifying the copy number of the DNA according to claim 2 or 3 in a microbial cell include, for example, a structural gene of the gene (216th to 974th positions in the base sequence described in SEQ ID NO: 1 in the Sequence Listing). Promoter sequences that function efficiently in the microorganism of interest, such as alcohol dehydrogenase of acetic acid bacteria (eg, “Journal of Bacteriology 175, 6857-6866, 1993”). Promoter derived from microorganisms other than the target microorganism, such as the promoter of each gene such as the ampicillin resistance gene of E. coli plasmid pBR322, the kanamycin resistance gene of plasmid pACYC177, the chloramphenicol resistance gene of plasmid pACYC184, and the β-galactosidase gene. A method of transforming a microorganism using a recombinant DNA obtained by substituting it with a tersequence may be used.

In addition, a multicopy vector containing the DNA of claim 2 or 3 may be introduced into a microorganism cell.
Here, examples of the multicopy vector include a plasmid and a transposon.
Examples of the plasmid include pUF106 (see, for example, “Cellulose 153-158, 1989”), pTA5001 (A), pTA5001 (B) (see, for example, JP-A-60-9488). Further, pMVL1 (see, for example, “Agricultural and Biological Chemistry,” 52, p. 3125-3129, 1988), which is a chromosome integration type vector, can also be used.
Examples of transposons include Mu and IS1452.

  The introduction of multi-copy vectors into cells of microorganisms can be carried out by the calcium chloride method (see, for example, “Agricultural and Biological Chemistry, Vol. 49, p. 2091-2097, 1985”), An electroporation method (for example, see “Bioscience, Biotechnology and Biochemistry, Vol. 58, p. 974-975, 1994”) can be used.

(4) Manufacturing method of vinegar of this invention As described in Claim 6, the manufacturing method of the vinegar of this invention is what uses alcohol which has alcohol oxidation ability among the microorganisms of Claim 4 or 5. It is a method for producing vinegar characterized by culturing in a medium containing it and acetic acid being produced and accumulated in the medium.

This method may be performed in the same manner as the conventional fermentation method using acetic acid bacteria. The medium containing alcohol is a medium containing alcohol such as ethanol, a carbon source, a nitrogen source, an inorganic substance, etc., and a medium having the same composition as the medium used in so-called acetic acid fermentation can be used. . If necessary, a synthetic medium or a natural medium may be used as long as it contains an appropriate amount of nutrients required by the strain used for growth.
As the carbon source, various carbohydrates including glucose and sucrose, various organic acids, and the like can be used. As the nitrogen source, natural nitrogen sources such as peptone and fermented bacterial cell decomposition products can be used.

Cultivation can be performed under aerobic conditions according to conventional methods such as stationary culture, shaking culture, and aeration and agitation culture. The culture temperature can be 20 to 40 ° C, preferably 25 to 35 ° C, and usually 30 ° C.
The pH of the medium is usually in the range of 2.5 to 7.0, preferably in the range of 2.7 to 6.5, and can be appropriately adjusted with various acids, bases, buffers and the like.
In this way, in the present invention, acetic acid can be produced and accumulated in a medium containing alcohol, and usually a high concentration of acetic acid is produced and accumulated in the medium by culturing for 1 to 21 days. It can be shown.

  As described above, the protein SCDH2 or acetic acid resistance-enhancing gene of the present invention has been deposited as FERM BP-491 in the Patent Organism Depositary at the Acetobacter altoacetigenes MH-24 strain, which is the gene source thereof. Therefore, the DNA of the gene according to the present invention can be easily obtained, and those skilled in the art can easily implement the present invention. Then, if desired, the protein SCDH2 or the acetic acid resistance enhancing gene of the present invention is transferred to a vector capable of autonomous replication in acetic acid bacteria, introduced into acetic acid bacteria, and the acetic acid bacteria are cultured, thereby acetic acid content. Can be produced easily.

  Furthermore, as described above, the cloning of the protein SCDH2 or acetate resistance enhancing gene of the present invention, the mode of PCR, the preparation of plasmid vectors, recombinant plasmids, the deposit of host bacteria, etc. are disclosed or implemented, and all are available. In addition, since the operation and treatment can be easily performed, the target acetic acid resistant transformant can be obtained by performing each operation and treatment with reference to the Examples. By using this, a high concentration of acetic acid can be obtained. Can be manufactured.

Hereinafter, the present invention will be specifically described by way of examples.
Example 1 (Cloning of acetic acid resistance enhancing gene)
(1) Preparation of chromosomal DNA library 6% of Acetobacter altoacetigenes MH-24 (FERM BP-491), which is a strain of Gluconacetobacter entanii, In YPG medium (containing 3% glucose, 0.5% yeast extract and 0.2% polypeptone) supplemented with acetic acid and 4% ethanol, shaking culture was performed at 30 ° C. for 240 to 336 hours.
After completion of the culture, the culture solution was centrifuged (7,500 × g, 10 minutes) to obtain bacterial cells.
Chromosomal DNA was prepared from the obtained cells by the method disclosed in JP-A-60-9489. That is, after washing the cells with TE buffer, the TES buffer solution was added to suspend the cells, lysozyme solution was added and allowed to stand, and EDTA solution was added and reacted at 37 ° C. for 20 minutes. After the reaction, sodium lauryl sulfate was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes. Then, brine was added, and the mixture was allowed to stand at 0 ° C. overnight.
Subsequently, centrifugation was performed to obtain a supernatant. Polyethylene glycol 6000 was added to the supernatant and allowed to stand overnight at 4 ° C., followed by centrifugation to obtain a precipitate. The precipitate was dissolved in UC buffer, ethidium bromide was added, cesium chloride was further added to adjust the density to 1.57, and density gradient centrifugation was performed. Then, the ultraviolet light of 365 nm was irradiated to the centrifuge tube, and the band which appeared under a chromosome band was fractionated as a plasmid fraction. The fraction was treated with isopropanol to remove ethidium bromide and dialyzed against TE buffer. This was used as a plasmid mixed nutrient solution.

The chromosomal DNA obtained as described above was partially digested with the restriction enzyme Sau3AI (manufactured by Takara Bio Inc.), and the E. coli-acetic acid shuttle vector pUF106 was digested with the restriction enzyme EcoRI and cleaved. These chromosomes and vector DNAs are blunt-ended with T ₄ DNA polymerase, mixed in appropriate amounts, and ligated using a ligation kit (TaKaRa DNA Ligation Kit Ver.2, manufactured by Takara Bio Inc.) to acetobacter altoacetogenes. A chromosomal DNA library of MH-24 was constructed.

(2) Cloning of acetic acid resistance-enhancing gene The chromosomal DNA library of Acetobacter altoacetylenes MH-24 obtained as described above can be obtained only on an agar medium under conditions up to an acetic acid concentration of about 1%. Acetobacter aceti no. 1023 strain (FERM BP-2287) was transformed.
Thereafter, transformed Acetobacter aceti no. Strain 1023 was cultured at 30 ° C. for 4 days on a YPG agar medium supplemented with 2% acetic acid and 100 μg / ml ampicillin.

Next, the resulting colonies were inoculated and cultured in a YPG medium containing 100 μg / ml ampicillin, and the plasmid was recovered from the resulting cells. As a result, as shown in the lower part of FIG. 1, it was possible to recover the plasmid in which the Sau3AI fragment of about 700 bp was cloned.
In this way, Acetobacter aceti No. 1 which can usually grow on an agar medium only to an acetic acid concentration of about 1%. From the strain 1023, an acetic acid resistance-enhancing gene fragment that enables growth on an agar medium containing 2% acetic acid was obtained.

(3) Determination of the base sequence and amino acid sequence of the acetic acid resistance enhancing gene The fragment cleaved with the restriction enzyme KpnI-XbaI containing the cloned Sau3AI fragment was inserted into the KpnI-XbaI cleavage site of pUC19. The nucleotide sequence was determined by the Sanger dideoxy chain termination method. The nucleotide sequence was determined for the entire region of both DNA double strands so that all the breakpoints overlapped.
As a result, the base sequence described in SEQ ID NO: 1 in the sequence listing was determined. The presence of an open reading frame (ORF) encoding an amino acid sequence consisting of 253 amino acids described in SEQ ID NO: 2 from the base numbers 216 to 974 is confirmed in the base sequence described in SEQ ID NO: 1 in the sequence listing. It was done. FIG. 1 shows a restriction map of the resulting acetate-resistance-enhancing gene and an outline of the inserted fragment into plasmid pSCDH2.

Example 2 (Transformation with an acetic acid resistance enhancing gene)
(1) Transformation to Acetobacter aceti Acetic acid resistance derived from Acetobacter altoacetigenes MH-24 (FERM BP-491) cloned as in Example 1 above The enhanced gene was amplified by PCR using KOD-Plus- (Toyobo Co., Ltd.). An acetic acid bacteria-E. Coli shuttle vector pUF106 (see, for example, “Cellulose 153-158, 1989”) was cleaved with the restriction enzyme EcoRI, blunt-ended with T ₄ DNA polymerase, and the amplified DNA fragment was placed at that site. Insertion made pSCDH2. An outline of the amplified fragment inserted into pSCDH2 is shown in FIG.

The PCR method was performed as follows. That is, the PCR method was performed using the genomic DNA derived from the acetic acid bacterium as a template, primers 1 and 2 as primers, and the following conditions. The base sequences of primers 1 and 2 are as shown in SEQ ID NOS: 3 and 4 in the sequence listing, respectively.
The cycle of the PCR method was 30 cycles, with 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 1 minute 30 seconds as one cycle.

  This pSCDH2 was designated as Acetobacter aceti No. The strain 1023 was transformed by electroporation (see “Bioscience, Biotechnology and Biochemistry, 58, p. 974-975, 1994”). Transformants were selected on YPG agar medium supplemented with 100 μg / ml ampicillin and 2% acetic acid.

  About the ampicillin resistant transformant grown on the selective medium, the plasmid was extracted and analyzed by a conventional method, and it was confirmed that the plasmid carrying the acetic acid resistance enhancing gene was retained.

(2) Acetic acid resistance of the transformed strain About the ampicillin resistant transformed strain having the plasmid pSCDH2 obtained as described above, the growth state in the case of aeration culture in a YPG medium supplemented with acetic acid was determined using only the shuttle vector pUF106. Former strain Acetobacter aceti No. Comparison with the growth of 1023 strain.
Specifically, 100 ml of YPG agar medium containing 3% ethanol and 100 μg / ml ampicillin, and 100 ml YPG medium containing 3% ethanol, 3% acetic acid and 100 μg / ml ampicillin, respectively, were transformed with pSCDH2. The strain and the original strain having the shuttle vector pUF106 were inoculated, cultured at 30 ° C. with shaking (150 rpm), and the growth of the transformed strain and the original strain in an acetic acid-added medium was measured by measuring the absorbance at 660 nm. FIG. 2 shows the results of measuring changes over time in the amounts of growth of the original strain and the transformed strain in a medium containing acetic acid by turbidity (optical density: OD).

  As a result, in the medium containing no acetic acid, the transformed strain and the original strain were able to grow in substantially the same manner. However, as is apparent from FIG. 2, the transformed strain can grow on the medium containing acetic acid, whereas the original strain Acetobacter aceti No. It was confirmed that the strain 1023 could not grow and the function of enhancing acetate resistance by the acetate resistance enhancing gene was confirmed.

It is the figure which showed the outline of the restriction enzyme map of the acetic acid tolerance enhancement gene of this invention, and the insertion fragment to plasmid pSCDH2. It is a figure which shows the time-dependent change of the growth amount of the original strain in a culture medium containing an acetic acid, and a transformed strain.

Claims (6)

Protein SCDH2 shown in the following (A) or (B).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A function comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and enhancing acetate resistance A protein having DNA of a gene encoding the protein SCDH2 shown in (A) or (B) below.
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A function comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and enhancing acetate resistance A protein having The DNA of the gene according to claim 2, which is DNA shown in the following (a) or (b).
(A) DNA comprising a base sequence consisting of base numbers 216 to 974 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 1 in the Sequence Listing, the nucleotide sequence consisting of nucleotide numbers 216 to 974, or the DNA of the nucleotide sequence that can be a probe prepared from at least a part of the nucleotide sequence, and stringent conditions DNA encoding a protein that hybridizes underneath and has a function of enhancing acetic acid resistance.   A microorganism having high acetic acid resistance, wherein the copy number of the DNA according to claim 2 or 3 is amplified in a microbial cell.   The microorganism according to claim 4, wherein the microorganism is an Acetobacter or Glucon Acetobacter acetic acid bacterium. A method for producing vinegar, comprising culturing a microorganism having an alcohol oxidizing ability among the microorganisms according to claim 4 or 5 in a medium containing alcohol to produce and accumulate acetic acid in the medium.

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