JP2005124479A - Gene involved in acetic acid tolerance, acetic acid bacterium bred by using the same, and method for producing vinegar using the acetic acid bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene involved in the acetic acid tolerance of a microorganism, to provide a method for improving the acetic acid tolerance of a microorganism, especially an acetic acid bacterium, using the gene, and to provide a method for efficiently producing vinegar of higher acetic acid concentration using an acetic acid bacterium with improved acetic acid tolerance. <P>SOLUTION: A protein SF represented by the protein(A) or (B) described below is provided. The gene encoding the protein and involved in acetic acid tolerance is provided. The acetic acid bacterium bred by using the gene is provided. The method for producing vinegar using the acetic acid bacterium is also provided. The protein(A) comprises a specific amino acid sequence. The protein(B) comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, added or inverted in the above-mentioned specific amino acid sequence and has the function of enhancing acetic acid tolerance. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a gene encoding a protein having a function of enhancing acetic acid resistance, a microorganism in which the copy number of the gene is amplified, in particular, an acetic acid bacterium belonging to the genus Acetobacter and the genus Gluconacetobacter, And a method for efficiently producing vinegar containing high concentrations of acetic acid using these microorganisms.

Acetic acid bacteria are microorganisms widely used for vinegar production. Particularly, acetic acid bacteria belonging to the genus Acetobacter and Gluconacetobacter are used for industrial acetic acid fermentation.
In acetic acid fermentation, ethanol in the medium is oxidized by acetic acid bacteria and converted to acetic acid. As a result, acetic acid accumulates in the medium, but acetic acid is also inhibitory to acetic acid bacteria. As the concentration of acetic acid increases and the concentration of acetic acid in the medium increases, the growth ability and fermentation ability of acetic acid bacteria gradually decrease.
Therefore, in acetic acid fermentation, it is required that growth ability and fermentation ability do not decrease even at higher acetic acid concentrations, that is, to develop acetic acid bacteria having strong acetic acid resistance. Attempts have been made to clone (acetic acid resistant gene) and to breed and improve acetic acid bacteria using the acetic acid resistant gene.

As for the knowledge about the acetic acid resistance gene of acetic acid bacteria so far, a cluster is formed as a complementary gene that can restore acetic acid sensitivity by mutating the acetic acid resistance of acetic acid bacteria of Acetobacter genus. Three genes (aarA, aarB, aarC) have been cloned (see, for example, Non-Patent Document 1).
Among them, the aarA gene is a gene encoding citrate synthase, and the aarC gene was estimated to be an enzyme encoding an enzyme related to acetic acid utilization, but the function of the aarB gene is unknown. (For example, refer nonpatent literature 2).

  A transformant obtained by cloning a gene fragment containing these three acetate-resistant genes into a multicopy plasmid and transforming it into an Acetobacter aceti subsp. Xylinum IFO3288 strain Has only a slight improvement level of acetic acid resistance, and it was unclear as to whether or not the actual performance of acetic acid fermentation was improved (see, for example, Patent Document 1).

  On the other hand, an example has been disclosed in which the final acetic acid concentration has been improved in acetic acid fermentation 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, since ALDH is an enzyme having a function of oxidizing aldehyde and not directly related to acetic acid resistance, it cannot be determined that the gene encoding ALDH is truly an acetic acid resistance gene.

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

As described above, there have been no reports of successful development of practical acetic acid bacteria having high acetic acid resistance by elucidating acetic acid resistance of acetic acid bacteria at the gene level. However, if acetic acid bacteria excellent in acetic acid resistance are developed, acetic acid fermentation at a higher concentration is performed than before, and high-concentration acetic acid and high-concentration vinegar can be efficiently produced. Again, we decided to elucidate the improvement in acetic acid resistance of acetic acid bacteria at the gene level.
And as a result of examining from various directions, the present inventors obtained a novel acetic acid resistant gene encoding a protein having a function capable of improving acetic acid resistance at a practical level, and using the obtained acetic acid resistant gene, In view of the importance of breeding acetic acid bacteria having strong acetic acid resistance, providing a novel gene involved in acetic acid resistance derived from microorganisms, and using the gene, acetic acid of microorganisms, particularly acetic acid bacteria Providing a method for improving resistance, and further providing a method for efficiently producing vinegar with higher acetic acid concentration using acetic acid bacteria having improved acetic acid resistance was newly set as a new technical problem.

  The present inventors hypothesized that acetic acid bacteria that can grow and ferment in the presence of acetic acid have a gene involved in specific acetic acid resistance that does not exist in other microorganisms. And if such a gene is used, the acetic acid tolerance of microorganisms can be improved more than before, and furthermore, an efficient manufacturing method of new vinegar containing high concentration of acetic acid that could not be obtained conventionally will be developed. I got a new idea that it would be possible.

As a conventional method for obtaining an acetic acid resistant gene, a method for cloning a gene that complements an acetic acid-sensitive mutant strain of acetic acid bacteria is generally used. However, it was difficult to find an industrially useful acetic acid resistance gene by such a method, and earnestly studied.
As a result, the present inventors constructed a chromosomal DNA library of acetic acid bacteria as a method for finding an acetic acid resistant gene from acetic acid bacteria, transformed the chromosomal DNA library into acetic acid bacteria, and usually about 1% acetic acid. We developed a method for obtaining strains that can only grow in the presence of 2 by screening for genes that can grow in the presence of 2% acetic acid.
By this method, we succeeded in cloning a novel acetic acid resistance gene having a function of improving acetic acid resistance at a practical level from glucoconacetobacter acetic acid bacteria actually used in vinegar production.

  Furthermore, the present inventors also succeeded in determining the base sequence of a novel acetate-resistant gene DNA and the amino acid sequence of the protein encoded by the gene. The coding region of the obtained acetate resistance gene has a characteristic base sequence common to genes encoding a group of proteins called RNA polymerase sigma factor (hereinafter sometimes referred to as sigma factor). Since it has a certain degree of homology with proteins, it was presumed to be a gene encoding a novel protein of acetic acid bacteria that resembles sigma factor to some extent.

  In addition, in the transformed strain in which the gene was linked to a plasmid vector and transformed into acetic acid bacteria and the copy number was amplified, the acetic acid resistance was remarkably improved, and as a result, the transformed strain was transformed in the presence of ethanol. In the case of aeration culture, it was found that the growth induction period was shortened, the growth rate and the raw acid rate were improved, and the final reached acetic acid concentration was further significantly improved, and the present invention was completed.

That is, the present invention according to claim 1 provides the protein SF 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 SF 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 611 to 1096 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the base sequences set forth in SEQ ID NO: 1 in the sequence listing, a base sequence consisting of base numbers 611 to 1096 or a base sequence DNA that can be a probe prepared from at least a part of the base 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 tolerance with respect to an acetic acid can be provided with respect to microorganisms, and an acetic acid tolerance can be reinforced. Resistance to microorganisms having alcohol oxidizing ability, particularly acetic acid bacteria, against acetic acid can be remarkably improved at a practical level, and the ability to efficiently accumulate a high concentration of acetic acid in a medium can be imparted.
Therefore, according to the present invention, high-concentration acetic acid and high-concentration vinegar can be efficiently produced, which is useful in industrial production of vinegar.

Hereinafter, the present invention will be described in detail.
(1) Protein of the present invention As described in claim 1, the protein SF 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 portion encoded by a characteristic base sequence common to a group of proteins called RNA polymerase sigma factors, Since it has a certain degree of homology with these proteins, it was presumed to be a novel protein of acetic acid bacteria that resembles sigma factor to some extent.
That is, when homology search was performed in DDBJ / EMBL / Genbank and SWISS-PROT / PIR, it was found to be 44% at the amino acid sequence level with the RNA polymerase sigma-E factor (sigma24) gene of Bradyrhizobium japonicum. 44% homology with the RNA polymerase sigma-70 factor gene of Brucella suiss 1330 strain at the amino acid sequence level.

  As described above, the protein SF 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 SF 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) (deposited with FERM BP-2287 at Patent Biological Depositary Center), Acetobacter aceti subspecies zylinum IFO3288 (Acetobacter aceti subsp. Xylinum IFO3288), Acetobacter aceti IFO3283 (Acetobacter aceti IFO3283) Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) strain (patented organism) Present in the deposit) as FERM BP-491 to the 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 SF shown in the above (A) or (B). 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 611 to 1096 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the base sequences set forth in SEQ ID NO: 1 in the sequence listing, a base sequence consisting of base numbers 611 to 1096 or a base sequence DNA that can be a probe prepared from at least a part of the base 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.

The DNA encoding the protein SF of the present invention can be obtained from the chromosomal DNA of Gluconacetobacter entanii as follows.
First, a chromosomal DNA library of Glucon Acetobacter enterii, for example, Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) strain (deposited as FERM BP-491 at the Patent Organism Depositary) is prepared. Chromosomal DNA can be obtained by the method disclosed in JP-A-60-9489.

  Next, a chromosomal DNA library is prepared in order to isolate an acetic acid resistance enhancing gene from the obtained chromosomal DNA. First, chromosomal DNA is partially decomposed with an appropriate restriction enzyme to obtain various DNA fragment mixtures. A wide variety of restriction enzymes can be used by adjusting the cleavage reaction time to adjust the degree of cleavage. For example, Sau3AI is partially 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 to produce a recombinant DNA. Specifically, a restriction enzyme that generates a terminal base sequence complementary to the restriction enzyme Sau3AI used for the cleavage of chromosomal DNA, such as BamHI, is heated for 1 hour at a temperature of 30 ° C. and at an enzyme concentration of 1 to 100 units / ml. As described above, the vector DNA is completely digested by acting on the vector DNA, and then cleaved and cleaved.

  The chromosomal DNA fragment mixture obtained as described above is mixed with the cleaved vector DNA, and T4 DNA ligase is added to this at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units / ml, preferably 1 hour or more. Is allowed to act for 6 to 24 hours to obtain recombinant DNA (chromosomal DNA library).

  Selection of the gene encoding the protein (A) from the obtained recombinant DNA (chromosomal DNA library), specifically, the DNA (a) can be performed, for example, as follows. Usually, acetic acid bacteria that cannot grow on an agar medium in the presence of acetic acid at a concentration higher than 1%, for example, Acetobacter aceti No.1023 strain (FERM BP- Is deposited with 2% acetic acid-containing agar medium 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, the base numbers 611 to 1096 DNA containing a base sequence consisting of 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 611 to 1096 is a coding region, and the amino acid sequence described in SEQ ID NO: 2 in the sequence listing corresponds to the coding region. It is.
As a result of homology search in DDBJ / EMBL / Genbank and SWISS-PROT / PIR, the gene encoding protein (A), specifically DNA (a), was found to be RNA polymerase sigma of Bradyrhizobium japonicum. 44% homology with the E factor (sigma24) gene at the amino acid sequence level and 44% homology with the RNA polymerase sigma-70 factor gene of the Brucella suiss 1330 strain.
From these facts, the gene encoding protein (A), specifically DNA (a), is a protein gene specific to acetic acid bacteria and encodes a novel protein similar to RNA polymerase sigma factor. It is estimated to be a gene.
Incidentally, the fact that the sigma factor protein gene is related to acetic acid resistance has never been known so far, and the present inventors have found it for the first time.

  Furthermore, the DNA encoding the protein SF of the present invention has a novel acetic acid resistance that is different from the acetic acid resistance genes (aarA, aarB, aarC) of acetic acid bacteria already acquired and the ADH gene having a function to enhance the acetic acid resistance. The gene was identified as having a function to enhance.

The gene encoding protein (A), specifically, DNA (a) is a gene encoding a protein comprising the amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing, that is, described in SEQ ID NO: 1 in the Sequence Listing. Of the nucleotide sequences of those having a base sequence consisting of base numbers 611 to 1096, for example, an oligonucleotide synthesized based on the base sequence using the genomic DNA of Acetobacter glucosacetobacter enterii as a template. It can be obtained by polymerase chain reaction (PCR reaction) used as a primer, or by hybridization using an oligonucleotide synthesized based on the base sequence as a probe, and its production method is not particularly limited.
Here, the oligonucleotide used as a primer or a probe by the said method is compoundable according to a conventional method, for example using various commercially available DNA synthesizers. Moreover, PCR reaction uses Applied Biosystems (Applied Biosystems) thermal cycler Gene Amp PCR System 2400, TaqDNA polymerase (made by Takara Shuzo), KOD-Plus- (made by Toyobo Co., Ltd.), etc., It can be carried out according to standard methods.

As described above, the DNA encoding the protein SF of the present invention 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) SEQ ID NO: in the sequence listing. 1 is a DNA comprising a base sequence consisting of base numbers 611 to 1096 among the base sequences described in 1. In the amino acid sequence described in SEQ ID NO: 2 in the sequence list (B), one or several, preferably 1 to A protein consisting of an amino acid sequence containing 5 amino acid substitutions, deletions, insertions, additions, or inversions and having a function of enhancing acetic acid resistance, ie, a protein substantially identical to the protein of (A) You may code.
The DNA encoding the protein (B) that is substantially the same as the protein having the function of enhancing the acetic acid resistance is substituted, deleted, inserted, reversed, etc., by, for example, site-specific mutagenesis. It can also be obtained by modifying the base sequence so as to be added or added. The DNA modified as described above can also be obtained by a conventionally known mutation treatment.

In general, it is known that the amino acid sequence of a protein and the base sequence encoding the protein are slightly different between species, strains, mutants, and variants. Therefore, substantially the same protein (B) The DNA encoding can be obtained from acetic acid bacteria in general, among them species, strains, mutants, and variants of the genus Acetobacter and Gluconacetobacter.
Specifically, from the acetic acid bacteria of the genus Acetobacter or Gluconacetobacter, or the acetic acid bacteria of the Acetobacter genus or Glucon acetobacter genus that have been mutated, natural mutants or variants thereof, for example, (b) Sequences Of the base sequence described in No. 1, it hybridizes under stringent conditions with a base sequence consisting of base numbers 611 to 1096 or a base sequence DNA that can be a probe prepared from at least part of the base sequence, and It can also be obtained by isolating a DNA encoding a protein having a function of enhancing acetate resistance.

  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 these conditions 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 of the hybridization conditions include normal hybridization washing conditions such as conditions in which washing is performed at 60 ° C. with 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 (FERM BP-2287), cultured in a medium containing 2% acetic acid, and examined for its ability to grow.

(3) Acetic acid bacterium of the present invention As described in claim 4, the microorganism of the present invention has high acetic acid resistance, wherein the copy number of the DNA according to claim 2 or 3 in the microbial cell is amplified. It has 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 obtaining such microorganisms having high acetic acid resistance is an acetic acid bacterium belonging to the genus Acetobacter or Glucon acetobacter, and examples of the acetic acid bacteria belonging to the genus Acetobacter include Acetobacter aceti. Specifically, 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 is exemplified.
Examples of the acetic acid bacteria belonging to the genus Gluconacetobacter include Gluconacetobacter europaeus DSM6160 (Gluconacetobacter europaeus DSM6160) and Gluconacetobacter entanii, specifically, Acetobacter altoacetigenes. MH-24 strain (Acetobacter altoacetigenes MH-24) (deposited as FERM BP-491 in the Patent Organism Depositary) is exemplified.

As a method for amplifying the copy number of the DNA according to claim 2 or 3 in a microbial cell, at least the structural gene of the DNA according to claim 2 or 3 (of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, Performing transformation of the microorganism using a recombinant DNA obtained by ligating a DNA fragment comprising SEQ ID NOs: 611 to 1096) to a promoter sequence that functions efficiently in the microorganism to which acetic acid resistance is desired. it can.
Here, the promoter sequence may be replaced with a promoter sequence derived from another microorganism as long as it functions efficiently in a microorganism to which acetic acid resistance is desired. For example, when the microorganism to be imparted with acetic acid resistance is an acetic acid bacterium belonging to the genus Acetobacter or Glucon acetobacter, the ampicillin resistance gene of plasmid pBR322 of E. coli, the kanamycin resistance gene of plasmid pACYC177, and the chloramphenicol resistance of plasmid pACYC184 Examples include promoters of genes such as genes and β-galactosidase genes.

Transformation using a recombinant DNA obtained by ligation to the promoter sequence can be performed by introducing a multicopy vector carrying the recombinant DNA into a microbial cell. That is, it can be carried out by introducing a multicopy vector such as a plasmid or transposon holding the recombinant DNA into cells of acetic acid bacteria belonging to the genus Acetobacter or Glucon acetobacter.
Examples of multi-copy vectors include pGI18 (see Japanese Patent Application No. 2003-350265), pTA5001 (A), pTA5001 (B) (see, for example, JP-A-60-9489), etc. PMVL1 (see, for example, “Agricultural and Biological Chemistry, Vol. 52, p. 3125-3129, 1988”) can also be used. Examples of transposons include Mu and IS1452.
Of these, pGI18 is preferable because it has high gene transfer efficiency into Acetobacter and Glucon Acetobacter acetic acid bacteria and is excellent in stability in cells.

Multicopy vectors can be introduced into cells of microorganisms by the calcium chloride method (see, for example, “Agricultural and Biological Chemistry, Vol. 49, p. 2091-2097, 1985”) or An electroporation method (for example, see “Bioscience, Biotechnology and Biochemistry, Vol. 58, p. 974-975, 1994”) can be used.
The microorganism thus obtained has high acetic acid resistance since the copy number of the DNA according to claim 2 or 3 in the microorganism cell is amplified. Among them, in an acetic acid bacterium belonging to the genus Acetobacter or Glucon acetobacter, when the DNA copy number according to claim 2 or 3 in the cell is amplified as described above, acetic acid resistance is selectively enhanced, Production volume and production efficiency can be increased.

(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.
That is, in the method for producing vinegar according to the present invention, the acetic acid belonging to the genus Acetobacter or Gluconacetobacter genus in which the copy number of the DNA according to claim 2 or 3 in microbial cells is amplified and the acetic acid resistance is selectively enhanced. Among bacteria, those having alcohol oxidizing ability are cultured in an alcohol-containing medium, and acetic acid is produced and accumulated in the medium, whereby vinegar is efficiently produced.

The culture in the medium containing alcohol in the production method of the present invention may be carried out in the same manner as acetic acid fermentation by conventional 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.
Examples of the carbon source include various carbohydrates including glucose and sucrose, and various organic acids. As the nitrogen source, natural nitrogen sources such as peptone and fermented bacterial cell decomposition products can be used.

  Cultivation is performed under aerobic conditions such as stationary culture, shaking culture, and aeration and agitation culture, and the culture temperature is usually 30 ° C. The pH of the medium is usually in the range of 2.5 to 7, preferably in the range of 2.7 to 6.5, and can be prepared with various acids, various bases, buffers and the like. Usually, acetic acid at a high concentration accumulates in the medium by culturing for 1 to 21 days.

Hereinafter, the present invention will be specifically described by way of examples.
Example 1 (Cloning of an acetic acid resistant gene from Gluconacetobacter enterii and determination of base sequence and amino acid sequence)
(1) Preparation of chromosomal DNA library Acetobacter altoacetigenes MH-24 strain (FERM BP-491), which is one strain of Gluconacetobacter entanii, 6% acetic acid And 4% ethanol in YPG medium (containing 3% glucose, 0.5% yeast extract, 0.2% polypeptone) and shaking culture at 30 ° C. After 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.

  The chromosomal DNA obtained as described above was partially digested with the restriction enzyme HaeI (Takara Shuzo), and the Escherichia coli-acetic acid shuttle vector pGI18 was completely digested with the restriction enzyme BamHI and cleaved. An appropriate amount of these DNAs was mixed and ligated using a ligation kit (TaKaRa DNA Ligation Kit Ver.2, manufactured by Takara Shuzo Co., Ltd.) to construct a chromoacetobacter / entany chromosomal DNA library.

(2) Cloning of acetic acid resistance gene The chromosomal Acetobacter chromosomal DNA library obtained as described above is usually Acetobacter aceti No. which can be grown on an agar medium only to an acetic acid concentration of about 1%. 1023 strain (FERM BP-2287) was transformed.
Thereafter, transformed Acetobacter aceti no. The strain 1023 was cultured on a YPG agar medium containing 2% acetic acid and 100 μg / ml ampicillin at 30 ° C. for 4 days.

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, a plasmid in which a HaeI fragment of about 2 kbp was cloned could be recovered.
FIG. 1 shows a restriction map of the cloned approximately 2 kbp HaeI fragment along with the location of the acetate resistance gene.

Furthermore, Acetobacter aceti No. 2 on YPG agar medium containing 2% acetic acid. It was confirmed that the DNA fragment enabling the growth of strain 1023 was a HaeI fragment of about 2 kbp.
In this way, Acetobacter aceti No. 1 which can usually grow on an agar medium only to an acetic acid concentration of about 1%. An acetic acid resistant gene fragment that enables the 1023 strain to grow even on an agar medium containing 2% acetic acid was obtained.

(3) Determination of the base sequence of the cloned DNA fragment The cloned HaeI fragment was inserted into the BamHI cleavage site of pUC19, and the base sequence of the fragment was determined by the Sanger dideoxy chain termination method. did. A part of the determined sequence is shown in SEQ ID NO: 1. Sequencing was performed for the entire region of both DNA strands, with all breakpoints overlapping.
In the base sequence described in SEQ ID NO: 1, the presence of an open reading frame (ORF) encoding 162 amino acids as described in SEQ ID NO: 2 was confirmed from base number 611 to base number 1096.

Example 2 (Enhancement of acetic acid resistance in a transformed strain transformed with an acetic acid resistant gene derived from Gluconacetobacter enterii)
(1) Transformation into Acetobacter aceti Acetic acid resistant gene derived from Acetobacter altoacetigenes MH-24 (FERM BP-491) cloned as described above, Amplification was carried out by PCR using KOD-Plus- (manufactured by Toyobo Co., Ltd.), and a plasmid pSF24 was prepared by inserting the amplified DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18. The outline of the amplified fragment inserted into the plasmid pSF24 and the position of the acetate resistance gene are shown in FIG.

The PCR method was performed as follows. That is, using the above genomic DNA derived from acetic acid bacteria as a template, primer 1 (primer # 1: refer to the nucleotide sequence described in SEQ ID NO: 3 in the sequence listing) and primer 2 (primer # 2: described in SEQ ID NO: 4 in the sequence listing) The PCR method was carried out under the conditions described below. That is, the PCR method was performed 30 cycles, with 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 50 seconds as one cycle.
This pSF24 was designated as Acetobacter aceti no. The strain 1023 was transformed by electroporation (see, for example, “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 acetate resistant gene was retained.

(2) Acetic acid resistance of the transformed strain For the ampicillin resistant transformed strain having the plasmid pSF24 obtained as described above, the growth in the YPG medium supplemented with acetic acid was carried out using the original strain acetoacetate into which only the shuttle vector pGI18 was introduced. Bacter aceti no. Comparison with 1023 strain.
Specifically, 100 ml of YPG 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 pSF24. The strain and the original strain having the shuttle vector pGI18 are inoculated, cultured at 30 ° C. with shaking (150 rpm), and the culture progress (growth state) of the transformed strain and the original strain in an acetic acid-added medium is measured for absorbance at 660 nm. Compared. The culture process of the transformed strain and the original strain is shown in FIG.

  As a result, as shown in FIG. 2, in the medium containing no acetic acid, the transformed strain and the original strain were able to grow in the same manner, whereas in the medium supplemented with 3% acetic acid and 3% ethanol, the trait was The transformed strain can grow, whereas the former strain Acetobacter aceti No. It was confirmed that the strain 1023 could not grow, and the acetic acid resistance enhancing function of the acetic acid resistance gene was confirmed.

  As described above, according to the present invention, a novel gene involved in acetic acid resistance is provided, and a breeding strain capable of producing vinegar with higher acetic acid concentration with high efficiency can be obtained using the gene. Furthermore, it has become possible to provide a method for producing vinegar having a higher acetic acid concentration with high efficiency using the breeding strain.

ADVANTAGE OF THE INVENTION According to this invention, the tolerance with respect to an acetic acid can be provided with respect to microorganisms, and an acetic acid tolerance can be reinforced. In particular, the resistance of microorganisms having alcohol oxidizing ability, particularly acetic acid bacteria, to acetic acid can be significantly improved at a practical level, and the ability to efficiently accumulate a high concentration of acetic acid in a medium can be imparted.
Therefore, according to the present invention, high-concentration acetic acid and high-concentration vinegar can be efficiently produced, and the present invention is useful in industrial production of vinegar.

It is a figure which shows the outline of the restriction enzyme map of the cloned gene fragment derived from Gluconacetobacter enterii, the position of an acetic acid resistant gene, and the amplified fragment inserted in the plasmid pSF24. It is a figure which shows the culture | cultivation progress of the transformant which amplified the copy number of the acetic acid tolerance gene derived from Gluconacetobacter enterii, and a former strain.

Claims (6)

Protein SF 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 SF 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 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 611 to 1096 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the base sequences set forth in SEQ ID NO: 1 in the sequence listing, a base sequence consisting of base numbers 611 to 1096 or a base sequence DNA that can be a probe prepared from at least a part of the base 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 acetic acid bacterium belonging to the genus Acetobacter or Glucon acetobacter. 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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