WO2003078622A1 - Squalene-hopene cyclase gene of acetic acid bacterium, acetic acid bacterium bred with the use of the gene, and process for producing vinegar using the acetic acid bacterium - Google Patents

Abstract

A novel acetic acid bacterium-origin gene participating in acetic acid-tolerance; a method of improving the acetic acid-tolerance of a microorganism, in particular, an acetic acid bacterium, using this gene; and a process of efficiently producing a vinegar having an elevated acetic acid concentration with the use of the acetic acid bacterium having the thus improved acetic acid tolerance. From a chromosomal DNA library of an acetic acid bacterium, a gene enabling the growth of the acetic acid bacterium in a medium containing acetic acid at such a high concentration as not allowing the growth in usual is obtained. By using this method, a novel gene participating in acetic acid-tolerance is cloned from an acetic acid bacterium for practical use belonging to the genus Gluconacetobacter. A transformant constructed by transferring this gene into an acetic acid bacterium has a remarkably improved acetic acid-tolerance. When this transformant is cultured under aeration in the presence of ethanol, the growth induction period is shortened and the growth speed and the acid-forming speed are elevated. Moreover, the final carryover acetic acid level thereof can be remarkably elevated thereby.

Description

 Description: Squalene-hopene cyclase gene of acetic acid bacterium, acetic acid bacterium bred using the gene, and method for producing vinegar using the acetic acid bacterium

Technical field to which the invention belongs

 The present invention relates to a gene encoding a protein derived from a microorganism and having a function of enhancing acetic acid resistance, a microorganism having an amplified copy number thereof,

The present invention relates to an acetic acid bacterium belonging to the genus (Acetobacter) and the genus Gluconacetobacter, and a method for efficiently producing vinegar containing acetic acid at a high concentration using these microorganisms. Conventional technology

 Acetic acid bacteria are microorganisms widely used in the production of vinegar. In particular, acetic acid bacteria belonging to the genus Acetobak and the genus Glucona acetobac are used for industrial acetic acid fermentation.

 In acetic acid fermentation, ethanol in the culture medium is oxidized by acetic acid bacteria and converted into acetic acid, which results in the accumulation of acetic acid in the medium, which is also inhibitory for acetic acid bacteria and accumulates acetic acid. As the amount increases and the acetic acid concentration 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 the growth ability and fermentation ability do not decrease even at a higher acetic acid concentration, that is, it is required to develop acetic acid bacteria having strong acetic acid resistance. Attempts have been made to clone a gene (acetate resistance gene) and to breed and improve acetic acid bacteria using the acetic acid resistance gene.

To date, knowledge of the acetic acid resistance gene of acetic acid bacteria has shown that mutation of the acetic acid resistance of acetic acid bacteria of the genus Acetobacter to restore the acetic acid-sensitive strain to its original resistance. Three genes (aarA, aarB, and aarC) that form a class have been cloned as complementary genes that can be used (for example, see Non-Patent Document 1). Of these, the aar A gene is a gene encoding citrate synthase, and the aar C gene was presumed to be a gene encoding an enzyme related to acetic acid utilization, but the aarB gene is functional. Was unknown (for example, see Non-Patent Document 2).

 The gene fragments containing these three acetate resistance genes were cloned into Multicopy Plasmid, and transformed into Acetobacter aceti subsp.xylin m IF03288 strain of Acetobacter aceti subsp. The obtained transformant showed only a slight improvement in acetic acid resistance, and it was not known whether or not the ability in actual acetic acid fermentation was improved (for example, see Patent Document 1). On the other hand, the introduction of a gene encoding a membrane-bound aldehyde dehydrogenase (ALDH) cloned from an acetic acid bacterium into an acetic acid bacterium showed an improvement in the final acetic acid concentration in acetic acid fermentation. It is disclosed (for example, see Patent Document 2). However, since ALDH is an enzyme having the function of oxidizing aldehydes and not directly related to acetic acid tolerance, it could not be determined that the gene encoding ALDH is truly an acetic acid resistance gene. Patent Document 1

 Japanese Patent Publication No. 3-2 19878

 Patent Document 2

 JP-A-2-2364

 Patent Document 3

 JP-A-60-9489

 Patent Document 4.

 JP-A-60-9488

 Non-patent document 1

"Journal of Bacteriology", 17 Vol. 2, 209 6-2 1 04, 1 1990 '

 Non-patent document 2

 "Journal of Fermentation and Bioengineermg, Vol. 76, 270-2 7 o Shell, 1 993"

 Non-patent document 3

 "Microbiology, Vol. 143, 123-5-1 242, 1997"

 Non-patent document 4

 "Applied of Environment and Microbiology (Applied of Environment and Microbiology) 55, 171-176, 1989" Non-Patent Document 5

 "Agricultural and Biological Chemistry", Vol. 52, p. 3 125-3 129, 1988 Non-Patent Document 6

 “Agricultural and Biological Chemistry”, Vol. 49, p.209-20997, 1985, Non-Patent Document 7

 "Bioscience, Biotechnology and Biochemistry, o 8 vol., .974-975, 1994"

 Non-patent document 8

 "Canadian Journal of Biochemistry ana Physiology; Vol. 7, 9 11 -9 1 7, 1959"

 Non-patent document 9

"Journal of Biological Chemistry, 226, 497-509, 1957" Non-patent document 10

 "Journal of Biological Chemistry, 272, 9809-98 17, 1997"

 Non-patent document 1 1

 "Journal of Biochemistry, Vol. 98, 327-3 31 1, 1985" Problems to be Solved by the Invention

 As described above, there has been no report on the case of elucidating the acetic acid resistance of acetic acid bacteria at the genetic level and successfully developing a practical acetic acid bacterium having high acetic acid resistance. However, if an acetic acid bacterium having excellent acetic acid resistance is developed, acetic acid fermentation at a higher concentration than before is performed, and high-concentration acetic acid mash can be efficiently produced. Decided again to elucidate the improvement of acetic acid tolerance in acetic acid bacteria at the genetic level.

 The present inventors have studied from various aspects, and as a result, obtained a novel acetic acid resistance gene encoding a protein having a function capable of improving acetic acid resistance at a practical level, and using the obtained acetic acid resistance gene, Providing a novel squalene-hopene cyclase gene involved in acetic acid resistance derived from a microorganism belonging to acetic acid bacteria, from the viewpoint that it is important to breed acetic acid bacteria having strong acetic acid resistance, and said gene A method for improving acetic acid tolerance of microorganisms using acetic acid, especially a method for improving acetic acid resistance of microorganisms belonging to acetic acid bacteria, and a method for efficiently producing vinegar with a higher acetic acid concentration using acetic acid bacteria having improved acetic acid resistance Is newly set as a new technical issue. Means for solving the problem

The present inventors hypothesized that acetic acid bacteria capable of growing and fermenting even in the presence of acetic acid have specific genes involved in acetic acid resistance that are not present in other microorganisms. If used, it is possible to improve the acetic acid resistance of microorganisms more than before, and it is also possible to obtain a novel acetic acid containing a high concentration of acetic acid. I got a new idea that it would be possible to develop an efficient production method of vinegar.

 Conventional methods for obtaining an acetic acid resistance gene generally include a method of cloning a gene that complements an acetic acid-sensitive mutant of acetic acid bacteria.

 However, it is considered difficult to find an industrially useful acetic acid resistance gene by such a method, and as a result of intensive studies, the present inventors have found that a method for finding an acetic acid resistance gene from acetic acid bacteria uses the chromosome of acetic acid bacteria. By constructing a DNA library, this chromosomal DNA library is transformed into acetic acid bacteria, allowing strains that can only grow on agar medium in the presence of 1% acetic acid to grow in the presence of 2% acetic acid. A method for obtaining the desired gene by screening was proposed.

 With this method, we succeeded in the first time to clone a novel acetic acid resistance gene that has the function of improving acetic acid resistance to a practical level from the acetic acid bacteria of the genus Dalcon acetobac, actually used in vinegar production. did.

 The acetic acid resistance genes obtained are: DB J / EMBL / Genbank search results show a certain degree of homology to a group of protein genes called squalene-hopene cyclase found in rhizobia, etc. Therefore, it was presumed to be a gene encoding squalene-hopenza cyclase of acetic acid bacteria.

 In addition, the results of a search by SWI SS-PRO T / PIR at the amino acid sequence level show that the protein has a motif (DXDD TA) conserved in squalene-hopene cyclase (for example, see Non-Patent Document 3). Therefore, it was considered to be a gene encoding squalene-hopen cyclase of acetic acid bacteria.

 However, the obtained squalene-hopene cyclase gene of the acetic acid bacterium had extremely low homology to the known squalene-hopene cyclase gene found in other microorganisms such as rhizobia. Although it is similar to other squalene-hopene cyclase genes to some extent, it was found to be a novel gene encoding a novel protein specific to acetic acid bacteria (hereinafter sometimes referred to as protein SHC).

Moreover, in a transformant in which the gene is ligated to plasmid and transformed into acetic acid bacteria and the copy number is amplified, metabolites, which are metabolites in their lipid composition, are used. The increase in the composition ratio of trahydroxypacterihopane confirms that the gene encodes a protein having squalene-hopene cyclase activity of acetic acid bacteria, and at the same time, is remarkable. It was confirmed that acetic acid resistance was improved.

 Furthermore, they found that when the transformed strain was cultured in aerated culture in the presence of ethanol, the growth induction period was shortened, the growth rate and the acid rate were improved, and the finally reached acetic acid concentration was significantly improved. Furthermore, the amino acid sequence of the protein and the base sequence of the gene DNA encoding the same were also successfully determined, and the present invention was completed.

BRIEF DESCRIPTION OF THE FIGURES

 Figure 1

 Restriction map of the gene fragment (pB1) derived from D. cerevisiae cloned using BamHI and the location of the SHC gene, and an outline of the fragment inserted into pSHC. FIG.

 Figure 2

 The figure which shows the culture progress of the transformant which amplified the copy number of SHC gene.

 Fig. 3

 Drawing showing temperature change and progress of acetic acid fermentation of a transformant in which the copy number of the SHC gene was amplified.

 Fig. 4

 The amino acid sequence of the protein encoded by the present acetate resistance gene (SEQ ID NO: 2) is shown.

 Fig 5

 Same as above.

 Fig. 6

Shows primer 1. _T indicating primer 1

That is, embodiments of the present invention are as follows.

 (1) A protein SHC shown in the following (A) or (B).

 (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing,

 (B) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, which comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and enhances acetic acid resistance Functional protein.

 (2) DNA of a gene encoding the protein SHC shown in (A) or (B) below.

 (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing,

 (B) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, which comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and enhances acetic acid resistance Functional protein.

 (3) A DNA of the gene according to (2), which is a DNA shown in (a) or (b) below.

 (a) DNA comprising a base sequence consisting of base numbers 406 to 2436 among the base sequences described in SEQ ID NO: 1 in the sequence listing.

 (b) a probe having a base sequence consisting of base numbers 406 to 2436 or a part thereof among the base sequences of SEQ ID NO: 1 in the sequence listing, and hybridizing under stringent conditions, and having acetic acid resistance DNA that encodes a protein that has the function of enhancing protein.

 (4) The microorganism according to the above (2) or (3), wherein the intracellular copy number of the DNA is amplified to thereby enhance acetic acid resistance.

(5) The microorganism according to the above (4), wherein the microorganism is an acetic acid bacterium belonging to the genus Acetobacter or the genus Glucone acetopactor. (6) Among the microorganisms according to (4) or (5), those having an alcohol oxidizing ability are cultured in a medium containing alcohol to produce and accumulate acetic acid in the medium. A method for producing vinegar, characterized by the fact that the acetic acid content obtained thereby is high (10 to 16%).

 (7) A recombinant plasmid pUSHC (FERM BP-7933) containing at least the DNA of (2) or (3) above.

 (8) A recombinant plasmid comprising a DNA fragment having at least the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, for example, an acetic acid bacterium-E. Coli shuttle vector (multicopy ぺ uichii) pMV24. Plasmid p SHC containing fragment A and / or this plasmid p SHC

(Acetobacter aceti) A transformant obtained by introducing No. 1023 (FERM BP-2287).

 ADVANTAGE OF THE INVENTION According to this invention, resistance to acetic acid can be provided and enhanced to microorganisms. Microorganisms having alcohol oxidizing ability, especially acetic acid bacteria, have remarkably improved resistance to acetic acid, and can impart the ability to efficiently accumulate a high concentration of acetic acid in a medium. Embodiment of the Invention

 Hereinafter, the present invention will be described in detail.

 (1) DNA of the present invention

The DNA of the present invention has an active region of squalene-hopene cyclase gene, has squalene-hopene cyclase activity, and has a function of improving acetic acid resistance. A base sequence capable of encoding a protein having a sequence; a regulatory element of the base sequence; and a structural portion of the gene. The DNA of the present invention can be obtained from the chromosomal DNA of Gluconacetobacter entanii as follows. First, the glucoconacetobacillus strain A., for example, the strain Acetobacter altoacetigenes MH-24 (FERM BP-49) Prepare the chromosomal DNA library in 1). The chromosomal DNA is obtained, for example, by the method disclosed in Patent Document 3.

 Next, to isolate the acetate resistance gene from the obtained chromosomal DNA, the chromosome! ) Make NA library. First, chromosomal DNA is partially digested with an appropriate restriction enzyme to obtain a mixture of various fragments. A wide variety of restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time. For example, Sau3AI is allowed to act on chromosome DNA at a temperature of 30 ° C or higher, preferably 37 ° C, and at an enzyme concentration of 1 to 10 units / ml for various times (1 minute to 2 hours) to digest it. I do. In the examples described later, BamHI was used.

 Next, the cut chromosomal DNA fragment is ligated to a vector DNA capable of autonomously replicating in acetic acid bacteria to prepare a recombinant DNA. Specifically, a restriction enzyme that generates a terminal base sequence complementary to the restriction enzyme BamHI used for cleavage of the chromosome DNA, for example, BamHI at a temperature of 30 ° C; an enzyme concentration of 1 to 100 units Under the condition of / ml, it is applied to vector DNA for 1 hour or more to digest it completely, and cut and cleave it.

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

 Using the obtained recombinant DNA, an acetic acid bacterium that can normally only grow on an agar medium with an acetic acid concentration of up to 1%, for example, Acetobacter aceti 1023 strain (Acetobacter ace No. 1023) Transform the strain (FERM BP-2287), spread on an agar medium containing 2% acetic acid, and culture. The resulting colony is inoculated into a liquid medium, cultured, and the plasmid is recovered from the resulting cells to obtain a DNA fragment containing the acetate resistance gene.

 Specific examples of the DNA of the present invention include a DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. Among them, the nucleotide sequence consisting of nucleotide numbers 406 to 2436 is a coding region.

The nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2 (FIGS. 4 and 5: Based on the homology search in DDBJ / EMBL / Genbank and SWISS-PROT / PIR, the nucleotide sequence of 406-2436 was compared with the SHC gene of Bradyrhizobium japonicum at the amino acid sequence level. 54.2%, and 53.5% homology with the SHC gene of Rhizobium sp. (Rizobium sp), all of which had low homology of the order of 50%, It was clear that this was a novel gene different from the gene encoding The above-mentioned SHC gene is not known at all to be related to acetic acid tolerance.

 Furthermore, the DNA of the present invention is a novel acetic acid resistant gene that is different from the acetic acid resistant genes (aa rA, aarB, aar C) of acetic acid bacteria that have already been obtained and the ADH gene that has the function of enhancing acetic acid resistance. It was identified as a gene having a function of enhancing.

 Since the nucleotide sequence of the DNA of the present invention has been clarified, for example, oligonucleotides synthesized based on the nucleotide sequence using genomic DNA of acetic acid bacteria Gluconacetobac Can be obtained by polymerase chain reaction using a primer as a primer (PCR reaction), or by hybridization using an oligonucleotide synthesized based on the nucleotide sequence as a probe. .

 Oligonucleotides can be synthesized, for example, using various commercially available DNA synthesizers according to a standard method. The PCR reaction was performed using Taq DNA polymerase (Takara Shuzo) and KOD-Plus— (Toyo Tokai) using a thermal cycler Gene Amp PCR System 2400 manufactured by Applied Biosystems. (Manufactured by Spinning Co., Ltd.).

 The DNA encoding the protein having the function of enhancing acetate resistance according to the present invention has one or several amino acids at one or more positions as long as the function of enhancing the acetate resistance of the encoded protein is not impaired. It may encode a deleted, substituted, inserted, or added protein.

A protein substantially the same as the protein having the function of enhancing acetic acid resistance. The DNA encoding the protein can also be obtained by, for example, modifying the nucleotide sequence such that the amino acid at a specific site is deleted, substituted, inserted, added or inverted by site-specific mutagenesis. The modified DNA as described above can also be obtained by a conventionally known mutation treatment.

 Also, it is generally known that the amino acid sequence of a protein and the nucleotide sequence encoding the same differ slightly between species, strains, mutants, and variants, and therefore DNA encoding a substantially identical protein Can be obtained from acetic acid bacteria in general, especially from species, strains, mutants, and varieties of the genus Acetobac.

 Specifically, acetobacilli of the genus Acetobacter glucoconacetobacillus or acetic acid bacterium of the genus Acetobabac genus glucoconacetobacillus, or a naturally-occurring mutant or variant thereof, for example, from It has a function to enhance acetic acid resistance by hybridizing under stringent conditions with DNA having a nucleotide sequence consisting of nucleotide sequence numbers 406-243 out of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. By isolating the DNA encoding the protein, a DNA encoding the protein substantially identical to the protein can be obtained. The stringent conditions referred to here are conditions under 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, an example is that DNAs with high homology, for example, DNAs with 70% or more homology, hybridize with each other and have lower homology Examples of the conditions include conditions under which DNAs do not hybridize, or conditions for washing ordinary hybridization, such as conditions for washing at 60 ° C. with lx SSC at a salt concentration equivalent to 0.1% SDS.

 (2) The acetic acid bacterium of the present invention

 The acetic acid bacterium of the present invention refers to a bacterium belonging to the genus Acetobacter and the genus Glucona acetobacillus, and is a bacterium belonging to the genus Acetobabac and the genus Daruconacetobacca having enhanced acetic acid resistance.

Acetobacter genus bacteria, specifically, Acetobacter aceti), and Acetobacter aceti No. 1023 (deposited as FERM BP-2287 in Patent Organism Depository Senyuichi).

 Gluconacetobacter species include Gluconacetobacter entanii, which is currently deposited as FERM BP-491 with the Patent Organism Depositary. Acetobacter yuichi · An example is Acetobacter altoacetigenes MH-24. The enhancement of acetic acid resistance can be achieved, for example, by amplifying the intracellular copy number of the acetic acid resistance gene, or by linking a DNA fragment containing the structural gene of the gene to a promoter sequence that functions efficiently in Acetobacter bacteria. The recombinant DNA can be used to enhance A. cerevisiae bacteria.

 In addition, the promoter sequence of the gene on the chromosomal DNA may be replaced with other promoter sequences that function efficiently in bacteria of the genus Acetobacter or the glucoconactor, such as the ampicillin resistance gene of Escherichia coli plasmid pBR322. By substituting a promoter sequence derived from a microorganism other than acetic acid bacteria, such as a promoter of each gene such as a kanamycin resistance gene of plasmid pACYC177, a kappa rampunicol resistance gene of plasmid pACYC184, and a 3-galactosidase gene. Can also enhance acetic acid tolerance.

 Amplification of the intracellular copy number of the gene can be carried out by introducing a multicopy vector carrying the gene into cells of a bacterium belonging to the genus Acetobacter. That is, it can be carried out by introducing a plasmid, transposon, or the like retaining the gene into cells of a bacterium belonging to the genus Acetocapyu and the genus Glucon acetopacu.

Examples of the multicopy vector include pMV24 (for example, see Non-Patent Document 4), PTA5001 (A), and pTA5001 (B) (for example, see Patent Document 4). PMVL 1 (for example, see Non-Patent Document 5). Examples of transposons include Mu and IS 1452. The introduction of DNA into the acetic acid bacteria of the genus Acetobacter genus Glucon acetobacillus can be carried out by the calcium chloride method (see, for example, Non-Patent Document 6) or the electrolysis method (for example, Non-Patent Document 7). See).

 In the acetic acid bacterium belonging to the genus Acetobabac and the genus Glucona acetobacca having the ability to oxidize alcohol, when the acetic acid tolerance is enhanced as described above, the production amount and production efficiency of acetic acid can be increased.

 (3) Vinegar production method

 As described above, a bacterium belonging to the genus Acetobacter var. Dalconacetopactor, whose acetic acid resistance is selectively enhanced by the amplification of the copy number of the acetic acid resistance gene, having alcohol oxidizing ability. The vinegar can be efficiently produced by culturing the product in an alcohol-containing medium and producing and accumulating acetic acid in the medium. The acetic acid fermentation in the production method of the present invention may be performed in the same manner as the conventional vinegar production method by the fermentation method of acetic acid bacteria. The medium used for acetic acid fermentation may be a synthetic medium or a natural medium as long as it contains a carbon source, a nitrogen source, an inorganic substance, and ethanol and, if necessary, contains an appropriate amount of a nutrient required by the strain used for growth. But it is good.

 Examples of the carbon source include various carbohydrates such as glucose-sucrose and various organic acids. As the nitrogen source, a natural nitrogen source such as peptone or a fermentation cell decomposition product can be used.

 The cultivation is performed under aerobic conditions such as a static culture method, a shaking culture method, and an aeration-agitation culture method, and the culture is usually performed at 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 the medium can be prepared with various acids, various bases, buffers and the like. A high concentration of acetic acid accumulates in the medium, usually after culturing for 1 to 21 days.

 (4) Embodiment of the present invention

The recombinant plasmid pUSHC obtained by inserting the ORF according to the present invention or the acetic acid resistance gene (SEQ ID NO: 1) containing the same into Escherichia coli vector pUC19 was obtained from Tsukuba-Higashi 1-chome, Ibaraki, Japan. No. 1 Established as the FEHM BP-7933 at the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (AIST) (2002) Since it was deposited on March 1, the DNA of the gene of the present invention can be easily obtained from this recombinant plasmid, and those skilled in the art can easily carry out the present invention. is there. Then, if desired, the SHC gene is taken out according to a conventional method, inserted into an appropriate vector, introduced into an acetic acid bacterium, and cultured to easily produce vinegar having a high acetic acid content. Can be manufactured.

 Furthermore, as described above and as will be apparent from the examples described below, the deposit number of the acetate-resistant gene source, the PCR embodiment, the plasmid vector, the preparation of the recombinant plasmid, the deposit number of the host bacteria, etc. Are embodied and clarified, and all are easy to obtain, operate, and process. Therefore, if each operation and process are performed according to the examples described in this specification, the desired acetic acid resistance can be obtained. A transformant can be obtained, and by using this, vinegar containing a high concentration of acetic acid can be produced. Therefore, implementation of the present invention is easy from this point as well. Hereinafter, the present invention will be described specifically with reference to examples.

 Example

 (Example 1) Determination of the nucleotide sequence and amino acid sequence of the acetic acid resistance gene from Glucon acetate

 (1) Preparation of chromosome DNA library

 Acetono, a strain of Gluconacetobacter entanii, is a strain of Gluconacetobacter entanii, a strain of Acetobacter altoacetigenes MH-24 (FERM BP- 49 1) was shake-cultured at 30 ° C in a YPG medium (3% glucose, 0.5% yeast extract, 0.2% polypeptone) supplemented with 6% acetic acid and 4% ethanol. After culture, centrifuge the culture

(7, 500 xg, 10 minutes) to obtain bacterial cells. Chromosomal DNA was prepared from the obtained cells by the method disclosed in Patent Document 3.

The chromosomal DNA and Escherichia coli-acetic acid bacteria shuttle vector PMV24 obtained as described above were cut with the restriction enzyme BamHI (Takara Shuzo). These DNAs are mixed in appropriate amounts, and ligated using the ligation kit (TaKaRa DNA Ligation Kit Ver.2, (Manufactured by Takara Shuzo Co., Ltd.) to construct a chromosome DNA lipase of Dalcon acetobacta.

 (2) Cloning of the acetate resistance gene

 The chromosome DNA library of Dalcon acetopathogen entannii obtained as described above can normally be grown only on an agar medium to an acetic acid concentration of about 1%. The strain was transformed and cultured on YPG agar medium containing 2% acetic acid and 100 μg / ml ampicillin at 30 ° C. for 4 days. The resulting colony was inoculated and cultured in YPG medium containing 100 zgZml of ampicillin, and plasmid was recovered from the resulting cells.The BamHI fragment of about 5 kbp shown in Figure 1 was cloned. This plasmid was recovered, and this plasmid was named pB1. Further, a DNA fragment capable of growing Acetobacter acetis No. 1023 strain on YPG agar medium containing 2% acetic acid was obtained from a BamHI fragment of about 5 kbp cloned into pBI. It was confirmed that the fragment was a BamHI-PstI fragment of about 2.7 kbp.

 In this way, an acetic acid resistance gene fragment is obtained that enables the Acetobacter aceti No. 1023 strain, which can normally grow only up to about 1% acetic acid concentration on an agar medium, to grow on an agar medium containing 2% acetic acid. did.

 (3) Determination of the nucleotide sequence of the cloned DNA fragment

 The above-mentioned cloned BamHI-PstI fragment was inserted into the BamHI-PstI site of Escherichia coli vector pUC19: recombinant plasmid pUSHC (FER MBP-7). 9 33) was prepared. Using this plasmid, the nucleotide sequence of the cloned BamHI-PstI fragment was determined by Sanger's didoxy chain termination method. As a result, the nucleotide sequence described in SEQ ID NO: 1 was determined. Sequencing was performed on all regions of both DNA strands, with all breaks overlapping.

In the nucleotide sequence described in SEQ ID NO: 1, an open reading encoding 677 amino acids (FIGS. 4 and 5) as described in SEQ ID NO: 2 from nucleotide number 406 to nucleotide number 2436 · The existence of a frame (ORF) was confirmed and squalene The DXDD TA motif, which is a conserved region of Hoppen cyclase, was also found to exist from amino acid number 415 to amino acid number 420, and this gene was designated as SHC gene.

(Example 2) Enhancement of acetic acid tolerance in a transformant transformed with an acetic acid resistance gene derived from Glucona acetobac

 (1) Transformation into Aspactor Asechi

 Gluconacetono, a strain of Gluconacetobacter entanii (Acetobacter altoacetigenes MH-24) (FERM BP-49), which is a strain of Gluconacetobacter entanii 1) A DNA fragment containing an acetic acid resistance gene derived from PCR was amplified by the PCR method. The resulting amplified fragment was digested with BamH EcoRI, and the fragment was subjected to acetic acid bacteria-E. Coli shuttle vector. Plasmid pSHC inserted into the restriction enzyme BamHI-EcoI cleavage site of pMV24 (see, for example, Non-patent Document 4) was prepared, and the outline of the amplified fragment inserted into pSHC is shown in FIG. Was.

 The PCR method was performed as follows. Specifically, genomic DNA derived from the acetic acid bacterium described above was used as type I, and primers 1 (the nucleotide sequence is shown in SEQ ID NO: 3 (FIG. 6)) and primer 1 (the nucleotide sequence was shown in SEQ ID NO: 4 (see FIG. The PCR method was performed under the following PCR conditions using the method described in 7)).

 The PCR condition is 94. 30 cycles were performed with one cycle of 15 seconds at C, 30 seconds at 60 ° C, and 2 minutes at 68 ° C.

のアンピシリン及び 2 %の酢酸を添加し fこ YP G寒天培地で選択 した。 This pSHC was transformed into Acetobacter aceti No. 1023 strain by the electroporation method (for example, see Non-Patent Document 7). 10 0 transformants

Of ampicillin and 2% acetic acid, and selected on a YPG agar medium.

Plasmid was extracted from the ampicillin-resistant transformant grown on the selection medium by a conventional method and analyzed, and it was confirmed that the plasmid possessed the SHC gene-containing plasmid. (2) Acetic acid resistance of the transformed strain

 The ampicillin-resistant transformant having the plasmid pSHC obtained as described above was grown on a YPG medium supplemented with acetic acid, and the original strain Acetobacter acetylacetone was introduced only with the shuttle vector pMV24. Compared to N 0.1023 strain.

 Specifically, shaking culture (15 ° rpm) at 30 ° C in 10 Oml YPG medium containing 3% acetic acid, 3% ethanol, and 100 μg / m1 of ampicillin The growth of the strain and that of the original strain in a medium containing acetic acid were compared by measuring the absorbance at 660 nm.

 As a result, as shown in FIG. 2, the original strain and the transformed strain showed almost the same growth in the YPG medium supplemented with 3% ethanol without acetic acid, whereas the original strain and the transformed strain exhibited the same growth in 3% acetic acid and 3% ethanol. Only the transformed strain was able to grow on the medium supplemented with evening water, and it was confirmed that it could not be grown on the original strain, Acetobacter yuuichi 'Aceti No. 1023. Was confirmed.

 (3) Temperature tolerance of the transformed strain

 The ampicillin-resistant transformant having the plasmid p SHC obtained in the above (1) was grown on a YPG medium at a different culture temperature, and the cells were transformed with only the Shuttle P. Yuichi pMV24. The strain was compared to the strain Acetobak Yuichi 'Aceti No. 1023.

 Specifically, using a 2 L mini jar (Chiyoda Seisakusho: TBR-2-1), 1 L YPG medium containing 1% acetic acid, 4% ethanol, and 100 μg / m 1 ampicillin was used. The mixture was cultured under aeration and agitation at 30 ° C, 400 rpm, and 0.20 vvm, and fermentation was performed until the acetic acid concentration was about 3%. After that, remove 200 ml of the culture solution in the mini jar, add 800 ml of YPG medium containing 100 μg / ml of acetic acid, ethanol and ampicillin, and add 4% of ethanol with 1% of acetic acid. After adjusting the concentration, the culture temperature was raised to 33 ° C and the fermentation was started again.

When the fermentation further progressed and the acetic acid concentration in the medium reached about 3%, the medium was removed again, the medium was added again, and the culture temperature was increased to 36 ° C. In the same manner, acetic acid fermentation was carried out by increasing the culture temperature by 1 ° C. The cell growth was measured by measuring the absorbance at 660 nm, and the degree of acetic acid fermentation was compared by measuring the concentration of acetic acid in the culture solution.

 As a result, as shown in Fig. 3, the transformed strain was capable of acetic acid fermentation and cell growth at 40 ° C, while the original strain Acetobacter 1'Aceti No. 1023 was

Acetic acid fermentation and cell growth were confirmed only up to 7 ° C, confirming the function of enhancing the temperature tolerance of the SHC gene.

 (Example 3) Acetic acid fermentation test and lipid analysis of a transformant transformed with the SHC gene derived from Yuichi N.

 (1) Acetic acid fermentation test

 The ampicillin-resistant transformant having the plasmid p SHC obtained in Example 2 was compared with the original strain Acetobac Yuichi Acetii No. 1023 strain having only Shuttle P. Yuichi pMV24 for acetic acid fermentation ability. .

 Specifically, using a 5 L mini-jar (manufactured by Sanka Rika Kogyo Co., Ltd .; KMJ-5A), a 2.5 L YPG medium containing 1% acetic acid, 4% ethanol, and 10 OzgZml ampicillin Then, aeration and stirring culture was performed at 30 ° C., 400 rpm, and 0.20 vvm, and fermentation was performed until the acetic acid concentration reached 3%. Then, remove the culture solution while leaving 700 ml of the culture solution in the mini jar, and add 1.8 L of YPG medium containing acetic acid, ethanol, and 100 / g / ml acetic acid to the remaining 700 ml, and add Adjust the concentration to 3% and ethanol to 4%, start acetic acid fermentation again, and continue aeration and stirring culture while adding ethanol so that the concentration of ethanol in the medium is maintained at 1%. The acetic acid fermentation ability of the transformed strain and the original strain was compared. Table 1 summarizes the results. table 1

表 1の結果から、 形質転換株の方が、 最終到達酢酸濃度、 比増殖速度、 生酸 速度、 増殖誘導期の何れにおいても、 顕著に優れていることが確認できた。

From the results in Table 1, it can be seen that the transformed strain has a higher final acetic acid concentration, specific growth rate, It was confirmed that both the speed and the proliferation induction period were remarkably excellent.

 (2) Analysis of lipid composition of bacterial cells

 The lipid composition of the cells of the transformant resistant to ampicillin having plasmid pSHC was compared with that of the original strain Asetobaku yuichi 'Aceti No. 1023 having only the shuttle vector-1 pMV24.

 Specifically, in the above (1), the fermentation broth fermented to a final acidity of 9.5% with the original strain and the fermentation broth fermented to a final acidity of 11.2% with the transformed strain are each separated. The cells were separated by heart (7,500 xg, 10 minutes) to obtain bacterial cells. The obtained cells were washed three times with 50 mM Tris-HCl buffer (pH 8.0). Immediately thereafter, total lipids were extracted according to the ply-diamond method (for example, see Non-Patent Document 8).

 Phospholipids in total lipids were quantified using a phospholipid-Test Co., Ltd. (manufactured by Wako Pure Chemical Industries, Ltd.), and tetrahydroxybacteriohopane was analyzed according to the following method. Calculated.

That is, all lipids were suspended in 0.4 N methanolic sodium hydroxide and kept at 37 ° C for 2 hours. The organic layer was collected from the reaction product by Folch partitioning (for example, see Non-Patent Document 9), concentrated to dryness in a single tally evaporator, and then dried in an appropriate amount of a mixture of chloroform-methanol (2: 1, v / v). Upon dissolution, an alkali-stable lipid was prepared.

 The thus obtained lipid-stable lipid is subjected to a penzyl derivatization treatment by the method of Nagiec et al. (For example, see Non-Patent Document 10), and is subjected to the method of Kito et al. (For example, see Non-Patent Document 11). Unreacted materials were removed.

The purified benzoyl derivative is concentrated to dryness on a rotary evaporator and dried, then dissolved in hexane-isopropanol (100: 1.5, v / v) and analyzed by high performance liquid chromatography (Shimadzu; SHIMADZU LC -6A). Hexane-isopropanol (100: 1.5, v / v) was eluted at a flow rate of 1 m1 / min using LiChrospher 100 CN (Merck; 4x250), and the detection wavelength was 230 nm. And Table 2 summarizes the above analysis results. (Table 2)

 T HB H: Tetrahydroxybacteriohopane

 From the results in Table 2, it was confirmed that in the transformed strain, THBH, which is a metabolite of SHC, is 1.28 times higher than the original strain, and the cloned SHC gene encodes squalene-hopene cyclase. Was done.

(Example 4) Enhancement of acetic acid resistance in a transformant transformed with an acetic acid resistance gene derived from Glucone acetobac

 (1) Transformation into Acetobacter

 Plasmid p SHC obtained in Example 2 was converted to Acetobac Yuichi, a strain of Gluconacetobacter entanii. ■ Acetobacter altoacetigenes MH-24 The strain was transformed into a strain (FERM BP-49 1) by the electroporation method (for example, see Non-Patent Document 7). Transformants were selected on a YPG agar medium containing 0.55% agar supplemented with 100 μg / ml of ampicillin, 4% acetic acid and 4% ethanol.

 Plasmids were extracted from the ampicillin-resistant transformant grown on the selective medium and analyzed by a conventional method, and it was confirmed that the plasmid possessed the SHC gene-containing plasmid.

 (1) Acetic acid fermentation test

 With respect to the ambicillin-resistant transformant having the plasmid pSHC obtained in Example 2, the acetic acid fermentation ability was compared with that of the original strain Asetobak yuichi-artacetogenes MH-24 strain having only the shuttle vector pMV24.

 Specifically, using a 5 L mini-jar (manufactured by Sanzaki Chemical Co., Ltd .; KM J-5A), 2.5 L of YPG containing 4% of acetic acid, 4% of ethanol, and 100 μg / ml of ampicillin Culture at 30 ° C, 500 rpm, 0.20 vvm with aeration and agitation in the medium.

E0 The fermentation was performed until the acetic acid concentration reached 6.3%. Then, leave the 700 ml culture in the mini jar, remove the culture, and add 1.8 L of YPG medium containing acetic acid, ethanol, and ampicillin 100/1111 to the remaining 700 ml. To adjust the concentration to 5.5% acetic acid and 4% ethanol, start acetic acid fermentation again, and add aeration while adding ethanol to maintain the concentration of ethanol in the culture medium at 1%. The culturing was continued, and the acetic acid fermentation ability of the transformed strain and the original strain was compared. Table 3 summarizes the results.

 Table 3

 From the results in Table 3, it was confirmed that the transformed strain was remarkably superior in all of the finally reached acetic acid concentration, specific growth rate, and raw acid rate. The invention's effect

According to the present invention, a novel gene involved in acetic acid resistance is provided, and a breeding strain capable of producing vinegar having a higher acetic acid concentration with high efficiency can be obtained by using the gene. Furthermore, a method for producing vinegar having a high acetic acid concentration with high efficiency could be provided. ·

Reference to deposited microorganisms in Rule 13bis

 pU S H C

(I) the name and address of the depositary institution that deposited the microorganism;

 Name National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary Sen-ichi Yuichi 305—8566 Tsukuba, Ibaraki, Japan

 Higashi 1-chome No. 1 1 Date of deposit with the depository at Central No. 6

 March 1, 2002

The accession number assigned to the deposit by the high depositary institution

 FERM BP— 7933

Claims

The scope of the claims 1 Protein SHC shown in (A) or (B) below.  (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing,  (B) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, which comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and enhances acetic acid resistance Functional protein.  2 DNA of the gene encoding the protein SHC shown in (A) or (B) below.  (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing,  (B) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, which comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and enhances acetic acid resistance Functional protein. .  3. The DNA of the gene according to claim 2, which is a DNA shown in the following (a) or (b).  (a) DNA comprising a base sequence consisting of base numbers 406 to 2436 among the base sequences described in SEQ ID NO: 1 in the sequence listing.  (b) a probe having a base sequence consisting of base numbers 406 to 2436 or a part thereof among the base sequences described in SEQ ID NO: 1 in the sequence listing, and hybridizing under stringent conditions, and having acetic acid resistance DNA that encodes a protein that has the function of enhancing chromosomes.  A microorganism whose acetic acid resistance has been enhanced by increasing the intracellular copy number of the DNA according to claim 2 or 3.  5. The microorganism according to claim 4, wherein the microorganism is an acetic acid bacterium belonging to the genus Acetopaku or the genus Glucone acetobac. (6) A vinegar characterized in that, among the microorganisms according to (4) or (5), those having the ability to oxidize alcohol are cultured in a medium containing alcohol to produce and accumulate acetic acid in the medium. Manufacturing method. 7 A recombinant plasmid pUSHC (FERM BP-7933) containing at least the DNA of claim 2 or claim 3.