WO2003078622A1 - Gene cyclase de squalene-hopene de bacterie acetique, bacterie acetique cultivee avec ce gene et procede de production de vinaigre au moyen de cette bacterie - Google Patents

Gene cyclase de squalene-hopene de bacterie acetique, bacterie acetique cultivee avec ce gene et procede de production de vinaigre au moyen de cette bacterie Download PDF

Info

Publication number
WO2003078622A1
WO2003078622A1 PCT/JP2003/002731 JP0302731W WO03078622A1 WO 2003078622 A1 WO2003078622 A1 WO 2003078622A1 JP 0302731 W JP0302731 W JP 0302731W WO 03078622 A1 WO03078622 A1 WO 03078622A1
Authority
WO
WIPO (PCT)
Prior art keywords
acetic acid
gene
dna
acid bacterium
protein
Prior art date
Application number
PCT/JP2003/002731
Other languages
English (en)
Japanese (ja)
Inventor
Hidetsugu Goto
Shigeru Nakano
Original Assignee
Mitsukan Group Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mitsukan Group Corporation filed Critical Mitsukan Group Corporation
Priority to JP2003576616A priority Critical patent/JP4312608B2/ja
Priority to AU2003221332A priority patent/AU2003221332A1/en
Publication of WO2003078622A1 publication Critical patent/WO2003078622A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
    • C12J1/00—Vinegar; Preparation or purification thereof
    • C12J1/04—Vinegar; Preparation or purification thereof from alcohol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid

Definitions

  • 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.
  • Acetic acid bacteria are microorganisms widely used in the production of vinegar.
  • acetic acid bacteria belonging to the genus Acetobak and the genus Glucona acetobac are used for industrial acetic acid fermentation.
  • 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.
  • 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.
  • 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).
  • the aar A gene is a gene encoding citrate synthase
  • the aar C gene was presumed to be a gene encoding an enzyme related to acetic acid utilization, but the aarB gene is functional.
  • Non-Patent Document 2 the aarA gene is a gene encoding citrate synthase
  • the aar C gene was presumed to be a gene encoding an enzyme related to acetic acid utilization, but the aarB gene is functional.
  • 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).
  • 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.
  • ADH membrane-bound aldehyde dehydrogenase
  • Patent Document 2 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 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 2
  • Patent Document 4 is a diagrammatic representation of Patent Document 4.
  • 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.
  • 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.
  • a method for finding an acetic acid resistance gene from acetic acid bacteria uses the chromosome of acetic acid bacteria.
  • 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.
  • 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.
  • 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).
  • metabolites which are metabolites in their lipid composition.
  • 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.
  • amino acid sequence of the protein encoded by the present acetate resistance gene (SEQ ID NO: 2) is shown.
  • embodiments of the present invention are as follows.
  • a DNA of the gene according to (2) which is a DNA shown in (a) or (b) below.
  • 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.
  • 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%).
  • a recombinant plasmid pUSHC (FERM BP-7933) containing at least the DNA of (2) or (3) above.
  • 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 ⁇ uichii) pMV24. Plasmid p SHC containing fragment A and / or this plasmid p SHC
  • 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.
  • 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.
  • 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.
  • chromosomal DNA is partially digested with an appropriate restriction enzyme to obtain a mixture of various fragments.
  • restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time.
  • 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.
  • BamHI was used.
  • the cut chromosomal DNA fragment is ligated to a vector DNA capable of autonomously replicating in acetic acid bacteria to prepare a recombinant DNA.
  • 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.
  • 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.
  • 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.
  • DNA of the present invention examples 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the carbon source examples include various carbohydrates such as glucose-sucrose and various organic acids.
  • 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.
  • 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.
  • 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.
  • the present invention will be described specifically with reference to examples.
  • Example 1 Determination of the nucleotide sequence and amino acid sequence of the acetic acid resistance gene from Glucon acetate
  • Acetono a strain of Gluconacetobacter entanii
  • 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
  • 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.
  • 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 ⁇ g / 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.
  • 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.
  • 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.
  • 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.
  • Example 2 Enhancement of acetic acid tolerance in a transformant transformed with an acetic acid resistance gene derived from Glucona acetobac
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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).
  • 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.
  • 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)
  • 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 ⁇ g / 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)

Abstract

L'invention concerne un gène, ayant pour origine une bactérie acétique, participant à la tolérance à l'acide acétique, un procédé d'amélioration de la tolérance à l'acide acétique chez un micro-organisme, en particulier, chez une bactérie acétique, au moyen de ce gène, ainsi qu'un procédé de production efficace de vinaigre à l'aide de la bactérie acétique dotée de la tolérance améliorée à l'acide acétique. A partir d'une banque d'ADN chromosomal de bactérie acétique, on obtient un gène permettant la croissance de bactérie acétique dans un milieu contenant de l'acide acétique à une concentration si élevée qu'elle ne permet normalement pas la croissance. Grâce à ce procédé, un gène conférant une tolérance à l'acide acétique est cloné à partir d'une bactérie acétique appartenant au genre Gluconaacetobacter, aux fins de production. Un transformant construit par transfert de ce gène dans une bactérie acétique permet d'obtenir une tolérance remarquable à l'acide acétique. Lorsque ce transformant est cultivé en aérobie en présence d'éthanol, la période d'induction de croissance est raccourcie et la vitesse de croissance et la vitesse de formation d'acide sont élevées. En outre, le niveau d'accumulation final d'acide acétique peut être remarquablement élevé.
PCT/JP2003/002731 2002-03-15 2003-03-07 Gene cyclase de squalene-hopene de bacterie acetique, bacterie acetique cultivee avec ce gene et procede de production de vinaigre au moyen de cette bacterie WO2003078622A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2003576616A JP4312608B2 (ja) 2002-03-15 2003-03-07 酢酸菌のスクアレン−ホペンサイクラーゼ遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
AU2003221332A AU2003221332A1 (en) 2002-03-15 2003-03-07 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

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-73115 2002-03-15
JP2002073115 2002-03-15

Publications (1)

Publication Number Publication Date
WO2003078622A1 true WO2003078622A1 (fr) 2003-09-25

Family

ID=28035222

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/002731 WO2003078622A1 (fr) 2002-03-15 2003-03-07 Gene cyclase de squalene-hopene de bacterie acetique, bacterie acetique cultivee avec ce gene et procede de production de vinaigre au moyen de cette bacterie

Country Status (3)

Country Link
JP (1) JP4312608B2 (fr)
AU (1) AU2003221332A1 (fr)
WO (1) WO2003078622A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005001095A1 (fr) * 2003-06-26 2005-01-06 Mitsukan Group Corporation Gene participant a la fonction de stimulation de la croissance des bacteries ascetiques et utilisations de ce gene
WO2006064746A1 (fr) * 2004-12-17 2006-06-22 Mitsukan Group Corporation Procede de reproduction de cellules ayant une meilleure tolerance envers les acides gras a chaine courte
JP2006246701A (ja) * 2005-02-10 2006-09-21 Mitsukan Group Honsha:Kk 中央代謝系の酵素活性が増強された酢酸菌、及び該酢酸菌を用いた食酢の製造方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS609488A (ja) * 1983-06-29 1985-01-18 Teruhiko Beppu 酢酸菌ベクタ−
JPS609489A (ja) * 1983-06-29 1985-01-18 Teruhiko Beppu 酢酸菌の形質転換方法
JPS60180581A (ja) * 1984-02-28 1985-09-14 Nakano Vinegar Co Ltd 新菌種アセトバクタ−・アルトアセチゲネス
EP0332120A2 (fr) * 1988-03-08 1989-09-13 Nakano Vinegar Co., Ltd. Gène structural pour aldéhyde-déhydrogénase lié à la membrane, plasmide le contenant, bactérie acido-acétique transformée avec ce plasmide, et fermentation acido-acétique utilisant cette transformante
JPH03219878A (ja) * 1989-02-15 1991-09-27 Nakano Vinegar Co Ltd 酢酸耐性遺伝子、それを含むプラスミド及び形質転換した酢酸菌
JPH05199887A (ja) * 1990-02-09 1993-08-10 Nakano Vinegar Co Ltd 酢酸菌ｒｅｃＡ遺伝子、それを含む プラスミド及び形質転換した酢酸菌

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS609488A (ja) * 1983-06-29 1985-01-18 Teruhiko Beppu 酢酸菌ベクタ−
JPS609489A (ja) * 1983-06-29 1985-01-18 Teruhiko Beppu 酢酸菌の形質転換方法
JPS60180581A (ja) * 1984-02-28 1985-09-14 Nakano Vinegar Co Ltd 新菌種アセトバクタ−・アルトアセチゲネス
EP0332120A2 (fr) * 1988-03-08 1989-09-13 Nakano Vinegar Co., Ltd. Gène structural pour aldéhyde-déhydrogénase lié à la membrane, plasmide le contenant, bactérie acido-acétique transformée avec ce plasmide, et fermentation acido-acétique utilisant cette transformante
JPH03219878A (ja) * 1989-02-15 1991-09-27 Nakano Vinegar Co Ltd 酢酸耐性遺伝子、それを含むプラスミド及び形質転換した酢酸菌
JPH05199887A (ja) * 1990-02-09 1993-08-10 Nakano Vinegar Co Ltd 酢酸菌ｒｅｃＡ遺伝子、それを含む プラスミド及び形質転換した酢酸菌

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FREIBERG C. ET AL.: "Molecular basis of symbiosis between rhizobium and legumes", NATURE, vol. 387, no. 6631, 1997, pages 394 - 401, XP002969316 *
FUKAYA M. ET AL.: "The aarC gene responsible for acetic acid assimilation confers acetic acid resistance on acetobacter aceti.", J. FERMENT. BIOENG., vol. 76, no. 4, 1993, pages 270 - 275, XP002969319 *
REIPEN I.G. ET AL.: "Zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, DNA sequence analysis and expression in escherichia coli", MICROBIOLOGY, vol. 141, no. 1, 1995, pages 155 - 161, XP002969317 *
SCHULLER G. ET AL.: "Gluconacetobacter entanii sp. nov., isolated from submerged high-acid industrial vinegar fermentations", INT. J. SYST. EVOL. MICROBIOL., vol. 50, no. 6, 2000, pages 2013 - 2020, XP002969320 *
TIPPELT A. ET AL.: "Squalene-hopene cyclase from methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids", BIOCHEM. BIOPHYS. ACTA, vol. 1391, no. 2, 1998, pages 223 - 232, XP002969318 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005001095A1 (fr) * 2003-06-26 2005-01-06 Mitsukan Group Corporation Gene participant a la fonction de stimulation de la croissance des bacteries ascetiques et utilisations de ce gene
US7541491B2 (en) 2003-06-26 2009-06-02 Mitsukan Group Corporation Gene involved in growth-promoting function of acetic acid bacteria and uses thereof
WO2006064746A1 (fr) * 2004-12-17 2006-06-22 Mitsukan Group Corporation Procede de reproduction de cellules ayant une meilleure tolerance envers les acides gras a chaine courte
JP2006246701A (ja) * 2005-02-10 2006-09-21 Mitsukan Group Honsha:Kk 中央代謝系の酵素活性が増強された酢酸菌、及び該酢酸菌を用いた食酢の製造方法

Also Published As

Publication number Publication date
JP4312608B2 (ja) 2009-08-12
AU2003221332A1 (en) 2003-09-29
JPWO2003078622A1 (ja) 2005-07-14

Similar Documents

Publication Publication Date Title
US7354751B2 (en) Alcohol dehydrogenase gene of acetic acid bacterium
JP4326967B2 (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP4312608B2 (ja) 酢酸菌のスクアレン−ホペンサイクラーゼ遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2006230329A (ja) 酢酸発酵能が増強された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
US7256025B2 (en) Gene improving temperature-tolerance of acetic acid bacterium, acetic acid bacterium bred using the gene and process for producing vinegar using the acetic acid bacterium
JP4083455B2 (ja) 酢酸菌のアコニターゼ遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2006246701A (ja) 中央代謝系の酵素活性が増強された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2004121021A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005040079A (ja) 酢酸菌のグルタチオン依存型ホルムアルデヒド脱水素酵素遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2004305209A (ja) 酢酸菌の増殖促進機能に関与する遺伝子
JP2005040065A (ja) タンパク質複合体ａｂｃ３、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005040019A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005013103A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005013101A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005013081A (ja) タンパク質ｐｖｄｃ、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005040020A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005013082A (ja) タンパク質ｐｖｋ、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005013083A (ja) タンパク質ｇｌｕｋ、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005013113A (ja) 酢酸耐性に関与する遺伝子、該遺伝子を用いて育種された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005040064A (ja) タンパク質ｓｃｄｈ２、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005124480A (ja) タンパク質ｈｔｐｇ、その遺伝子、高度な温度耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005040080A (ja) タンパク質ｇａｄｈ、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2006230260A (ja) 酢酸耐性が増強された酢酸菌、及び該酢酸菌を用いた食酢の製造方法
JP2005040078A (ja) タンパク質ｏｍｐ１、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法
JP2005040063A (ja) タンパク質ｆｕｒ１、その遺伝子、高度な酢酸耐性を有する微生物、及び該微生物を用いた食酢の製造方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2003576616

Country of ref document: JP

122 Ep: pct application non-entry in european phase