JPH1175860A - Esterase structural gene, its transformed strain and production of vinegar using the structural gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene comprising the DNA of a structural gene coding for an esterase protein having a specific amino acid sequence and capable of giving recombinant Acetobacter, etc., for producing excellent vinegar containing high amounts of ethyl acetate and isoamyl acetate. SOLUTION: This invention relates to a new DNA of an esterase structural gene coding for a protein expressed by an amino acid sequence expressed by the formula and vinegar containing high amounts of ethyl acetate and isoamyl acetate and excellent in flavor can be produced by using recombinant Acetobacter introduced with fragments of the gene. This esterase structural gene is obtained by preparing a genome library of Acetobacter, Acetobacter pasteurianus, with Escherichia coli as a host, replicating the genome library on filtration paper, lysing the bacteria by immersing in a bacteriolytic solution, selecting colonies, which are changed to red color by a color developing method, as a clone strain of the esterase gene and harvesting the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an esterase structural gene, a transformant thereof, and a method for producing vinegar using the structural gene.

[0002]

2. Description of the Related Art Esterase is known as an enzyme that catalyzes a reaction for producing an ester from an acid and an alcohol and a reaction for decomposing the ester. ing. Three types of esterases have been partially purified from Acetobacter pasteurianus, one of the acetic acid bacteria, and their properties have been examined. (Japanese brewing society conference abstracts, p.9, 199
6 years).

[0003] The flavor of food is one of the important factors influencing its quality. Even in vinegar, various flavor components are generated during the brewing process. In particular, esters such as ethyl acetate reduce the flavor of acetic acid. It is considered important to make it mild. However, to date, no detailed studies have been made on the production of ethyl acetate by acetic acid bacteria.

[0004]

In order to improve the flavor of vinegar, it is essential to study the decomposition pathway and biosynthesis pathway of the flavor components of acetic acid bacteria belonging to the genera Acetobacter and Gluconobacter. Until very little research. Furthermore, destruction and amplification of key enzymes involved in aroma generation using genetic recombination technology as an advanced technology have not been attempted because of their complexity.

[0005] Therefore, efforts to improve the flavor of vinegar are:
At present, it is responding by changing the raw materials and leaving the scent obtained there as much as possible in the product, or removing the unpleasant odor with activated carbon. However, in the former,
Unless inexpensive raw materials or inexpensive vinegar fermentation starting material (vinegar fermentation raw material) can be obtained, there is a problem that the product becomes expensive, and in the latter case, activated carbon is not only unpleasant odor but also preferable Because it has the property of removing scent components,
From the viewpoint of producing fragrant vinegar, this method was difficult to use.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies from various angles to solve the above-mentioned problems, and as a result, have focused on a genetic recombination technique that has not been attempted before, and have developed an esterase gene. Was cloned, and the change in scent quality due to ester degradation was observed. However, it was discovered that only one of the three esterase genes significantly unexpectedly increased the amount of ester production. After various studies based on this discovery, it was possible to produce high-quality vinegar from inexpensive materials, and completed the present invention.

[0007] That is, the present inventors first, Escherichi
A genomic library of Acetobacter pasteurianus was prepared using a coli as a host, and three types of clones were obtained from the library by a conventional method. Was confirmed.

Next, one esterase gene of the three clones was inserted into a vector pUC119, and the plasmid pU119 was inserted.
E122 was prepared and introduced into E. coli to obtain a transformant. Further, an SphI fragment (containing an esterase gene) of plasmid pUE122 was inserted into an acetic acid bacteria-E. Coli shuttle vector (for example, pMV24) to prepare an expression plasmid pME122. Then, this plasmid was introduced into Acetobacter aceti, and it was discovered that the obtained transformant produced a large amount of ethyl acetate.As a result of further research, the gene encoding esterase was identified from the inserted DNA fragment contained in plasmid pUE122. The base sequence was also successfully determined, and these useful new findings were combined to finally complete the present invention.

That is, the present invention relates to a structural gene for the esterase represented by the amino acid sequence of SEQ ID NO: 1, a transformant having the gene, and a method for producing vinegar using the transformant. Hereinafter, the present invention will be described in detail.

(1) Cloning of esterase structural gene The acetic acid bacterium (hereinafter referred to as Acetobacter
Or acetic acid bacteria belonging to Gluconobacter)
A strain isolated from vinegar moromi and identified as Acetobacter pasteurianus by the Barges manual.
Year). That is, the present bacterium has three esterases, and the enzymatic properties of the partially purified enzyme are as already reported. The gene of the present invention corresponds to the structural gene of (esterase-1) protein in the above abstract.

[0011] The gene fragment containing the structural gene of the enzyme is
Any microorganism can be used as long as it produces the enzyme. For example, it can be isolated from the total DNA of acetic acid bacteria. Total DNA can be prepared, for example, by the method disclosed in JP-A-60-9489. This total DNA digested with a restriction enzyme and a commercially available E. coli vector digested with a restriction enzyme are ligated with T4 DNA ligase, and the ligated product is introduced into an E. coli host (commercially available). Transformation of Escherichia coli may be performed according to a conventional method. Among the obtained transformants, a strain having a gene of interest can be detected by a method of developing a red color when it has an esterase activity (Analytical Biochemistry, vol. 66, p.
206, 1975). If the target strain is not selected by the above method, it is necessary to change the type of restriction enzyme. The nucleotide sequence of the obtained gene may be determined according to a conventional method, for example, the dideoxy method.

(2) Expression of esterase gene in acetic acid bacteria In order to produce an esterase protein using the gene fragment containing the esterase structural gene thus isolated, a gene fragment containing the enzyme gene is usually used. It is necessary that the gene be linked to a gene having promoter activity that functions in the host in an expressible manner. The promoter used to produce the esterase protein in the cells of the acetic acid bacterium includes not only the original promoter of the esterase gene, but also a gene fragment having another promoter activity derived from the acetic acid bacterium, or an acetic acid bacterium that can be expressed in the acetic acid bacterium. Other promoters derived from microorganisms can also be used. As a promoter derived from acetic acid bacteria, for example, a membrane-bound alcohol dehydrogenase (A
Examples of the promoter of the DH) gene derived from Escherichia coli include a promoter of an ampicillin resistance gene of pUC18 and a lac promoter. Too much esterase protein is produced and the host grows,
When the fermentation performance is adversely affected, it is necessary to select another appropriate promoter in order to suppress the gene expression level to an appropriate level.

As a vector for retaining a gene fragment containing an esterase structural gene in an acetic acid bacterium, a vector constructed based on a plasmid of a host acetic acid bacterium, or a vector derived from another acetic acid bacterium or a bacterium that can be replicated in a host. Vectors can be used. For example, pTA5001 disclosed in JP-A-60-9488, RP4, pR
K2013 etc. can be used.

The expression level of the esterase protein in the host can be controlled not only by changing the promoter, but also by changing the copy number of the plasmid to be used, the connection and type of terminator, and the distance between the SD sequence and the translation initiation codon. . When it is intended to stably maintain the esterase gene in the recombinant acetic acid bacteria for a long period of time, it can be achieved, for example, by incorporating the esterase gene into the chromosome of the host acetic acid bacterium. The gene may be introduced into acetic acid bacteria by a conventional method. For example, electroporation (Bios
ci. Biotech. Biochem. 58, p. 974, 1994
Year).

(3) Fermentation Using Esterase Gene-Introduced Acetic Acid Bacteria As described above, the esterase structural gene was introduced,
By expressing it in a host, an esterase protein can be produced in an acetic acid bacterium in a significant amount. This makes it possible to produce vinegar excellent in preferred aroma with alcohols and acids that are contained in the culture medium and have a property of crossing the cytoplasmic membrane of the acetic acid bacterium. Vinegar prepared to contain ethyl acetate in the range of 200 ppm to 800 ppm and isoamyl acetate in the range of 7 ppm to 30 ppm was clearly identified compared to vinegar that was not, and was highly preferred by the majority of consumers. . Of course, there is also an inexpensive method of simply adding ethyl acetate and isoamyl acetate to the above range, in which both are added.

On the other hand, as an exceptional use, the concentration of alcohols remaining during fermentation is always low (for example, 0.1%).
By controlling the concentration of alcohols to zero when the fermentation is stopped or the fermentation is terminated, it is possible to tilt the esterase reaction equilibrium toward the decomposition direction and reduce the residual ester to almost zero level. is there. Vinegar for special uses may be produced by such a method.

Since acetic acid and ethanol, which are substrates of ethyl acetate, are necessary for conducting acetic acid fermentation, the raw materials are not particularly limited. However, among acetic acid and isoamyl alcohol which are substrates of isoamyl acetate, isoamyl alcohol is used. Since a large amount of alcohol is contained in the fermented broth of yeast, the fermented broth is preferably used as a substrate source for ethanol and isoamyl alcohol.

The acetic acid fermentation method may be performed without any difference from the conventional method. Static fermentation method, deep fermentation method, bioreactor method, etc. can be used.
Techniques such as a semi-continuous fermentation method and a continuous method are used. Generally, the acetic acid fermentation is completed at 25 to 40 ° C for one day to one month. When it is produced by deep fermentation and it is desired to accumulate the ester at a high concentration, the amount of aeration should be relatively small in order to suppress the decrease of the ester as much as possible. The bacterium removal and sterilization methods of the acetic acid fermentation termination solution may be performed in a conventional manner.

In the present invention, the acetic acid concentration was measured by a conventional method using high performance liquid chromatography or a commercially available enzyme kit. The concentrations of various alcohols and various esters were measured by gas chromatography. The specific condition is GC-17 manufactured by Shimadzu Corporation.
A gas chromatograph, TC-WA manufactured by GL Sciences
X column 0.53 mm x 30 m; D. 0.1 μm, injection 200 ° C., detection 220
° C, column temperature condition: 40 ° C, 5min hold, 4 ° C / m
The temperature was raised to 220 ° C. at 220 ° C., and the temperature was maintained at 220 ° C. for 10 minutes. 1 μl of the sample was used. Next, the present invention will be described in more detail with reference to examples.

[0020]

[Example 1] (A) Identification of isolated acetic acid bacteria The acetic acid bacteria were separated from vinegar fermented moromi at a vinegar factory. Since this bacterium is a bacterium having the properties shown in Table 5 below, Acetobacter pasteu
rianus. This strain is Acetobacter
Pasteuriana TUAY-1 (Acetobacter pasteurianus TU
AY-1).

[0021]

[Table 5]

(B) Purification of esterase The present bacterium was used in a GYP medium (glucose 3%, yeast extract 1).
%, Peptone 1%) and a GYP medium supplemented with ethanol to 3% (GYPE medium).
The obtained cells were washed with 10 mM Tris-HCl buffer (pH 7.
After suspension in 5), the suspension was crushed by a French press (20,
000 psi), ammonium sulfate was added to the obtained cell lysate so as to be 30% saturated, and the supernatant was used as Butyl-Toyope.
Apply to an arl column and run from 1.3M to 0% ammonium sulfate.
Eluted with a linear gradient of M. Esterase could be separated from each other by this column chromatography. Hereinafter, the Basal buffer of the column chromatography was 10 mM Tris-HCl buffer (pH 7.5). Each of the three active fractions obtained was dialyzed and then applied to a DEAE-Toyopearl column, and eluted with a linear gradient from 0 M to 0.3 M of sodium chloride. The obtained active fraction was then applied to a Hydroxyapatite column, and a potassium phosphate buffer (pH 7) was added to the suspension.
Elution was with a linear gradient from 01M to 0.4M. Finally, Sephadex G-150 and G-100 were subjected to gel filtration to collect the active fraction.

The three partially purified esterases thus obtained were obtained by gel filtration and SDS gel electrophoresis. (1) Esterase-1 was converted to a dimer having a molecular weight of about 35 kDa, and (2) Esterase-2 was converted to , About 40kD
A dimer (3) esterase-3 having a molecular weight of a
It was found to be a monomer having a molecular weight of about 45 kDa. Esterase-2 expresses activity regardless of the presence or absence of ethanol in the medium.
Means that when ethanol is present,
No. 3 was found to be active in the absence of ethanol.

The measurement of the esterase activity was carried out according to a conventional method. However, due to the reactivity with the substrate, esterase-1 and esterase-2 were obtained from Agric. Biol. Chem., 4
7, p. According to 1865, 1983, esterase-3 is available from Arch. Biochem. Biophys. 159, p. 61,
The activity was carried out according to 1973.

(C) Cloning of esterase gene Acetobacter pasteur obtained by culturing in the same manner as above
From the cells of ianus TUAY-1, a conventional method (JP-A-60-948)
No. 9), total DNA was prepared. The prepared total DNA was cleaved with restriction enzyme EcoRI, and this was randomly ligated with a commercially available vector pKF3 (manufactured by Takara Shuzo) cleaved with restriction enzyme EcoRI using T4 DNA ligase. Transfer the ligated product to an E. coli TH2 competent host
A clone (colony) into which a foreign gene was inserted into the cell and obtained from Takara Shuzo Co., Ltd., was replicated on a filter paper, and a lysis solution (50 mM Tris-HCl buffer, 0.1% Triton X-100, lysozyme 2 mg / ml (pH 7.5)) )), 37 ℃
For 1 hour, and then the color development method (Analytical Biochemit
stry, volume 66, p. (206, 1975) was selected as a clone of the esterase gene.

(D) Subcloning of esterase gene One of the obtained clones was examined.
Insertion of the foreign DNA fragment of b was confirmed, and this recombinant plasmid was designated as pKE1. Restriction enzyme Kp
The 3.3 kb fragment obtained by cutting with nI was used as a commercial vector.
Once subcloned into pUC119 (Takara Shuzo) (the recombinant plasmid obtained here is named pUE12), the restriction enzyme S
A DNA fragment containing esterase cut to 2.7 kb by digestion with phl was inserted into the SphI site of pUC119 to obtain a recombinant plasmid (designated pUE122). This recombinant plasmid (pUE122) was introduced into E. coli (E. coli HB101), and was transformed into Escherichia coli EST-122 (Escherichia coli EB).
ST-122) deposited under the strain name as FERM BP-6095 with the National Institute of Advanced Industrial Science and Technology. FIG. 3 shows a restriction enzyme map of pUE122.

When the remaining clones were examined, two clones having different restriction enzyme maps from the above-mentioned DNA fragment were included, and it was determined that they were different esterase genes. In the same manner as described above, the recombinant plasmids prepared by inserting different (two) esterase genes into pUC119 were ligated with pUE222 and pU222.
Named E322.

[0028]

Example 2 (A) Introduction of Gene Fragment Containing Esterase Structural Gene into Acetic Acid Bacteria A 2.7 kb SphI fragment containing an esterase gene cut out from pUE122 of Example 1 with SphI was transferred to an acetic acid bacteria-E. Coli shuttle vector. pMV24 (Applied and Environmental M
icrobiology, vol. 55, p. 171, 1989) pUC
Insert into the SmaI site derived from 18 and insert the recombinant plasmid (pME12
2) got. pME122 was introduced into Acetobacter acetiIFO3283 by electroporation (Biosci. Biotech. B
iochem. 58, p. 974, 1994). Selection of the transformant of this acetic acid bacterium was performed using ampicillin.
GY containing 100 μg / ml sodium and 1.5% agar
Performed in P medium. Similarly, for pUE222 and pUE322, the esterase gene fragment was inserted into pMV24, and
Named ME222 and pME322, they were introduced into A.aceti IFO3283 to obtain transformed strains.

(B) Properties of acetic acid bacteria transformant The esterase activity of the transformant of A.aceti IFO3283 obtained above was measured. All cells were cultured in a GYPE liquid medium containing 50 μl of ampicillin sodium.
g / ml was added, and shaking culture was performed at 30 ° C. for 16 hours. After the culture, the cells were collected by centrifugation, suspended in 10 mM Tris-HCl buffer (pH 7.5), disrupted by a French press (20,000 psi), and the enzyme activity was measured. As described later, pME122, pME222, pME3
22 each contain a gene encoding only -1, -2 and -3 of the esterase.

Specific activity of pMV24 (vector) -introduced strain (units
/ mg protein) was 0.4 as esterase (-1 and -2) and 0.1 as esterase-3. Here, the specific activity of (-1 and -2) is the same as described above because the method for measuring the activity of esterase-1 and -2 is the same. Because it is difficult. In contrast, when the esterase gene is introduced,
4.2 for pME122-introduced strain, 2.9 for pME222-introduced strain, pM
The E322-introduced strain exhibited a specific activity of 0.2, and in each case, a remarkable increase in esterase activity was confirmed.

The A. acetiIFO3283 transformant into which the above three recombinant plasmids containing the esterase gene have been introduced is inoculated into GYPE medium supplemented with 0.05% isoamyl alcohol, and reciprocally shaken at 30 ° C. Culture was performed. FIG. 4 shows the progress of fermentation of the pME122-introduced strain. After 17 hours of cultivation, the production and accumulation of ethyl acetate and isoamyl acetate reached the maximum, with ethyl acetate being 400 ppm and isoamyl acetate being 10 ppm. After 20 hours, when it can be said that the acetic acid fermentation was almost completed, both the esters slightly decreased, but maintained a high value. On the other hand, the vector (pM
V24) introduced A.aceti IFO3283 transformant
At most, ethyl acetate did not reach 100 ppm, and isoamyl acetate also reached about 2 ppm. Although not shown in FIG. 4, the process of ester formation of A.aceti IFO3283 was the same as that of the vector (pMV24) -introduced strain. Also, pME222
The ester production process of the transfected strain and the pME322 transfected strain was almost the same as that of the vector (pMV24) transfected strain.
ppm, isoamyl acetate was less than 0.5 ppm.

As described above, each of the three esterase genes of Acetobacter pasteurianus was ligated to a plasmid vector (pMV24), and Acetobacter aceti IFO3283 was ligated.
Although the esterase activity was remarkably increased by introduction into the strain, remarkable production and accumulation of ester during acetic acid fermentation was unexpectedly observed only in the strain into which pME122 was introduced, as described above. From these results, it was found that the remaining two esterases acted on the decomposition of the ester in the cells as expected, as expected. The host acetic acid bacterium into which the esterase gene is introduced can be used without any particular limitation as long as it does not have a strong restriction system. For example, acetic acid bacterium used in a vinegar factory can be used.

As a special use, it is also possible to obtain a fermentation broth containing very few esters by devising fermentation conditions. In the fermentation using pME122-introduced A.aceti in this example, when the fermentation is continued until after 30 hours,
The residual ethanol concentration was almost zero, but at this time, ethyl acetate was reduced to 10 ppm and isoamyl acetate was reduced to 1 ppm. Thus, by changing the fermentation conditions,
The ester remaining in a high concentration can be decomposed in reverse.

A. aceti introduced with pME122 and vector (pMV24)
Vinegar obtained by disinfecting the above-mentioned fermented solution (fermented solution for 17 hours and 20 hours) obtained using the introduced A.
When the aroma was evaluated by several evaluators, all 20 preferred the vinegar produced by the transformed strain into which pME122 had been introduced.

[0035]

Example 3 (A) Production of Rice Vinegar A. aceti A. aceti introduced with IFO3283 and pME122
Rice vinegar was produced using a transformant of IFO3283. 300ml of rice spirit fermented liquid (about 15% ethanol)
Commercial pure rice vinegar (acidity 4.5%, ethanol 0.2%) 2
00 ml and water 450 ml were prepared in a 2 liter mini jar (manufactured by Aple), and the rice saccharified solution (glucose concentration 5
%) Of a culture solution in which each of the above two acetic acid bacteria was grown.
Was added to start the acetic acid fermentation, and the acetic acid fermentation was performed until the residual ethanol became 0.4% (temperature: 29
° C, number of revolutions: 500 rpm, ventilation amount: 0.15 vvm). Centrifuged and sterilized by 0.45 μm filter,
The product was diluted with water so as to have an acidity of 4.5% to obtain a rice vinegar product.
When this product was analyzed, A. aceti IFO3283
In the A. aceti IFO3283 transformant into which pME122 and pME122 were introduced, ethyl acetate was 80 ppm and 310 pp, respectively.
m and isoamyl acetate were 1 ppm and 8 ppm, respectively.
When the aroma was evaluated by 20 people, 18 of them preferred rice vinegar produced by the transformed strain.

(B) Effect of Ester Addition to Rice Vinegar A rice vinegar experimentally produced by continuous fermentation of a jar using raw materials having the same composition as described above contains 100 ppm of isovaleric acid. Recognized as rice vinegar with an aroma. Ethyl acetate (special grade reagent) and isoamyl acetate (special grade reagent) were added to the rice vinegar to the concentrations shown in Table 6 below, and evaluated.

[0037]

[Table 6]

As apparent from the results, 200 to 800 ppm of ethyl acetate and 7 to 30 ppm of isoamyl acetate were used.
Rice vinegar added to contain pm contained a higher concentration of isovaleric acid as an unpleasant odor substance, but the scent was improved compared to the original rice vinegar and was evaluated as a rich rice vinegar .

[0039]

Example 4 (A) Determination of Nucleotide Sequence of Esterase Structural Gene The nucleotide sequence of the inserted DNA fragment contained in pUE122 was determined by the dideoxy method according to a conventional method. Open reading frame (ORF) capable of encoding a protein having a molecular weight of 35 to 45 kDa based on the determined base sequence
Was searched, and O as shown in the base sequence of FIG.
RF was found. The molecular weight of the protein translated into amino acids and encoded was calculated to be 35 kDa, which was consistent with that of purified esterase 1. Further, the amino acid sequence translated from the base sequence to the amino acid is as shown in FIG. 1, and the N-terminal amino acid sequence (19 amino acids) in this figure is the N-terminal amino acid sequence of purified esterase 1 (proline-lysine-serine- Valine-valine-asparagine-proline-glutamic acid-phenylalanine-leucine-proline-isoleucine-leucine-glutamic acid-lysine-methionine-proline-serine-phenylalanine-). It was determined that it was a structural gene.

A similar experiment was conducted, and it was found that pUE222 contained a structural gene for esterase-2 and pUE322 contained a structural gene for esterase-3. That is, pME122, pME222, and pME322 are:
Each of them is meant to contain the structural genes of -1, -2 and -3 of esterase.

The esterase-1 gene was compared at the amino acid sequence level to previously reported genes (searched in database). The closest homologue was Streptomyces hygroscopicus esterase, but its homology was only 31.4% at the amino acid level, which was very low. In addition, the results showed that not only the esterase-1 gene was novel, but also no related gene having a homology of more than 80 to 90%.

[0042]

Industrial Applicability According to the present invention, a structural gene of an esterase produced by an acetic acid bacterium can be isolated and introduced into an acetic acid bacterium to reduce the efficiency of acetic acid fermentation without reducing ethyl acetate or isoamyl acetate. It has become possible for the first time to generate and accumulate representative esters at high concentrations. This has made it possible to produce vinegar containing a large amount of isoamyl acetate known as a characteristic flavor of Ginjo sake, that is, aromatic vinegar with Ginjo flavor, using inexpensive raw materials.

[0043]

[Sequence List] The amino acid sequence of the esterase protein and the base sequence of the esterase structural gene are shown in SEQ ID NO: 1, respectively.
And 2. Tables 1 to 4 below show the sequences shown in SEQ ID NOS: 1 and 2.

[0044]

[Table 1]

[0045]

[Table 2]

[0046]

[Table 3]

[0047]

[Table 4]

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of an esterase protein.

FIG. 2 shows the nucleotide sequence of an esterase structural gene.

FIG. 3 is a restriction map of a recombinant plasmid pUE122 prepared by inserting a gene fragment containing an esterase structural gene into an E. coli vector pUC119.

FIG. 4 shows the progress of acetic acid fermentation of a recombinant acetic acid bacterium (A. aceti IFO3283) into which a vector or an esterase gene has been introduced, and the amounts of various esters produced (accumulated).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:02) (C12J 1/04 C12R 1:19) (C12N 1/21 C12R 1:19) (C12P 7/54 C12R 1 : 19) (72) Inventor Yukimichi Koizumi 39-45, Umedaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Shigezo Unaka 1-2-4-3, Ueno-cho, Meto-ku, Nagoya-shi, Aichi Prefecture 4-28-6 Minamikarasuyama, Setagaya-ku, Tokyo

Claims (8)

[Claims] 1. A structural gene DNA encoding a protein represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. The DN of the structural gene encoding the protein according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2.
A. 3. A DNA of a structural gene, wherein the structural gene encoding the protein according to claim 1 is derived from acetic acid bacteria. 4. The D according to claim 1, wherein
A plasmid incorporating NA. 5. A transformed microorganism obtained by transforming the plasmid of claim 4. 6. The transformed microorganism is Escherichia coli EST.
The transformed microorganism according to claim 5, which is -122 (FERM BP-6095). 7. A method for producing vinegar, comprising using the transformed microorganism according to claim 5. 8. A vinegar containing 200 to 800 ppm of ethyl acetate and 7 to 30 ppm of isoamyl acetate.