JP3363198B2 - Production of alcoholic beverages with reduced ester flavor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[Background of the Invention]

BACKGROUND OF THE INVENTION The present invention relates to a method for producing an ester having an ability to produce an ester, which is transformed using a DNA chain capable of producing a biotechnological alcohol acetyltransferase (hereinafter referred to as AATase) as produced by Saccharomyces cerevisiae. It is about decreasing yeast. The present invention also relates to a method for producing alcoholic beverages with reduced ester flavor using the transformed yeast.

[0002]

2. Description of the Related Art It is well known that the amount of various ester components greatly affects the evaluation of alcoholic beverages such as sake, beer, wine, and whiskey. These esters are generally present in the fermentation broth by the yeast producing various lower or higher alcohols during the fermentation process and further converting them into esters.

Among the esters, isoamyl acetate is an ester having a fruity scent, and it is known that its content ratio with its precursor, isoamyl alcohol, has a high correlation with the sensory evaluation of the scent. For example, in sake, the higher the ratio, the higher the value of ginjo aroma, which has been prized as being higher (brewing test report No. 145, p. 26 (1973)).

As previously reported by Yoshioka et al. (Agric.
Biol. Chem. 45 , 2183 (1981)), AATase is an enzyme that plays a central role in the production of isoamyl acetate and has the property of condensing isoamyl alcohol with acetyl-CoA to produce isoamyl acetate. Also, A
ATase is known to have a wide range of substrate specificities, and has been known to produce many other acetates, such as ethyl acetate, by a similar mechanism.

[0005] Therefore, as a method for increasing the amount of esters such as isoamyl acetate in the production of alcoholic beverages, it is effective to enhance the activity of AATase in the yeast to be used, which has been conventionally used in the production of alcoholic beverages. As a result of selection of raw materials and management of fermentation conditions, many of them have enhanced AATase activity as a result.

On the other hand, in beer, although ester is an important flavor component, it is also a fact that excess ester is hated as an ester odor, and AATase is considered to be involved in the formation factor of the ester odor. .

As described above, although it was clear that AATase was important for ester formation, AATase was important.
There are few reports on itself. So far in the partially purified reported (Japan Agricultural Chemistry Journal 63. 435 (1989), Agric . B
iol. Chem., 54. 1485 (1990), the Brewing Society of Japan Journal of 87.33
4 (1992)), but its purification is difficult due to the extreme instability of this enzyme, and as far as the present inventors know, there has been no successful example of complete purification. There were no reports on DNA chains producing enzymes. [Summary of the Invention]

[0008]

SUMMARY OF THE INVENTION The present invention relates to a yeast AA.
An object of the present invention is to obtain a transformed yeast having reduced AATase-producing ability by elucidating the structure of Tase and utilizing its DNA sequence, and further to produce alcoholic beverages having reduced ester flavor.

<Summary> The present invention substantially relates to FIGS.
2, 3 to 4, any of A to B and 19 to 20
The present invention relates to a technique for eliminating or reducing the AATase-producing ability inherent to yeast using the DNA sequence from A to C or from B to C.

[0010] Accordingly, the present invention, in its first aspect, relates to a yeast which has been transformed and has lost or reduced the ability to produce alcohol acetyltransferase.

[0011] That is, the yeast transformed and lost or reduced in the ability to produce alcohol acetyltransferase according to the present invention can be prepared by using any of the following DNA sequences (1a) to (1d). Characterized in that the expression of an acetyltransferase-producing gene is suppressed. (1a) The DNA sequence from A to B of the DNA sequence substantially as shown in FIGS. 1 and 2. (1b) The DNA sequence from A to B of the DNA sequence substantially as shown in FIGS. 3 and 4. (1c) D from A to C or from B to C of the DNA sequence substantially as shown in FIGS.
NA sequence. (1d) the DNA of any of the above (1a) to (1c)
A DNA strand that hybridizes to the sequence or its complement.

[0012] Here, "to suppress the expression of an alcohol acetyltransferase-producing gene specific to a host yeast" specifically means that the gene is AATA
or the AA of the gene
Tase-producing ability is not substantially destroyed, but means that the expression process is impaired. Specific examples of the latter include, for example, a foreign DN that expresses RNA complementary to the mRNA of the gene, that is, a so-called antisense RNA.
Introducing the A chain into yeast cells to cause inhibition of translation by mRNA and, consequently, suppression of gene expression. Such a so-called "negative" transformation can be achieved only when the nucleotide sequence of AATase in yeast has been clarified. That is, in the first embodiment, "using" a DNA sequence encoding a polypeptide having a predetermined amino acid sequence specifically means, for example, that the DNA sequence corresponds to an AATase specific to a host yeast. The homology is conserved, but the AATase-producing ability is modified so as to disappear or decrease, and this modified DNA sequence is replaced with the AATase-producing gene of the host yeast by homologous recombination to lose the AATase-producing ability inherent to the host yeast. Or reduce or mR of the AATase producing gene specific to the host yeast
Making RNA complementary to NA from the DNA sequence,
Using it as said antisense RNA,
Means

[0013] From the fact that it was possible to obtain a yeast in which the AATase producing ability was eliminated or reduced,
ADVANTAGE OF THE INVENTION According to this invention, manufacture of liquor with reduced ester flavor becomes possible.

Accordingly, the present invention, in a second aspect thereof, relates to a method for producing liquors with reduced ester flavor.

That is, according to the method for producing alcoholic beverages having reduced ester flavor according to the present invention, in a method for producing alcoholic beverages by fermenting sugar with yeast, the yeast used is transformed by the transformation shown in the first embodiment. That the yeast has lost or reduced the ability to produce alcohol acetyltransferase,
It is characterized by the following.

In the present invention, "DNA strand", "DN"
"A sequence" and "gene" are used as substantially equivalent.

<Effects> The AATase gene is modified by modifying the yeast AATase gene using genetic engineering techniques.
It is possible to produce a foreign DNA strand that disrupts a gene or suppresses its expression. That is, by introducing this DNA chain into a yeast cell as an extranuclear and / or intranuclear gene, the ability of the yeast to produce AATase can be suppressed, and the yeast is used to ferment sugar to reduce the ester flavor. Alcoholic beverages can be produced.

[Specific Description of the Invention] <AATase> In the present invention, A specific to host yeast
DN used to suppress the expression of ATase-producing gene
The A sequence is a DNA sequence capable of producing AATase.

AATase, ie, alcohol acetyltransferase, is an enzyme capable of producing an acetate ester by a reaction of transferring an acetyl group from acetyl-CoA to alcohols.

In this case, the alcohols are mainly linear or branched alcohols having 1 to 6 carbon atoms. According to the study by the present inventors,
It is known that AATase can use an alcohol having a higher carbon number such as 2-phenylethyl alcohol as a substrate.

Therefore, when it is necessary to discuss the substrate alcohol of AATase in the present invention, it is to be understood that "alcohols" includes a wide range of alcohols.

AATase in the present invention is derived from yeast. It is specifically obtained from S. cerevisiae and is substantially the amino acid sequence from A to B of the amino acid sequence shown in FIGS. 1 and 2, and FIGS. The amino acid sequence from A to B of the amino acid sequence shown over, the amino acid sequence from A to C of the amino acid sequence shown over FIGS. 19 and 20, and FIGS. 19 and 2
A polypeptide having any one of the amino acid sequences B to C of the amino acid sequence shown over zero. It is evident from genetic or protein engineering that the biological activity of a polypeptide may not be altered by the addition, insertion, deletion, deletion or substitution of some amino acids. The amino acid sequence A to B shown in the present invention "substantially" is shown in FIGS.
The amino acid sequence described above is not limited to the case where the amino acid sequence referred to in the present invention is exactly the same as the amino acid sequence of AB in the amino acid sequence shown in FIGS. 1 and 2, for example, and the AATase-producing ability is preserved. As far as possible, it is intended to include the amino acid modification as described above.

The Saccharomyces cerevisiae referred to in the present invention is the yeasts, a taxonomic study 3rd.
on (ed.by NJWKreger-van Rij, Elsevier Science Pub
lishers BV, Amsterdam (1984), p379) and Saccharomyces cerevisiae and synonyms or mutants thereof.

[0024] AATase, including also the purification method, has already been reported (Journal of the Japan Society for Bioscience, Biotechnology, and Agrochemistry 63.435 (1989),
Agric. Biol. Chem., 54. 1485 (1990), Japan Brewing Association Journal 87. 334 (1992), etc.). However, to the knowledge of the present inventors, the AATase is not sufficiently high in purity,
The amino acid sequence could not be determined.

The present inventors have proposed 1-AATase purification.
It has been found that the use of an affinity column using hexanol as a ligand is effective, and
The ase was completely purified from Saccharomyces cerevisiae, and its properties could be elucidated here. The amino acid sequences shown in the accompanying FIGS. 1 and 2 were obtained for AATase from Saccharomyces cerevisiae thus sufficiently purified.

Among the properties of AATase that have become apparent this time, the molecular weight of AATase can be increased as a representative one. That is, the molecular weight of AATase estimated so far is about 45,000 to 5
The molecular weight of AATase purified this time was about 60,000 according to the result of SDS-PAGE (SDS polyacrylamide electrophoresis) (FIG. 10).
It was suggested that the protein was different from the previously reported proteins. The AAT estimated from the base sequence
ase had a molecular weight of about 61,000.

The AATase of the present invention is defined enzymatically as follows. a Action: Acts on various alcohols such as ethyl alcohol and acetyl-CoA to produce esters of acetic acid. b Substrate specificity: For alcohols, it acts on various alcohols having 2 to 5 carbon atoms. In the range of 2 to 5 carbon atoms, the greater the number of carbon atoms, the better the action. Also,
It works better on linear alcohols than on branched alcohols. c Molecular weight: about 60000 d Optimum and stable pH: Optimum pH: 8.0 Stable pH: 7.5-8.5 e Optimum and stable temperature: Optimum temperature: 25 ° C. Stable temperature: 4 ° C. f Inhibitor Effects: Parachloromercuric benzoic acid (PCM
B) and dithiobisbenzoic acid (DTNB). g Effect on the activity of various fatty acids: Not significantly inhibited by saturated fatty acids, but strongly inhibited by unsaturated fatty acids. h Km value for isoamyl alcohol or acetyl-CoA: isoamyl alcohol: 29.8 mM acetyl-CoA: 190 μM

This AATase is obtained from Saccharomyces
It can be obtained by a method comprising culturing K. cerevisiae Kyokai Yeast No. 7 and recovering and purifying a crude enzyme from the contents of bacterial cells. AATA consisting of these unit processes
The actual acquisition of the AATase of the present invention was carried out as described in the examples below, including the affinity column chromatography using 1-hexanol as a ligand.
Is easy for those skilled in the art.

<DNA Sequence or DNA Chain / Gene Producing AATase> In the present invention, the DNA sequence or DNA chain capable of producing AATase refers to a DNA sequence or DNA strand encoding a polypeptide having AATase activity. And expressed by the amino acid sequence of the polypeptide encoded by it, that is, AATase:
It is selected from the group consisting of (2d), and specific examples thereof are those selected from the group consisting of the following (3a) to (3d). (2a) An alcohol acetyltransferase gene comprising a DNA sequence encoding a polypeptide having an amino acid sequence from A to B of the amino acid sequence shown in FIGS. 1 and 2. (2b) An alcohol acetyltransferase gene comprising a DNA sequence encoding a polypeptide having an amino acid sequence from A to B of the amino acid sequence shown in FIGS. 3 and 4. (2c) DNA encoding a polypeptide having the amino acid sequence from A to C or from B to C of the amino acid sequence substantially as shown in FIGS.
An alcohol acetyltransferase gene comprising a sequence. (2d) a DNA sequence encoding a polypeptide having a mutant amino acid sequence resulting from addition, insertion, deletion, deletion or substitution of any of the amino acids of the above-mentioned polypeptides (2a) to (2c). The alcohol acetyltransferase gene. (3a) An alcohol acetyltransferase gene substantially consisting of the nucleotide sequence from A to B of the nucleotide sequence shown in FIGS. 1 and 2. (3b) An alcohol acetyltransferase gene substantially consisting of the nucleotide sequence from A to B of the nucleotide sequence shown in FIGS. 3 and 4. (3c) An alcohol acetyltransferase gene substantially consisting of the nucleotide sequence from A to C or from B to C in the nucleotide sequence shown in FIGS. 19 and 20. (3d) the DNA of any of the above (3a) to (3c)
A DNA strand that hybridizes to the sequence or its complement and is capable of producing a polypeptide having alcohol acetyltransferase activity.

Here, the meaning of "substantially" in the definition of the polypeptide encoded by the DNA sequence is as described above, and the DNA sequence also varies according to the variation of the polypeptide encoded by the DNA sequence. In Although,
The term "encoding DNA sequence" is also to be understood in its broadest sense according to the knowledge of the so-called genetic code. Therefore, the degeneracy (de
generacy), there may be more than one triplet of nucleic acid encoding a given amino acid, and may correspond to amino acid additions, insertions, deletions, deletions or substitutions, as long as the AATase-producing ability of the encoded polypeptide is preserved. Thus, some of the triplets and / or some of their constituent nucleic acids may be altered. Note that “coding” is synonymous with “having coding ability”.

The DNA sequence of interest in the present invention may be derived from a natural product, may be totally synthesized, or may be synthesized using a part of the natural product, that is, a semi-synthetic sequence. May be.

One of the typical methods for obtaining such a DNA strand is plaque hybridization, colony hybridization using a part of the DNA strand provided by the present invention having the ability to produce AATase as a probe. , PCR and the like. All of these methods are well known to those skilled in the art and can be easily implemented.

Further, AATase can be obtained by such means.
Bacteria, yeasts, plants and the like are considered as useful as a gene source for obtaining a DNA chain having the ability to produce yeast, and especially yeasts used for the production of fermented foods such as sake and soy sauce are There is a high possibility that the DNA strand has the above conditions.

The DNA sequence may have a length corresponding to AATase, or may have a structure in which a DNA sequence is bound upstream and / or downstream thereof. A specific example of the latter is in the form of a plasmid integrated into a suitable vector.

<Transformation> In order to suppress the expression of AATase-producing ability of the host yeast using the AATase gene, a transformation technique may be used.

Procedures and methods for producing transformants are those commonly used in the field of genetic engineering, and in the present invention, except for the following, conventional standards (ANALYTICAL BIOCHEMITSTRY 163.391 (1)
987) etc.).

Typical transformations include foreign genes,
That is, in the case of the present invention, the AATase-producing gene is introduced into the nucleus and / or outside the nucleus of a host cell, ie, yeast, to express the trait.

However, the host yeast has a unique AATase
When the gene has a production gene, by suppressing the expression of the gene, it is possible to produce a yeast with a reduced or reduced AATase-producing ability. That is,
(A) The gene can be replaced with a DNA strand that disrupts or inactivates the AATase, thereby reducing or reducing AATase-producing ability itself.
With the ATase-producing gene retained, the expression of the gene can be suppressed by suppressing the translation by introducing a foreign DNA strand that expresses a so-called antisense RNA.

As the vector used at this time, those known for yeast, for example, YRp system (multicopy vector for yeast using the ARS sequence of yeast chromosome as a replication origin), YEp system (replication of 2 μm DNA of yeast) A multicopy vector for yeast having an origin, a YCp system (a single copy vector for yeast having an ARS sequence of the yeast chromosome as a replication origin, and having a centromere DNA sequence of the yeast chromosome), and a YIp system (having a replication origin of yeast) All known vectors can be used. These vectors are not only known in the literature (Asakura Shoten, ABC Series of Japanese Society for Agricultural Chemistry, “Genetic engineering for substance production”, p68), but can also be easily prepared.

The former method (a) specifically utilizes, for example, so-called “homologous recombination”, and converts an AATase-producing gene specific to a host yeast into an AATase.
It consists of destruction to the extent that it loses productivity.

The method of disrupting the AATase gene using homologous recombination is performed in two steps.

First, the AATA to be destroyed as the first step
A part of the center of the se gene is cut off with a restriction enzyme, and a DNA fragment into which another appropriate gene (such as a G418 resistance gene) is inserted is prepared instead. This fragment is AATas
The AATase gene cannot be expressed because part of the e gene has been deleted and replaced with another fragment.

Next, the fragment prepared in the second step is replaced with the yeast AATase gene. This method is carried out by exactly the same procedure as the usual transformation. That is, this fragment can replace the AATase gene of yeast using homologous base sequences of the AATase gene present at both ends (homologous recombination), and the replaced yeast is
The ATase gene has been lost.

[0044] The details of the method according to the homologous recombination, for example, Proc.Natl.Acad.Sci.USA. 76 .4951 (19
79), Methods in Enzymology. 101 , 202 (1983) and the like.

The latter method (b) is described, for example, in AATA
Although it does not substantially destroy the se-producing ability, it impairs the process of expressing the AATase-producing gene, and specifically uses antisense RNA.

The method of reducing the expression level of the AATase gene by using antisense RNA is described in US Pat.
It is carried out by expressing mRNA complementary to mRNA of the ATase gene with an appropriate promoter. In order to express the complementary mRNA, a method is generally employed in which the mRNA portion of the gene is connected to the promoter in the reverse direction. The gene connected in the opposite direction to the original direction is the DN of the opposite side of the double-stranded DNA.
The A chain, that is, the DNA that does not encode the AATase protein, is transcribed. The resulting antisense RNA is AA
It forms a hybrid in the cell with the mRNA of the Tase gene, and significantly prevents the ribosome from binding to the mRNA of the gene to synthesize the AATase protein.

In order to express the antisense RNA of the AATase gene according to the present invention in yeast or to increase or decrease the expression, a promoter which is a unit for controlling transcription and translation is used in the present invention.
The terminator may be incorporated in the 5'-upstream region of the NA chain and in the 3'-downstream region, respectively. Examples of the promoter and terminator include those derived from the AATase gene itself, an alcohol dehydrogenase gene (J. Biol. Chem., 257 , 3018 (1982)), and a phosphoglycerate kinase gene (Nucleic Acids Res., 10 , 7791).
(1982)), glycerol aldehyde-3-phosphate dehydrogenase gene (J. Biol. Chem., 254 , 9839 (1979)).
It is possible to use those derived from all known genes or artificially improved ones. For details of the antisense RNA method, see
See, for example, Science, 229. , 345 (1985).
See also Curr. Genet., 13 , 283 (1988) for the method of using antisense RNA in yeast.

In the present invention, the yeast to be transformed, that is, the host yeast, may be any taxonomically any of the yeasts, but for the purpose of the present invention, the production of alcoholic beverages belonging to Saccharomyces cerevisiae Yeast for use, specifically, brewer's yeast, sake yeast, wine yeast and the like are preferable. Specifically, for example, beer yeast: ATCC26292
, ATCC2704, ATCC32634 and AJL2155,
Sake yeast: ATCC4134, ATCC26421 and IFO
2347, wine yeast: ATCC38637, ATCC38638 and IFO2260.

Another preferred group of host yeasts is baker's yeast. Specifically, ATCC32120 can be exemplified.

<Production of Alcoholic Beverages>
Since the transformed yeast having reduced ATase-producing ability has a trait unique to the host yeast in addition to the introduced trait, it can be used for various uses focusing on the unique trait.

When the host yeast is a yeast for the production of alcoholic beverages, the transformed yeast also has the ability to utilize sugar to produce alcohol. Reduced alcoholic beverages can be produced.

Sake, wine, whiskey and beer are typical examples of alcoholic beverages, and their production methods using yeast are well known.

[0053]

【Example】

(1) Production of AATase The enzyme of the present invention belongs to the genus Saccharomyces, and can be obtained from a culture obtained by culturing a microorganism that produces an enzyme having the above characteristics. An example of a preferred production method is as follows.

(1)-(i) Measurement of alcohol acetyltransferase activity AATase reaction buffer (25 mM imidazole hydrochloride buffer (pH 7.5), 1 mM acetyl-CoA, 0.1 mM
% Triton X-100, 0.5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol, or 10 mM phosphate buffer (pH 7.5), 1 mM acetyl-CoA, 0.1% Triton X-100, 0.5% isoamyl alcohol,
Solution 1 containing 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol) and this enzyme
After sealing the mixture in a 20 ml vial, the reaction was carried out at 25 ° C. for 1 hour, and then opened and the reaction was stopped by adding 0.6 g of sodium chloride. As an internal standard,
n-butanol was added to a concentration of 50 ppm,
After covering with a Teflon stopper again, isoamyl acetate produced by the headspace method was quantified by gas chromatography (Shimadzu GC-9A, HSS-2A). Analytical conditions: Column: glass column 2.1 m × 3 mm Filler: 10% polyethylene glycol 1540 DiaSolid L (60/80 mesh) Column temperature: 75 ° C. Injection temperature: 150 ° C. Carrier-gas: nitrogen flow rate: 50 ml / min sample Amount: 0.8ml

(1)-(ii) Preparation of Crude Enzyme Solution Yeast No. 7 was converted to YPD medium (yeast extract 1).
%, Bactopeptone 2%, glucose 2%) 500ml
And pre-cultured at 15 ° C. for 3 days. 1
Twenty 2,000 ml Erlenmeyer flasks each containing 2,000 ml of YPD medium were inoculated with 25 ml each, and cultured at 30 ° C. for 12 hours. Next, the cells were recovered by centrifugation (3000 rpm / min, 10 minutes), and a buffer (50 mM Tris-HCl buffer (pH 7.5), 10 times the cell weight) was collected.
Suspended in 0.1 M sodium sulfite, 0.8 M potassium chloride), "Zymolyase 100T" (a yeast cell lysing enzyme commercially available from Seikagaku Corporation) having a 1 / 1000th of the cell weight (Patent No. 702095) , USPatent No.3917
510)) was added. This was shaken at 30 ° C. for 1 hour, and the resulting protoplast was collected by centrifugation at 3000 rpm for 5 minutes. 400 ml of collected protoplasts
Suspended in a buffer (25 mM imidazole-hydrochloric acid buffer (pH 7.5), 0.6 M potassium chloride, 1 mM sodium ethylenediaminetetraacetate (abbreviated as EDTA)), and “Polytron PT10 type” (KINEMAT).
The cells were disrupted using a cell disrupter (ICA). After crushing, the crushed residue was removed by centrifugation at 45000 rpm to obtain a crude enzyme solution.

(1)-(iii) Preparation of Microsomal Fraction The crude enzyme solution obtained in (1)-(ii) was subjected to 100,000 × G
And the resulting precipitate (microsome fraction)
40 ml of buffer (25 mM imidazole-hydrochloric acid (pH
7.5) 1 mM dithiothreitol). If not used immediately, this was stored at -20 ° C.

(1)-(iv) Preparation of solubilized enzyme The microsomal fraction obtained in (1)-(iii) was transferred to an Erlenmeyer flask, and 1/100 volume of Triton X-100 was added. At 4 ° C., take care not to foam
The mixture was stirred using a magnetic stirrer for 0 minutes. Thereafter, the mixture was centrifuged at 100,000 × G for 2 hours, and the supernatant was subjected to buffer A (25 mM imidazole-hydrochloric acid buffer (pH 7.
2) against 0.1% Triton X-100, 0.5% isoamyl alcohol, 1 mM dithiothreitol, 20% glycerol) overnight.

(1)-(v) Purification of Enzyme Purification of the enzyme was carried out by repeating the operations of (1)-(ii) and (1)-(iii) 20 times and stored at -20 ° C. The fractions were subjected to the operations (1) to (iv), and the fractions were subjected to the solubilized enzyme solution. First, the solubilized enzyme solution was purified by column chromatography using Polybuffer Exchanger 94 (Pharmacia) (adsorption: buffer A, elution: buffer A 0.0-
0.6M sodium chloride concentration gradient).

The active fraction was repeatedly purified by means of a polybuffer exchanger 94. The active fraction was further purified as shown in Table 1. Ion exchange column chromatography DEAE Toyopearl 55 (TOSOH) Adsorption: Buffer A, Elution: Buffer A 0.0-0.2 M sodium chloride concentration gradient Gel filtration column chromatography Yopearl HW
60 (TOSOH) Buffer B (10 mM phosphate buffer (pH 7.5),
0.1% Triton X-100, 0.5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol) Hydroxyapatite column chromatography (Wako Pure Chemical Industries) Adsorption: buffer B, elution: Buffer B 10-50 mM phosphate buffer (pH 7.5) Concentration gradient Octyl Sepharose column chromatography
(Pharmacia) Adsorption: 50 mM imidazole-hydrochloric acid (pH 7.5),
0.5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol, elution: 50 mM imidazole-hydrochloric acid buffer (pH
7.5), 0.1% Triton X-100, 0.5% isoamyl alcohol, 1 mM dithiothreitol,
(1M sodium chloride, 20% glycerol).

As shown in Table 1, it was confirmed at this stage that the product was purified to a specific activity of about 2000 times.
When S-polyacrylamide electrophoresis and silver staining were performed, a plurality of bands were still observed, and complete purification was not achieved.

Therefore, the present inventors carried out affinity chromatography of 1-hexanol, utilizing the fact that 1-hexanol, which is a substrate of AATase, has a strong affinity for AATase. Hexanol Sepharose 4B was prepared using CNBr-activated Sepharose 4B (Pharmacia) with 6-amino-1-hexanol (Wako Pure Chemical Industries) as a carrier according to the Pharmacia manual. Affinity chromatography was performed using this carrier (adsorption: 5 mM phosphate buffer (pH 7.2), 0.1% Triton X-10).
0, 20% glycerol, 1 mM dithiothreitol,
Elution: 0.0-0.2 M sodium chloride concentration gradient). The active fraction obtained as shown in FIG. 9 was subjected to SDS polyacrylamide gel electrophoresis and stained with silver. The active fraction is shown in FIG. Thus, it was determined that the enzyme preparation gave a single band and that AATase was completely purified.

Table 1 Purification of AATase (Part 1) Volume Activity Total activity (ml) (ppm / ml) (ppm) Solubilizing enzyme 505 119 60 100 PBE 94 1st. 395 77 30400 PBE 94 2nd. 86 304 26 100 DEAE Toyoparl 24 580 13900 Toyopearl HW60 24 708 17000 Hydoroxy apatite 7. 6 1020 7750 Octyl sepharose 1.0 2390 2390 Table 1 Purification of AATase (Part 2) Protein amount Specific activity Yield Purification rate ppm (mg / ml) mg prot. (%) Solubilizing enzyme 5.432 22 100 1 PBE 94 1st. 0.515 150 517 PBE 94 2nd. 1.086 280 43 13 DEAE Toyoparl 0.96 604 23 27 Toyopearl HW60 0.262 2700 28 123 Hydoroxy apatite 0.119 8570 13 266 Octyl sepharose 0.056 42700 4 1940

(2) Characteristics of AATase (2)-(i) Substrate specificity The substrate specificity for various alcohols was examined by the above-mentioned analytical instrument and method. did. The higher the number of carbons, the better. It also worked better on linear alcohols than on branched alcohols (FIG. 11).

(2)-(ii) Optimum and stable pH To examine the effect of pH on the stability of the enzyme,
Range from 5 to 9 (pH 5 to 6: 50 mM citrate-phosphate buffer, pH 6 to 8: 50 mM phosphate buffer, pH
8-9: 50 mM Tris-phosphate buffer).
After maintaining at 4 ° C. for a time, the pH was adjusted to 7.5 with 0.2 M disodium phosphate, and the enzyme activity was measured by the method of (1)-(i).

To examine the effect of pH on the activity of the enzyme, the pH was adjusted to 5-9 (pH 5-6: 50 mM citric acid-
Phosphate buffer, pH 6-8: 50 mM phosphate buffer, p
H8-9: 50 mM Tris-phosphate buffer), and the enzyme activity was examined by the method of (1)-(i). The pH stability was stable in the range of pH 7.5 to 8.5. Further, the optimum pH was 8.0.

(2)-(iii) Optimum and stable temperature In order to investigate the effect of temperature on the activity of the enzyme, (1)
The activity was measured at each temperature by the method of-(i).

In order to examine the effect of temperature on the stability of the enzyme, the enzyme solution was kept at each temperature for 30 minutes.
(1) The enzyme activity was measured by the method of (i). The optimum temperature was 25 ° C. As for thermal stability, 4 ° C
Was stable, but was extremely unstable at higher temperatures.

(2)-(iv) Influence of Inhibitors, etc. In order to examine the results of the effects of various inhibitors on enzyme activity, various inhibitors were added to the reaction mixture of (1)-(i). After the addition at the concentrations shown in the table, the activity was measured by the method of (1)-(i). The results are shown in Table 2. Parachloromercuric benzoic acid (PCMB), dithiobisbenzoic acid (DTN)
Since the enzyme was strongly inhibited by B), this enzyme is considered to be an SH enzyme.

Table 2 Effect of inhibitors Inhibitors specific activity inhibitors specific activity (1mM) (%) (1mM ) (%) None _{100 ZnCl 2 12.7 KCl 98.6 MnCl 2} 53.3 MgCl 2 86.2 HgCl 2 0 CaCl 2 87.7 SnCl ^{_{2 52.0 BaCl 2 73.7 TNBS * 16.8}} FeCl 3 54.5 PCMB * 0 CoCl 2 37.6 DTNB * 0 CdCl 2 3.1 PMSF * 70.2 NiCl 2 22.3 1,10- Fenan CuSO ₄₀ suloline 87.9 * 1 mM TNBS: Trinitrobenzoic acid 0.1 mM PCMB: Parachloromercuric benzoic acid 0.1 mM DTNB: Dithiobis (2-nitrobenzoic acid) 1 mM PMSF: Phenylmethanesulfonyl fluoride

(2)-(v) Effect on fatty acid activity Various fatty acids were added to the reaction buffer of (1)-(i) at a concentration of 2 mM.
And the effect on enzyme activity was examined (Table 3).

Table 3 Effect of fatty acids on enzyme activity Fatty acid Specific activity (2 mM) (%) None 100 Myristic acid C ₁₄ H ₂₈ O ₂ 60.5 Palmitic acid C ₁₆ H ₃₂ O ₂ 88.1 Palmito oleic acid C ₁₆ H ₃₀ O ₂ 16.7 Stearic acid C ₁₈ H ₃₆ O ₂ 80.5 Oleic acid C ₁₈ H ₃₄ O ₂ 59.6 Linoleic acid C ₁₈ H ₃₂ O ₂ 4.3 Linolenic acid C ₁₈ H ₃₀ O ₂ 32.0

(3) Determination of Partial Amino Acid Sequence The determination of the partial amino acid sequence was carried out according to Iwamatsu (Biochemical 63 , p13).
9 (1991)) of polyvinylidene difluoride (PV
DF) was performed according to a method using a membrane. (1)
-The AATase enzyme obtained in (v) was replaced with 10 mM formic acid 3
After dialysis against 1 liter for 1 hour, it was freeze-dried. This was added to an electrophoresis buffer (10% glycerol, 2.5% SDS,
The suspension was suspended in 2% 2-mercaptoethanol, 62 mM Tris-HCl buffer (pH 6.8) and subjected to SDS polyacrylamide electrophoresis. After the electrophoresis, the enzyme was transferred from the gel to a 10 cm × 7 cm PVDF membrane (“ProBlot” (Applied Biosystems)) by electroblotting. Electroblotting was performed at 160 mA for 1 hour according to "Method for Pretreating Sample of Protein Sequencer (1)" edited by Shimadzu Corporation using a Sartoblot IIs type (Sartorius) as an electroblotting apparatus.

After the transfer, the membrane where the enzyme was transferred was cut off, and about 300 μl of a reducing buffer (6 M guanidine hydrochloride-0.5 M Tris hydrochloride buffer (pH 3.
5), 0.3% EDTA, 2% acetonitrile), 1 mg of dithiothreitol (abbreviated as DTT) was added, and reduction was performed at 60 ° C./about 1 hour under argon. To this, a solution prepared by dissolving 2.4 mg of monoiodoacetic acid in 10 μl of a 0.5 N sodium hydroxide solution was added, and the solution was added under light shielding.
Minutes. The PVDF membrane was taken out, sufficiently washed with 2% acetonitrile, and then stirred in 0.1% SDS for 5 minutes. Next, the PVDF membrane was lightly washed with water, and then immersed in 0.5% polyvinylpyrrolidone-40-100 mM acetic acid.
Let stand for minutes. Thereafter, the PVDF membrane was sufficiently washed with water, and cut into about 1 mm square. Add this to digestion buffer (8
% Acetonitrile, 90 mM Tris-HCl buffer (pH
9.0)) and soak in Achromobacter protease I
(Wako Pure Chemical Industries, Ltd.) was added at 1 pmol and digested at room temperature for 15 hours. The digest was applied to a C8 column (Nippon Millipore Limited, μ-Bondasphere 5C8, 300A, 2.1 ×
Separation was performed by reversed-phase high-performance liquid chromatography (Hitachi L6200) using 150 mm) to obtain ten or more peptide fragments. Solvents A (0.05% trifluoroacetic acid) and B (0.02%
Using 2-propanol / acetonitrile 7: 3) containing trifluoroacetic acid, elution was performed with a linear concentration gradient of 2 to 50% at a flow rate of 0.25 ml / min for 40 minutes with respect to the B solvent. The obtained peptide fragment was subjected to an amino acid sequencing test by an automated Edman degradation method using a gas-phase protein sequencer 470 of Applied Biosystems according to the manual.

As a result, the following amino acid sequence was determined. Peak 1 Lys Trp Lys Peak 2 Lys Tyr Val Asn Ile Asp Peak 3 Lys Asn Gln Ala Pro Val Gln Gln Glu Cy
s Leu Peak 4 Lys Gly Met Asn Ile Val Val Ala Ser Peak 5 Lys Tyr Glu Glu Asp Tyr Gln Leu Leu Ar
g Lys Peak 6 Lys Gln Ile Leu Glu Glu Phe Lys Peak 7 Lys Leu Asp Tyr Ile Phe Lys Peak 8 Lys Val Met Cys Asp Arg Ala Ile Gly Ly
s Peak 9 Lys Leu Ser Gly Val Val Leu Asn Glu Gl
n Pro Glu Tyr Peak 10 Lys Asn Val Val Gly Ser Gln Glu Ser
Leu Glu Glu Leu Cys Ser Ile Tyr Lys

(4) Cloning of AATase-producing DNA Chain from Sake Yeast Association No. 7 (i) Preparation of Sake Yeast Library D. 600 = 10
After the cells were collected, the cells were washed with autoclaved water. 2 ml of SCE solution (1 M sorbitol, 0.125 MEDTA, 0.1 M trisodium citrate (pH 7), 0.75% 2-mercaptoethanol, 0.01% “Zymolyase 100T” (Seikagaku Corporation) The suspension was then gently shaken at 37 ° C. for about 2 hours to completely transform the yeast into protoplasts, and 3.5 ml of Lysis Buffer per cell was added.
(0.5 M Tris-HCl buffer (pH 9), 0.2 MED
TA and 3% sodium dodecyl sulfate (abbreviated as SDS) were added, followed by gentle stirring. This was kept at 65 ° C. for 15 minutes to completely lyse the cells. After lysis, this was cooled to room temperature, and 23.5 ml of a 10% -40% sucrose density gradient solution (0.8 M sodium chloride, 0.02 M Tris-HCl buffer) previously prepared in Hitachi Ultracentrifuge Tube 40PA. Liquid (pH 8), 0.01MEDTA, 10%
(-40% sucrose) gently. Using a Hitachi ultracentrifuge SCP85H at 4 ° C
And centrifuged at / 26000 rpm / 3 hr. After centrifugation, about 5 ml of the liquid was collected from a place as close to the tube bottom as possible using a Kogame pipette. Collect 1 sample
Dialyzed overnight with 1 liter of TE solution.

Next, the obtained chromosomal DNA was
Methods in Enzymology; 152 , 183
, Academic press 1987), restriction enzyme Sau
Partially decompose with 3AI, re-load on 10% -40% sucrose density gradient solution, 20 ° C / 25000 rpm / 2
Centrifugation was performed for 2 hours. After completion of the centrifugation, a hole was made in the bottom of the ultracentrifuge tube with a syringe needle, and 0.5 ml of the density gradient solution was collected in a sampling tube. A portion of each fraction was subjected to electrophoresis on an agarose gel to confirm the molecular weight of the chromosomal DNA contained in the density gradient solution. DNA of 15 to 20 kb was collected, and collected by ethanol precipitation. 1 μg of this chromosomal DNA and λ
-Λ-E of EMBL3 / BamHI vector kit (Stratagene, purchased from Funakoshi Co., Ltd.)
1 μg of the MBL3 vector was ligated at 16 ° C. overnight. This was packaged using GIGAPACK GOLD (manufactured by Stratagene, purchased from Funakoshi Co., Ltd.). Ligation method is λ-E
The manual attached to the MBL3 vector and packaging were performed according to the manual attached to GIGAPACK GOLD.

Next, 50 μl of the packaging solution was added to λ-E
Host strain P included in MBL3 vector kit
Infected 2392. P2392 One platinum loop was placed in 5 ml of TB medium (1% bactotryptone (Difco), 0.5% sodium chloride, 0.2% maltose, pH 7.4) at 37 ° C.
, Overnight, and inoculated with 1 ml of this culture in 50 ml of TB medium. D. Culture until 600 = 0.5,
After cooling on an ice bath, the cells were collected and suspended in 15 ml of an ice-cooled 10 mM magnesium sulfate solution. To 1 ml of this bacterial solution was added 0.95 ml of SM solution (0.1 M sodium chloride, 10 mM magnesium sulfate, 50 mM Tris-HCl buffer (pH 7.5), 0.01% gelatin) and 50 μl.
Was mixed gently, and the mixture was kept at 37 ° C. for 15 minutes. This was added to a BBL soft agar medium (1% Tripticase peptone (BBL
200 ml each in 7 ml of 0.5% sodium chloride and 0.5% agarose (Sigma), mixed gently, and then prepared in advance to a 15 cm diameter BBL agar medium (1% tripticase peptone, 0.1%). 5% Sodium chloride, 1.5% Bactogar (Difco)).

This was incubated at 37 ° C. for 8 hours, and the agar medium 1
A phage library containing 0, about 30,000 yeast chromosomal DNA fragments was completed. Next, the library was transferred to a membrane for cloning. A hybridization transfer membrane (NEN) having a diameter of 15 cm was brought into contact with an agar medium overlaid for about 2 minutes, and two sets of phage were transferred to the membrane, for a total of 20 sheets. This membrane was allowed to stand on a filter paper impregnated with an alkali denaturing solution (1.5 M sodium chloride, 0.5 N sodium hydroxide) with the surface in contact with the agar medium facing up for about 5 minutes. Subsequently, the membrane is neutralized with a neutralizing solution (3 M sodium acetate (pH 5.8)).
Was transferred onto a filter paper soaked with the swatch and soaked for about 5 minutes. Thereafter, the membrane is dried at room temperature, and further dried at 80 ° C. for 1 hour.
Vacuum dried for hours. Further, the agar medium after the transfer was stored at 4 ° C.

(Ii) Synthesis and Labeling of Probe Based on the information of peaks 5 and 2 in the partial amino acid sequence obtained in (3), a DNA synthesizer “Model 380B” manufactured by Applied Biosystems was used as follows. A novel synthetic probe was prepared. Probe 5 Lys Tyr Glu Glu Asp Tyr (peak 5) 5′-AAA TAT GAA GAA GAT TAT CA-3 ′ GCGGCC Probe 2 Lys Tyr Val Asn Ile (peak 2) 5′-AAA TAT GTA AAT ATT GA-3 ′ GCGCCCAT

All the synthetic reagents such as phosphoramidite were from the company and used according to the attached operator's manual.

The obtained synthetic DNA was treated with 3 ml of 28% aqueous ammonia at 60 ° C. for 4 hours, and this was treated with Oligonucleotide Purification C, manufactured by Applied Biosystems.
Purified by artridges.

Each of the two synthesized probes was independently labeled with [γ- ³² P] ATP (up to 6000 Ci / mM). About 250 ng of each probe DNA was added to 10 units of T4
Polynucleotide kinase, 500 μCi [γ-
37 P in a 200 μl reaction containing ³² P] ATP and a phosphorylation buffer (0.1 mM spermidine, 0.1 mM EDTA, 10 mM magnesium chloride, 5 mM DTT, 50 mM Tris-HCl buffer (pH 7.6)).
After the reaction for 1 hour, the mixture was kept at 70 ° C. for 10 minutes. This was purified by Whatman DE52 to remove unreacted [γ- ³² P] ATP.

(Iii) Cloning by Plaque Hybridization Cloning by plaque hybridization was performed by screening the first, second and third steps as follows.

First, as the first screening, (4)
-Transfer 20 membranes containing the yeast library prepared in (i) to the hybridization solution (6 × SSP).
E (1.08 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA, pH 7.4), 5 × Denhardt's solution (0.1% polyvinylpyrrolidone, 0.1% ficoll, 0.1% bovine serum albumin), 0.5% SD
S, 10 μg / ml single-stranded salmon sperm DNA) 200 ml
, And gently shaken at 60 ° C for 3 hours to perform prehybridization.

Next, [γ-produced in (4)-(ii)]
Probe 5 labeled with [ ³² P] ATP was kept at 95 ° C. for 5 minutes and quenched with ice water. Twenty membranes after the hybridization were immersed in a solution mixed with the hybridization solution (400 ml) and gently shaken at 30 ° C. overnight to hybridize the membrane and the labeled probe 5.

The hybridization solution is discarded and 2 × S
Gently shake the membrane in 400 ml of SC (0.3 M sodium chloride, 0.03 M sodium citrate) at 30 ° C. for 20 minutes, and probe 5 in excess of the membrane.
The operation except for was repeated twice. After this, the membrane is
And exposed at -80 ° C. overnight. Plaque 49 exposed to either of the two membranes
Individuals were subjected to a second screening as positive clones.

In the second screening, first, these plaques were suspended in 1 ml of SM each from the original agar medium with a sterile pasteur. 1 / of this solution
Prepare 1000 dilutions, and use 100 μl of each as 100 μl of the bacterial solution of P2392 as in the library preparation.
, and mixed with 3 ml of BBL soft agar medium, and spread on a 9 cm diameter BBL agar medium. After the appearance of the plaque,
In the same manner as described in (3)-(iii), 49 sets of two membranes were prepared for one clone. (4)-
Using the probe 2 labeled with [γ- ³² P] ATP prepared in (ii), the same operation as in the first screening was repeated. Fifteen plaques were subjected to a third screening as positive clones.

In the third screening, [γ
- ³² P] using a probe 5 labeled with ATP, was repeated the same procedure and a second screening. By the above operation, 14 positive clones were finally obtained.

Next, DNA was obtained from the obtained positive clones.
Was extracted. After P2392 was cultured overnight in a TB medium, it was concentrated 4-fold in a TB medium containing 10 mM magnesium sulfate. 10 ⁹ -10 ¹⁰ plaques / 5 ml of this bacterial solution
20 μl of each positive clone prepared to a concentration of
and maintained at 37 ° C. for 15 minutes. This was transferred to 50 ml of TB medium containing 10 mM magnesium sulfate, and
For 6 hours. 2 ml of chloroform was added thereto, and the mixture was shaken at 37 ° C. for 30 minutes to lyse P2392, and then centrifuged at 10,000 rpm for 10 minutes to collect a supernatant. DNase I (Takara Shuzo) and RNase I (Boehringer Mannheim) were added thereto at a concentration of 10 μg / ml, respectively, and the mixture was kept at 37 ° C. for 30 minutes. Polyethylene glycol liquid (20% polyethylene glycol 600
(0, 2.5 M sodium chloride) was added and left at 4 ° C. overnight. Centrifuge at 10,000 rpm / 10 minutes,
The supernatant was discarded and the precipitate was suspended in 3 ml of SM. EDT
A (pH 7.5) and SDS at 20 mM and 0.1 mM, respectively.
% And kept at 55 ° C. for 4 minutes. Phenol liquid (phenol (25): chloroform (24):
Isoamyl alcohol (1)), and slowly stirred for 10 minutes, and centrifuged at 10,000 rpm for 10 minutes.
The A layer (aqueous layer) was collected. After this operation was repeated once, 0.33 ml of 3M sodium acetate was added to the aqueous layer.
5 ml of ethanol was added and stirred, and the mixture was allowed to stand at -80 ° C for 30 minutes. After centrifugation at 10,000 rpm / 10 minutes, the precipitate was rinsed with 70% ethanol, the ethanol was removed, and the precipitate was dried and dissolved in 500 μl of TE.
Each phage DNA thus obtained was cut with various restriction enzymes, electrophoresed and compared. The 14 positive clones not only contained the entire DNA chain capable of producing AATase, but also appeared to be partially missing, but all cloned from the same site on the yeast chromosome. . FIG. 5 shows a restriction map of the 6.6 kb XbaI fragment containing the full length of the DNA chain. The nucleotide sequence was determined using the XbaI fragment
C119 (Takara Shuzo) was subcloned and performed by the dideoxy method. The nucleotide sequence of the portion encoding AATase is shown in FIGS.

(5) Acquisition of a DNA strand to be hybridized from brewer's yeast The sake yeast AATase gene obtained in (4)-(iii) is used as a probe to produce a sake yeast (Kyoto 7).
A DNA strand hybridizing with the ATase gene was cloned from brewer's yeast. 1.6 kb shown in FIG.
50 ng of HindIII (range of arrow) fragment of
Using a multi-prime labeling kit (Amersham Japan Co., Ltd.), 100 μCi of [α- ³² P] dCT was used.
The reaction was carried out with P (ｍＭ3000 Ci / mM). Using this as a probe, cloning by plaque hybridization was performed using 30,000 brewer's yeast libraries prepared exactly as in (4)-(i).
The hybridization temperature was set to 50 ° C. After hybridization, the membrane was removed at 50 ° C. in 2 × SSC at 50 ° C. to remove excess probe from the membrane.
And gently shaken in 0.2 × SSC (0.03 M sodium chloride, 3 mM sodium citrate) for 20 minutes. In the first screening, 60 positive clones were obtained. These positive plaques were subjected to the second screening in the same manner as in (4)-(iii), and hybridization was repeated using the same probe under the same conditions as at the beginning. As a result, 30 positive clones were obtained. DNA was extracted from these positive clones and subjected to restriction enzyme analysis. As a result, these clones consisted of two kinds of DNAs, and the restriction enzyme maps of both clones were completely different. Therefore, it was considered that these clones cloned another site on the yeast chromosome. AATAs of these clones
FIG. 6 (a) shows a restriction map of a portion containing the e gene,
(B). These clones are hereinafter referred to as brewer's yeast AATase1 gene and brewer's yeast AATas, respectively.
Called the e2 gene.

Determination of the base sequences of the brewer's yeast AATase1 gene and the brewer's yeast AATase2 gene
(4) Performed in the same manner as in (iii). AATase1
The nucleotide sequence of the gene is shown in FIGS.
The nucleotide sequence of the ATase2 gene is shown in FIGS. It should be noted that AATase2 is an AATase regardless of the DNA chain from A to C or the DNA chain from B to C.
It was a DNA chain producing a polypeptide having aSe activity.

(6) Preparation of vector containing AATase gene and breeding of yeast transformed with this vector (i) Construction of expression vector for Saccharomyces cerevisiae Sake of FIG. 5 obtained in (4)-(iii) Yeast AATase
A 6.6 kb XbaI fragment of the gene (AAT-K7) was obtained. This is used as the origin of replication for yeast 2 μm DNA and LEU.
YEP13K, a yeast vector having two genes as markers
(FIG. 12, JP-A-62-228282)
I and ligated to the expression vector YATK1
1 was produced (FIG. 13).

Similarly, a 6.6 kb XbaI fragment (AAT-1) of the brewer's yeast AATase1 gene shown in FIG. 6 (a) obtained in (5) was obtained and ligated to a fragment obtained by cutting YEp13K with NheI, and expressing it. The vector YATL1 was produced (FIG. 14). In addition, the brewer's yeast AAT shown in FIG.
5.6 kb BglII fragment of Aase2 gene (AAT
-2) was obtained and ligated to a fragment obtained by cutting YEp13K with BamHI to prepare an expression vector YATL2 (FIG. 15).

(Ii) Construction of Expression Vector for Sake Yeast Association No. 9 Plasmid pUC4k (Pharmacia) having a G418 resistance gene was digested with restriction enzyme SalI to obtain G
YA obtained by cutting a fragment containing the 418 resistance gene with SalI
By linking with TK11, a vector YATK11G for introducing the AATase gene into sake yeast was prepared (FIG. 16).

(Iii) Construction of Expression Vector for Beer Yeast (iii-a) Obtaining G418 Resistance Marker A 2.9 kb HindIII fragment containing the PGK gene (JP-A-2-265488) was cloned into pUC18 (Takara Shuzo). . From this fragment, a plasmid pUCPGK21 having a PGK promoter and a terminator was prepared by the means shown in FIG. pUCPG
The G418 resistance gene was inserted into K21 and the plasmid pNEO was used.
(Pharmacia) by the means shown in FIG. 18 to produce pPGKneo2.

(Iii-b) Construction of Expression Vector pPGKNEO2 was digested with SalI, and a fragment of about 2.8 kb containing the PGK promoter, G418 resistance gene and PGK terminator was ligated to YATL1 digested with XhoI. YATL1G was produced (FIG. 17).

(7) Acquisition of transformed yeast by AATase gene AAT cloned in (4)-(iii) and (5)
Using various vectors prepared in (6), AAase was used to confirm that the Aase gene could produce AATase.
Transformed yeast was obtained using the Tase gene, and the AATase activity of the transformation was examined. Saccharomyces cerevisiae TD4 (a, his, leu, ura,
The introduction of the plasmid into the trp) is carried out by the lithium acetate method (J. Bacte
riol., 153 , 163 (1983)).
1 / TD4, YATL1 / TD4 and YATL2 / T
D4 (SKB105 strain) was obtained.

The transformant of Sake Yeast Association No. 9 (SKB1
(06 shares) was obtained as follows. That is, after introducing the plasmid using the lithium acetate method,
It was spread on a YPD agar medium containing g / ml of G418.
The agar medium was kept at 30 ° C. for 3 days, and the growing colonies were again inoculated on a YPD agar medium containing 500 μg / ml G418, and cultured at 30 ° C. for 2 days to obtain a transformant.

Beer yeast Alfred Jorgensen Laboratory
(Denmark), introduction of YATL1G into 2155 strains (hereinafter referred to as AJL2155 strain) was carried out as follows. The yeast was grown in 100 ml of YPD medium in O.D. D. 60
Shaking culture was performed at 30 ° C. until 0 = 16. After collection, the cells were washed once with sterile water. Subsequently, the cells were washed once with 135 mM Tris-HCl buffer (pH 8.0) and suspended in the same buffer so that the bacterial concentration was 2 × 10 ⁹ cells / ml. 10 μg of YATL1G and 20 μg of cal thymu as carrier DNA were added to 300 μl of this bacterial solution.
sDNA (Sigma) was added. Add 1200μl to this
35% PEG 4000 (filtered aseptically with a filter)
And stirred well. 750 μl of the stirred liquid was transferred to a cuvette dedicated to Gene Pulser (Bio-Rad), and subjected to electric pulse treatment once at 1 μF / 1000 V using the Gene Pulser. The bacterial solution was transferred from the cuvette to a 15 ml tube and allowed to stand at 30 ° C. for 1 hour.
The cells were collected by centrifugation at 3000 rpm for 5 minutes,
and suspended at 30 ° C. for 4 hours.
After harvesting, the cells were suspended in 600 μl of sterilized water and applied to a YPD agar medium containing 100 μg / ml G418 in 150 μl portions. The agar medium was kept at 30 ° C. for 3 days to obtain a transformant (SKB108 strain).

The AATase activity of the transformant into which the AATase gene was introduced and the control strain were measured as follows. That is, in the case of Saccharomyces cerevisiae TD4, the transformant was cultured in an SD liquid medium (0.65%
Yeast nitrogen base (excluding amino acids, Difco), 2% glucose) was used. In the case of Sake Yeast Association No. 9, a YPD liquid medium containing 400 μg / ml G418 was used. 1 for beer yeast AJL2155 strain
A YPD liquid medium containing 0 μg / ml G418 was used. 25 ml of a culture cultured with shaking at 30 ° C. for about 16 hours in 1000 ml of medium was added, and the mixture was incubated at 30 ° C. for 12 hours to
The culture was allowed to stand for hours.

Preparation of the crude enzyme solution and measurement of the activity were performed according to (1)-(ii) and (1)-(i), respectively. The protein was quantified using a Bio-Rad Protein Assay Kit (Bio-Rad) according to the manual.

The results of Saccharomyces cerevisiae TD4 are shown in Table 4, the results of Sake Yeast Association No. 9 are shown in Table 5, and the results of brewer's yeast AJL2155 are shown in Table 6. AAT
The ase activity was 2 to 15 times higher than the control strain. Therefore, by using the AATase gene according to the present invention, it is possible to easily breed strains that produce large amounts of acetate such as isoamyl acetate.

Table 4 Enzyme activities of recombinant yeast cells and crude enzyme solution Crude enzyme solution ppm mg Protein YEp13K / TD4 7.8 YATK11 / TD4 84.0 YATL1 / TD4 116.2 YATL2 / TD4 (SKB105) 50.6

Table 5 Enzyme activities of recombinant yeast cells and crude enzyme solution Crude enzyme solution ppm mg protein K9 3.4 YATK11G / K9 (SKB106) 11.6

Table 6 Enzyme activity of recombinant yeast cells and crude enzyme solution Crude enzyme solution ppm mg protein AJL2155 4.1 YATL1G / AJL2155 (SKB108) 23.4

(8) Acquisition of DNA strand hybridizing with sake yeast AATase gene from wine yeast Primers A and B homologous to two places in the sake yeast AATase gene shown in FIG.
Primer annealing was performed at 50 ° C for 2 hours using a CR Regent kit (Takara Shuzo) and a DNA thermal cycler (Perkin-Elmer Cetus Instruments).
As minutes, 30 cycles of reaction were performed. The reaction solution was subjected to agarose electrophoresis, and a DNA strand of 1.17 kb amplified from primer A to primer B was cut out from the gel and purified. 0.5 μg of this DNA chain was lysed using Nick Translation Kit (Takara Shuzo).
Labeled with μCi [ ³² P] dCTP. Sake yeast DN
Hybridization was performed with 20,000 genomic libraries of wine yeast W-3 (Yamanashi Industrial Technology Center) prepared in the same manner as in A. Hybridization was performed at 65 ° C. and the membrane was 2 × SSC2 at 65 ° C.
0 minutes, 2 x SSC for 10 minutes, and finally 0.1 x SSC
(15 mM sodium chloride, 1.5 mM sodium citrate) for 10 minutes with gentle shaking to wash. Initially, 14 positive plaques were obtained, and these plaques were similarly hybridized to obtain 7 positive plaques that strongly hybridized to a 1.17 kb DNA strand.
The phage DNA of the positive plaque was extracted and purified, and subjected to restriction enzyme analysis. As a result, all seven clones were one kind of DNA, and the restriction enzyme map was as shown in FIG.

(9) Acquisition of AATase gene-deficient yeast (i) Preparation of gene disruption vector The gene disruption vector was prepared by the method shown in FIG. That is, the brewer's yeast AATA shown in FIG.
The 6.6 kb XbaI fragment of the se1 gene was cloned into pUC19
Plasmid pATL16 inserted into the XbaI cleavage site of (Takara Shuzo) was cut with BamHI and self-ligated to prepare pATL196B. On the other hand, from YIp5, about 1.
A 1 kb HindIII fragment was excised, and the pUC19 H
Insertion into the indIII cleavage site created pUCura3. Approximately 1.1 kb by cutting pUCura3 with SmaI
URA3 fragments were removed. Next, pATL196B was cut with ClaI, and cut with exonuclease III.
ng bean nuclease and klenow treatments were performed and the ends of both ends were blunt-ended, and a 1.1 kb URA3 fragment was ligated to prepare pdel-AAT1.

(Ii) Preparation of AATase Gene-Disrupted Strain Saccharomyces cerevisiae TD4 was transformed by the lithium acetate method using pdel-AAT1 prepared in (9)-(i), which had been cut with EcoRI and NruI. AATase gene disrupted strain (SKB109 strain)
I got Transformants were selected using L-histidine hydrochloride,
L-tryptophan and L-leucine were each 40 pp
This was performed using an SD agar medium containing m. Also, pUCu
Saccharomyces cerevisiae TD4 was transformed in exactly the same manner using ra3 cut with SmaI, and the resulting transformant was used as a control strain.

(Iii) Measurement of AATase activity of the transformed strain The SKB109 strain (AATase gene-disrupted strain) and the control strain obtained in (9)-(ii) were subjected to L-histidine hydrochloride, L-tryptophan and L-leucine. S containing 40ppm each
Shaking culture was carried out at 20 ° C. for 48 hours in a D liquid medium. 1 ml of this culture was added to a YM15 medium (yeast nitrogen base 1.25% (Difco), malt extract 1.25, respectively).
% (Difco), 100% of glucose 15%) was transferred to a 500 ml Erlenmeyer flask and incubated at 20 ° C. for 24 hours. After collecting about 1 × 10 ⁹ cells each, the cells were washed twice with distilled water and further once with an AATase reaction buffer. This was suspended in 0.5 ml of an AATase reaction buffer. Add glass beads (425-600)
1.5 g (Sigma, Sigma) and the cells were disrupted with a vortex mixer. The suspension was collected and 15,000 g /
The mixture was centrifuged for 20 minutes to recover the supernatant, which was used as a crude enzyme solution. The measurement of AATase activity was performed by the method shown in (1)-(i).

The results of the AATase activity measurement were as shown in Table 7. In the SKB109 strain (AATase gene-disrupted strain), the AATase activity was reduced to about 1/5 compared to the control strain.

Table 7 AATase activity of AATase gene-deficient yeast Strain AATase activity (ppm / mg protein) SKB109 1.6 TD4 (control strain) 10.3

(10) Production of Alcohol with Low Ester Using AATase Gene-Disrupted Strain The SKB109 strain (AATase gene-disrupted strain) and the control strain obtained in (9)-(ii) were replaced with L-histidine hydrochloride and L-tryptophan. Containing 40 ppm each of L-leucine and L-leucine
Shaking culture was carried out at 20 ° C. for 48 hours in a liquid medium. 1 ml of this culture solution was added to 500 ml each containing 100 ml of YM15 medium.
The mixture was transferred to an Erlenmeyer flask and cultured at 20 ° C. for 7 days. The analysis results of the supernatant were as shown in Table 11. Compared to the control strain, isoamyl acetate was suppressed to about 1/5 and ethyl acetate was suppressed to about 3/5, and it was confirmed that liquor having a small amount of ester can be produced by using the SKB109 strain.

Table 8-1 Strains Ethanol (%) Acetaldehyde (ppm) Ethyl acetate (ppm) SKB109 7.28 11.51 6.70 TD4-URA3 7.29 9.19 11.13 ^{(Control strain)} Table 8-2 Strain n- butanol isobutanol acetate Isoa Isoamiruaru Nord (ppm Lumpur (ppm) mils (ppm) call (ppm) SKB109 19.50 35.80 0.06 54.23 TD4-URA3 24.75 36.58 0. 28 56.47 ^{(control strain)}

<Deposit of Microorganism> The following microorganisms related to the present invention have been deposited with the Research Institute of Microorganisms, Ministry of International Trade and Industry and have obtained the following deposit numbers. (1) SKB105, Microfabrication, No. 3828 (2) SKB106, Microfabrication, No. 3829 (3) SKB108, Microfabrication, No. 3830 (4) SKB109, Microfabrication, No. 4166 (5) ) EKB101 Microfabrication Kenjo No.4165

YATL2, YATK11G and YAT
To acquire L1G, SKB105, SKB, respectively
106 and SKB108 were cultured under predetermined conditions, and total DNA of yeast was extracted therefrom (Reference Metbods in yea).
st genetics, Cold SpringHarbor Laboratory. 198
6), after transforming Escherichia coli with this total DNA, the alkaline method (reference literature Molecular Cloning, Cold Spring Ha
rbor Laboratory. 1989). To obtain pdel-AAT1, use EK
B101 may be cultured under predetermined conditions, and the plasmid may be extracted therefrom by an alkaline method. EKB101
Shows that E. coli DH5 was transformed with pdel-AAT1. Transformation was performed using Nippon Gene's Tran
The measurement was carried out according to the attached manual using a formation kit DH5.

A DNA strand containing a part of the DNA strand A to B shown in FIGS. 1 and 2 disclosed in the present invention can be obtained by digesting YATK11G with an appropriate restriction enzyme. An example of a desirable DNA strand is a 1.6 kb HindIII fragment indicated by an arrow in FIG.

[0117]

As described above in the section of the Summary of the Invention, it is possible to produce alcoholic beverages with reduced ester flavor by obtaining the AATase gene.

[Brief description of the drawings]

FIG. 1 is an amino acid and base sequence diagram of AATase according to the present invention. FIG. 1 and FIG. 2 below are continuous.

FIG. 2 is an amino acid and base sequence diagram of AATase according to the present invention. 1 and 2 above are continuous.

FIG. 3 is an amino acid and nucleotide sequence diagram of another AATase according to the present invention. FIG. 3 and FIG. 4 below are continuous.

FIG. 4 is an amino acid and nucleotide sequence diagram of another AATase according to the present invention. 3 and 4 described above are continuous.

FIG. 5 is a restriction map of the AATase gene (derived from sake yeast) according to the present invention.

FIG. 6 is a restriction map of two AATase genes (derived from brewer's yeast) according to the present invention.

FIG. 7 is a restriction map of the AATase gene (derived from wine yeast) according to the present invention.

FIG. 8 is an explanatory diagram showing a method for preparing a probe used for obtaining an AATase gene from wine yeast.

FIG. 9 is an explanatory diagram showing an elution pattern of an AATase active fraction by affinity chromatography in the purification method according to the present invention.

FIG. 10 is an SDS polyacrylamide electrophoretogram of an AATase-active fraction after affinity chromatography elution according to the present invention.

FIG. 11 is an explanatory diagram showing the substrate specificity of AATase according to the present invention for various alcohols.

FIG. 12 is a restriction map of a yeast vector YEp13K.

FIG. 13 is a restriction map of an AATase gene (derived from sake yeast) expression vector YATK11 according to the present invention.

FIG. 14 is a restriction map of an AATase1 gene (derived from brewer's yeast) expression vector YATL1 according to the present invention.

FIG. 15 is a restriction map of an AATase2 gene (derived from brewer's yeast) expression vector YATL2 according to the present invention.

FIG. 16 shows an expression vector YATK11G for sake yeast of the AATase gene (derived from sake yeast) according to the present invention.
FIG.

FIG. 17 is an expression vector YATL for brewer's yeast of the AATase1 gene (derived from brewer's yeast) according to the present invention.
It is a restriction map of 1G.

FIG. 18 is a diagram showing a part of the process of constructing an expression vector for brewer's yeast.

FIG. 19 is an amino acid and nucleotide sequence diagram of the brewer's yeast AATase2 gene according to the present invention. FIG. 19 and the following FIG. 20 are continuous.

FIG. 20 is an amino acid and nucleotide sequence diagram of the brewer's yeast AATase2 gene according to the present invention. 19 and 20 are continuous.

FIG. 21 is a view showing a process of constructing an AATase gene disruption vector.

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Claims (3)

(57) [Claims] The transformed AATas comprises suppressing the expression of an alcohol acetyltransferase (AATase) producing gene specific to the host yeast.
e) a method for producing a yeast in which the productivity has been lost or reduced, wherein (1) the homology with the AATase-producing gene specific to the host yeast is preserved, but the AATase-producing ability of the host yeast is reduced by homologous recombination. DNA having any one of the following nucleotide sequences (a) to (d), which has been modified so as to be eliminated or reduced, is transformed into a host yeast A by homologous recombination.
(2) DNA having a base sequence complementary to any one of the following base sequences (a) to (d) or a partial sequence thereof, or (a) to (d) ) In any of the base sequences
2 × SSC (0.03 M sodium chloride, 3 mM sodium citrate), a DNA having a base sequence that hybridizes under the conditions of 50 ° C. for 20 minutes and that hybridizes with mRNA of the AATase gene and can prevent the synthesis of AATase protein A method for producing a host yeast, wherein the expression of an AATase-producing gene specific to the host yeast is suppressed by transforming the host yeast to express antisense RNA. (A) (B) (C) (D) 2. The method according to claim 1, wherein the yeast is Saccharomyces cerevisiae. 3. A method for producing alcoholic beverages by fermenting sugar with yeast, characterized in that yeast produced by the method according to claim 1 or 2 having lost or reduced the ability to produce alcohol acetyltransferase is used. A method of producing alcoholic beverages with reduced ester flavor.