JPH10276788A - Ferulic acid decarboxylase - Google Patents

Abstract

(57) [Problem] To provide a ferulic acid decarboxylase useful for producing distilled liquor having excellent flavor. SOLUTION: The protein comprises an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence wherein amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and has ferulic acid decarboxylase activity. A protein having the same, a gene encoding the protein,
Recombinant vector containing the gene, a transformant containing the recombinant vector, 4-vinylguaiacol, vanillin or vanillic acid or distilled spirit using an enzyme source derived from the transformant and having ferulic acid decarboxylase activity The present invention relates to a method for producing distilled spirits, a method for producing distilled spirits using yeast having an increased acid decarboxylase activity, and a spirit produced by the above method for producing spirits.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a distilled liquor having excellent flavor using yeast into which a ferulic acid decarboxylase gene has been introduced.

[0002]

2. Description of the Related Art Development of distilled spirits such as shochu, whiskey, brandy, vodka, rum, gin, white liquor and the like, which have excellent flavor, has always been desired. Distilled liquor having an excellent flavor contains a large amount of 4-vinylguaiacol, vanillin or vanillic acid.

[0003] Vanillin and vanillic acid are produced by oxidizing 4-vinyl guaiacol [Journal of the Japanese Society of Agricultural Chemistry, 70 , 684-686, (1996)]. As a method for producing a distilled liquor having an excellent flavor, a hydroxycinamic acid ester hydrolase or a koji mold producing the enzyme at a high level (Japanese Patent Laid-Open No.
-115957) or ferulic acid esterase [Japanese Journal of Agricultural Chemistry, 70 , 684-686 (1996)] to release ferulic acid into moromi.

A method for obtaining a yeast having a high ferulic acid decarboxylase activity by cell fusion has been proposed [Abstracts of the 1995 Annual Meeting of the Society of Biotechnology, 41 (1995)]. However, yeast having an enhanced ferulic acid decarboxylase activity has not been obtained, and a method for producing distilled liquor with excellent flavor using such yeast has not been known. Ferulic acid decarboxylase is an enzyme that catalyzes a reaction to decarboxylate ferulic acid to produce 4-vinylguaiacol.

[0005] Ferulic acid is a kind of phenylacrylic acid together with cinnamic acid, coumaric acid and the like, and is present as an ester bond to the arabinose side chain of arabinoxylan contained in a hemicellulose fraction constituting a cell wall of a plant such as a grain. . Bacillus pumilus (Bachillus pumilus) [Applied And Environmental Micro Biology (Appl. Environ. Microbiol.) , 61, 326-332
(1995) and Pseudomonas fluorescens ( Pseud
omonas fluorescens ) [Journal of Bacteriol., 176 , 5912-5918 (1994)] has been isolated and purified. However, ferulic acid decarboxylase of yeast belonging to the genus Saccharomyces and the like has not been isolated and purified because only the presence of the enzyme activity is known.

[0006] A gene encoding B. pumilus ferulic acid decarboxylase (hereinafter referred to as FDC)
Genel) [Appl. Environ. Microbiol., 61, 448
4-4486 (1995)] is known. Also, Saccharomyces Serebije (S. cerevisiae) the gene encoding the decarboxylase phenyl acrylate (hereinafter, PAD1
Gene, 142 , 107-112 (199
4)] is known, but the FDC gene is not known.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide ferulic acid decarboxylase which is useful for producing distilled liquor having an excellent flavor.

[0008]

The present invention provides a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or a deletion of an amino acid in the amino acid sequence represented by SEQ ID NO: 1.
A protein consisting of a substituted or added amino acid sequence and having ferulic acid decarboxylase activity (hereinafter, referred to as the protein of the present invention), a gene encoding the protein (hereinafter, referred to as a gene of the present invention), containing the gene Recombinant vector (hereinafter, referred to as the recombinant vector of the present invention), and a transformant containing the recombinant vector (hereinafter, referred to as a recombinant vector).
A transformant of the present invention), in the presence of an enzyme source derived from the transformant and having ferulic acid decarboxylase activity.
A method for producing 4-vinyl guaiacol, vanillin or vanillic acid, characterized by reacting ferulic acid in an aqueous medium and collecting 4-vinyl guaiacol, vanillin or vanillic acid produced in the aqueous medium (hereinafter referred to as the present invention). A process for producing 4-vinylguaiacol, vanillin or vanillic acid according to the present invention), which comprises adding an enzyme source derived from the transformant and having ferulic acid decarboxylase activity to moromi. A method (hereinafter referred to as a method for producing a distilled liquor using the protein of the present invention), and a method for producing a distilled liquor characterized by using a yeast having an increased ferulic acid decarboxylase activity (hereinafter, using the yeast of the present invention) And a distilled liquor obtained by the above-described method for producing a distilled liquor.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1 in which amino acid residues are deleted, substituted or added, as long as it has ferulic acid decarboxylase activity. Or a protein consisting of The number of amino acid residues to be deleted, substituted or added is not particularly limited.
It is preferable that the number of amino acid residues is from one to several.
Further, when a simple homology analysis was performed on the entire amino acid sequence with the amino acid sequence represented by SEQ ID NO: 1 using DNASIS ver3.0 (manufactured by Hitachi Software Engineering), the homology was at least 20%, particularly at least 40%. It is preferably an amino acid sequence.

[0010] Isolation of the gene of the present invention, determination of the nucleotide sequence of the gene, recombinant vector of the present invention, transformants containing the recombinant vector of the present invention, and genetic engineering for producing the protein of the present invention. Or, for basic operations of biotechnology, refer to commercially available experimental manuals such as the Gene Manual.
Kodansha, Yasutaka Takagi Ed. Gene manipulation experiment method Kodansha, Molecular Cloning (Molecular Cloning) Cold Spring Harbor Laboratory (Cold Sprin)
g Harbor Laboratory) (1982), Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory (Cold Spring)
Harbor Laboratory) (1989), Methods in Enzymol., 194 (1991), Experimental Medicine Separate Volume, Gene Experiment Using Yeast Performed according to the method described in Yodosha (1994), etc. Can be.

[0011] The gene of the present invention is a yeast having no ferulic acid decarboxylase activity or a yeast having extremely low enzymatic activity (hereinafter, also referred to as a yeast having substantially no ferulic acid decarboxylase activity), such as Saccharomyces -It can be isolated as a gene imparting the enzyme activity to S. cerevisiae strain K9H14 (hereinafter referred to as strain K9H14).
That is, the K9H14 strain is transformed with a yeast DNA library having an FDC gene, and DNA is obtained from yeast to which ferulic acid decarboxylase activity has been imparted, whereby the FDC gene can be isolated.

A yeast DNA library having an FDC gene is obtained by cutting a chromosomal DNA of a yeast having ferulic acid decarboxylase activity, for example, a Saccharomyces cerevisiae W3 strain (hereinafter referred to as a W3 strain), which is a wine yeast, with a restriction enzyme. It can be prepared by ligating the obtained DNA fragment to a vector that can be maintained in yeast.

Any restriction enzyme can be used as long as it can cleave the chromosomal DNA. Preferably, a restriction enzyme that generates a DNA fragment of 10 Kbp or less is used. In addition, chromosomal DNA
May be completely or partially cleaved by a restriction enzyme. Examples of vectors that can be maintained in yeast include a YCp-type vector, a YEp-type vector, a YRp-type vector, a YIp-type vector, and a YAC (yeast artificial chromosome) vector.

The transformation of the DNA library into the K9H14 strain is performed by the spheroplast method [for example, Proceedings of National Academy of Science USA (Proc. Natl. Acad. Sci. USA), 75 , 1929- 1933
(1978)], lithium acetate method [for example, J. Bacteriol., 15
3 , 163-168 (1983)] and an electroporation method [eg, Methods in Enzymol., 194, 182-187 (1991)].
The method can be performed according to a method commonly used in the field of genetic engineering or biotechnology.

As a method for selecting yeast to which ferulic acid decarboxylase activity has been imparted, for example, the following method can be mentioned. The transformant obtained by the above method is cultured overnight (20 to 24 hours) in a YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose), and 1 g / 0.1 ml of 1 ferulic acid solution is added, and the cells are cultured overnight (20 to 24 hours). After the cultivation, a culture solution with a smoke odor is sensuously selected and further subjected to high performance liquid chromatography [Journal of the Japanese Society of Agricultural Chemistry, 69 , 1587-159].
6, (1995)], a culture solution in which 4-vinylguaiacol can be detected. The transformed strain is recovered from the selected culture solution, and the obtained strain is selected as yeast to which ferulic acid decarboxylase activity has been imparted.

As the ferulic acid, either trans-ferulic acid or cis-ferulic acid may be used, but trans-ferulic acid is preferably used. As a method of recovering a plasmid from yeast to which ferulic acid decarboxylase activity has been imparted and a method of transforming the recovered plasmid into Escherichia coli, a method commonly used in the field of genetic engineering can be used. Examples of a method for recovering a plasmid from the yeast include, for example, Experimental Medicine Separate Volume, Gene Experiment Method Using Yeast, and a method described in Yodosha (1994) .A method for transforming the recovered plasmid into Escherichia coli includes, for example, Molecular Cloning, 2nd ed., Cold Spring
Harbor Laboratory (1989)).

The DNA clone selected by the above method is cleaved with an appropriate restriction enzyme, for example, PstI, and the obtained DNA fragment is subjected to a dideoxy method and a DNA sequencer (ALF DNA Sequencer II, manufactured by Pharmacia LKB). By performing the analysis, the nucleotide sequence of the FDC gene can be determined. The nucleotide sequence of the FDC gene derived from the W3 strain was determined by the results of the yeast genome project [for example, on the Internet http: // genome-www.
stanford.edu/sacchdb/].

The nucleotide sequence of the FDC gene determined as described above includes, for example, the nucleotide sequence shown in SEQ ID NO: 1. Once it has been determined that the base sequence shown in SEQ ID NO: 1 encodes ferulic acid decarboxylase,
Thereafter, by chemical synthesis or by PCR (Polymera
The gene of the present invention can be obtained by hybridization using a DNA fragment having the nucleotide sequence as a probe by the Se Chain Reaction) method.

The gene of the present invention can also be obtained by artificially modifying, for example, deleting, substituting, or inserting a part of the nucleotide sequence of the gene prepared as described above. The choice of codon for an amino acid may be arbitrarily determined, for example, it can be determined in consideration of the codon usage of the host to be used [for example, Nucleic Acids Res., 9, 43-74 (1981) )
]. In addition, the modification of the nucleotide sequence can be performed by cytospecific mutagenesis [for example, Proc. Natl. Acad. Sci.
USA, 81 , 5662-5666 (1984)].

The gene of the present invention can be obtained, for example, from Methods in Enz
By using the method described in Ymol., 194, 594-597 (1991), gene disruption, expression control, expression level modification, and the like can also be performed. The recombinant vector of the present invention can be prepared by preparing a DNA fragment containing the gene of the present invention using a restriction enzyme or the like, and then inserting the DNA fragment downstream of a promoter in an expression vector.

In the recombinant vector of the present invention, it is preferable to use the nucleotide sequence of the gene of the present invention by substituting the base as necessary so that the codon is optimal for expression in a host cell. In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for expression of the gene of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host cell compatible with the above expression vector. As a host cell, any one that can express a gene of interest, for example, Escherichi
a coli , Bacillus subtilis , Bacillus amylolique
faciens , Brevibacterium flavum , Brevibacterium
lactofermentum, Corynebacterium glutamicum, Micr
bacteria belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, etc., such as bacterium ammoniaphilum ; Sa
ccharomyces cerevisiae , Schizosaccharomyces pom
be , Kluyveromyces lactis , Trichosporon pullulan
and yeast cells such as s . Schwanniomyces alluvius , animal cells such as Namalba cells, COS cells and CHO cells, and plant cells such as tobacco cells and carrot cells.

As the expression vector, those capable of autonomous replication in the above-mentioned host cells or integration into the chromosome, and containing a promoter so that the sequence encoding the recombinant protein of the present invention can be transcribed are used. . When a bacterium such as Escherichia coli is used as a host cell, the recombinant vector of the present invention is capable of autonomous replication in the microorganism, and is composed of a promoter, a ribosome binding sequence, a DNA of the present invention, and a transcription termination sequence. Is preferred. A gene that controls the promoter may be included.

As an expression vector, for example, pBTr
p2, pBTac1, pBTac2 (both are Boehring
er Mannheim), pKYP200 [Agric. Biol. C
hem., 48 , 669-675 (1984)], pLSA1 [Agric. Bio
l. Chem., 53 , 277 (1989)], pGEL1 [Proc. Nat.
l. Acad. Sci., USA, 82, 4306 (1985) ", pBluescript.
(Stratagene) or the like is used.

Any promoter can be used as long as it can be expressed in a host cell such as Escherichia coli.
For example, trp promoter (P trp), lac promoter (P lac), P _L promoter, such as P _R promoter, promoters derived from Escherichia coli or phage, etc. used. P trp two series is not a promoter (P tr
p x2), may be used artificially designed and modified promoters such as the tac promoter.

Any ribosome-binding sequence can be used as long as it can be expressed in a host cell such as Escherichia coli, but the distance between the ribosome-binding sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). It is preferable to use them. As a method for introducing a recombinant vector into bacteria,
As a method for introducing DNA into bacteria, for example, a method using calcium ions [Proc. Natl. Acad. Sci., US
A, 69, 2110-2114 (1972)], and the protoplast method (JP-A-63-2483942).

When yeast is used as a host cell, as an expression vector, for example, YEp13 (ATCC37115)
), YEp24 (ATCC37051), YCp50 (ATCC374)
19) etc. are used. As the promoter, if it can be expressed in yeast, for example, a promoter of a glycolytic gene such as hexose kinase, a gal 1 promoter, a gal 10 promoter, a heat shock protein promoter, an MFα1 promoter, a CUP 1 promoter, etc. May be used.

As a method for introducing a recombinant vector into yeast, any method for introducing DNA into yeast, for example, an electroporation method [Methods in Enzymol., 194 ,
182-187 (1990)], spheroplast method [Proc. Natl.
Acad. Sci., USA, 84, 1929-1933 (1978)] and lithium acetate method [J. Bacteriol., 153 , 163-168 (1983)].

When an animal cell is used as a host cell, pcDNAI / Am may be used as an expression vector.
p, pcDNAI, pcDM8 (all manufactured by Funakoshi) and the like are used. In this case, any promoter may be used as long as it can be expressed in animal cells, such as the promoter of the IE (immediate early) gene of human CMV. In addition, human CMV
An IE gene enhancer may be used with a promoter.

As a method for introducing a recombinant vector into animal cells, a method for introducing DNA into animal cells includes, for example, electroporation [Cytotechnology
(Cytotechnology), 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc.
Acad. Sci., USA, 84, 7413 (1987)].

When a plant cell is used as a host cell, pBI121 [Nucl
eic Acids Res., 12 , 8771-8721 (1984)]. In this case, any promoter may be used as long as it can be expressed in plant cells, such as the cauliflower mosaic virus 35S promoter.

As a method for introducing a recombinant vector into plant cells, a method for introducing DNA into plant cells, for example, Agrobacterium tumefaciens ( Agroba
cterium tumefaciens ) [Methods in Enzaymol., 11
8, 627-640 (1986)], high-speed microparticle method [Plant Molecular Biology,
11 , 433-439 (1989)] and the protoplast method [Nature, 319, 791-793 (1986)].

The transformant of the present invention is used for production of the protein of the present invention, production of flavors such as 4-vinylguaiacol, vanillin or vanillic acid, and the like. Also,
The transformant of the present invention in which the host cell is yeast is suitably used for the production of distilled liquor having excellent flavor. The protein of the present invention can be produced by culturing the transformant of the present invention in a medium, producing and accumulating the protein of the present invention in a culture, and collecting the protein from the culture.

The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing host cells of the transformant. When culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host cell, the culture medium contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be assimilated by the microorganism. Either a natural medium or a synthetic medium may be used as long as the medium can be efficiently used.

As the carbon source, carbohydrates such as glucose, fructose, sucrose, molasses, starch, and hydrolyzed starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.
Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, a soybean meal hydrolyzate, various fermentation cells or digests thereof are used.

As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. Cultivation is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
Perform at 5-40 ° C. for 16-96 hours. During the cultivation period, the pH is kept at 3.0 to 9.0.

During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D
When culturing a microorganism transformed with thiogalactopyranoside (IPTG) or the like using an expression vector using a trp promoter, indoleacrylic acid (IAA) or the like may be added to the medium.

When a transformant obtained by using an animal cell as a host cell is cultured, a medium containing a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the animal cell, so that the transformant can be efficiently cultured. Any of a natural medium and a synthetic medium may be used, but the RPMI1640 medium, Eagle's M
An EM medium or a medium obtained by adding fetal bovine serum or the like to such a medium is usually used.

Cultivation is usually performed in the presence of 5% CO ₂ for 35 to 3
Perform at 7 ° C for 3-7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture. When culturing a transformant obtained by using a plant cell as a host cell, if the medium contains a carbon source, a nitrogen source, inorganic salts, and the like that can be utilized by the plant cell, a medium that can efficiently culture the transformant can be used. Either a natural medium or a synthetic medium may be used, but Murashige-Skoog (MS) medium, white (Wh
ite) A medium or the like is usually used.

The cultivation is usually carried out at 15 to 40 ° C. for 1 to 30 days under aerobic conditions such as shaking culture or deep aeration stirring culture. During the cultivation period, the pH is kept at 3.0 to 9.0.
If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture. After completion of the cultivation, to isolate and purify the protein of the present invention produced inside or outside the cells of the transformant of the present invention, usual enzyme isolation,
A purification method may be used. When the protein of the present invention is produced in the cells, the cells obtained by centrifuging the culture solution are collected, and after washing the cells, the cells are treated with an ultrasonic crusher, French press, Mantongaulin homogenizer, Dynomill, or the like. Crush the body to obtain a cell-free extract. The supernatant obtained by centrifuging the cell-free extract is subjected to salting out with ammonium sulfate or the like, anion exchange chromatography such as diethylaminoethyl (DEAE) -Sepharose, or hydrophobic chromatography such as butyl sepharose or phenyl sepharose. A purified enzyme preparation of the protein of the present invention can be obtained by using a method such as gel filtration using a molecular sieve or electrophoresis such as isoelectric focusing. When the protein of the present invention is produced extracellularly,
A purified enzyme preparation of the protein of the present invention can be obtained by using a method similar to that for purifying from the culture solution from a cell-free extract.

The method for producing 4-vinylguaiacol, vanillin or vanillic acid according to the present invention comprises a crude purification obtained by the above method as an enzyme source derived from the transformant of the present invention and having ferulic acid decarboxylase activity. Alternatively, in addition to the purified protein of the present invention, cultures, cells, treated cells, etc. of the transformant of the present invention may be used. Examples of the treated cells include physicochemically and biochemically treated cells such as washed cells, freeze-dried cells, and acetone-treated cells.

The enzyme source is usually used in an aqueous medium in a concentration range of 0.1 to 1000 units / ml as an enzyme amount unit of ferulic acid decarboxylase. The unit of the amount of ferulic acid decarboxylase is 50 mM using ferulic acid as a substrate.
The amount of the enzyme that produces 1 nmol of 4-vinyl guaiacol when reacted at 30 ° C. for 1 hour in a phosphate buffer (pH 5.0) is indicated as one unit.

When a culture or a cell is used as an enzyme source, the culture or the cell may be added with a surfactant such as cetylpyridium chloride or cetyltrimethylammonium bromide or an organic solvent such as toluene or xylene. . When a surfactant or an organic solvent is added, the amount of addition is 0.05 to 1.0% (w /
v) or 1 to 20% (v / v).

Ferulic acid is usually present in aqueous media at 0.0
It is used in a concentration range of 1 to 10 g / l. The reaction varies depending on the culture, the amount of the cells or the treated cells, and the amount of ferulic acid, but at 20 to 60 ° C., pH 2.5 to 10.0.
For 1 to 72 hours. 4-vinyl guaiacol is present in the aqueous medium thus obtained.
After completion of the reaction, the culture, the cells or the treated cells are crushed as necessary from the aqueous medium, the precipitate is removed by means such as centrifugation, and the resulting supernatant is subjected to extraction, distillation, By using ordinary methods such as various types of chromatography and recrystallization, 4-vinylguaiacol can be isolated and purified.

Vanillin or vanillic acid can be obtained by forcibly oxidizing 4-vinylguaiacol isolated and purified in or from the aqueous medium, for example, by aeration, or by using Bacillus subtilis ( B. subtilis ) or corynebacterium. Umm glutamicum ( Corynebacterium Glu
tamicum ) [Journal of Industrial Microbiology (J. In
d. Microbiol.), 15 , 457-471 (1995)].

Vanillin or vanillic acid can be isolated and purified by using a method similar to that for isolating and purifying 4-vinylguaiacol. As a method for producing a distilled liquor using the protein of the present invention, an enzyme source derived from the transformant of the present invention and having ferulic acid decarboxylase activity is contained in moromi as an enzyme unit of ferulic acid decarboxylase in an amount of 0%. Except for adding to a concentration of 0.1 to 50 units / ml, a conventional method for producing a distilled spirit is used, in which a carbon source is saccharified with a koji mold or a saccharifying enzyme, yeast is added, alcohol fermentation is performed, and distillation is performed.

In the present invention, moromi refers to a culture obtained through alcohol fermentation. As a method for producing distilled spirits using the yeast of the present invention, except that yeast having increased ferulic acid decarboxylase activity is used, a carbon source is saccharified with Aspergillus or saccharifying enzyme, and yeast is added and alcohol fermentation is performed. Distillation is carried out using a conventional method for producing distilled spirits.

A yeast having an increased ferulic acid decarboxylase activity is defined as a host cell or a parent yeast which is obtained by subjecting the yeast to a gene recombination technique, a mutation technique, or the like, to a host cell or a parent yeast. Yeast with increased ferulic acid decarboxylase activity. When using genetic recombination technology,
Yeast having increased ferulic acid decarboxylase activity is a recombinant vector containing a gene encoding ferulic acid decarboxylase, for example, the gene used in the present invention, the Bacillus pumilus FDC gene, etc. This can be produced by transforming a host cell by the above method.

When the mutation technique is used, the yeast having an increased ferulic acid decarboxylase activity can be treated with a mutagenic agent such as UV irradiation or ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. , Etc., and mutated by ordinary mutation means, and using the same method as the above-described method for selecting yeast to which ferulic acid decarboxylase activity has been imparted, the resulting mutant strain is 4-vinyl as compared with the parent strain. It can be produced by selecting a strain with improved ayacol productivity. After mutagenizing the parent strain, a medium containing phenylacrylic acid at a concentration such that the parent strain cannot grow sufficiently, for example, a medium containing 1 mM or more of ferulic acid,
If a mutant strain that can grow significantly compared to the parent strain is selected, a yeast having an increased ferulic acid decarboxylase activity can be efficiently produced.

As a host cell or a parent strain used for producing a yeast having an increased ferulic acid decarboxylase activity, any yeast can be used as long as it is a yeast used for producing distilled liquor, and preferably Saccharomyces sp. And more preferably yeast belonging to Saccharomyces cerevisiae. As the host cell, yeast belonging to Saccharomyces cerevisiae having substantially no ferulic acid decarboxylase activity, for example, Japan Brewery Association 7, Japan Brewery Association 9, K9H14 strain (
Sake, shochu yeast), IFO 2112, IFO 2114,
When IFO2115 (the above is whiskey yeast) is used, a remarkable effect can be obtained.

For example, when a yeast having substantially no ferulic acid decarboxylase activity is used as a host cell, a transformant prepared by the same method as the method for preparing a transformant of the present invention may be used as a substrate. Ferulic acid was added as a reagent, and 4-vinyl guaiacol was detected functionally or quantitatively using a device such as high performance liquid chromatography, and a strain in which the production of 4-vinyl guaiacol was confirmed was selected. The yeast of the invention can be made.

The yeast of the present invention produced by the above method includes, for example, Saccharomyces cerevisiae YS
A7 strain (hereinafter referred to as YSA7 strain). YS
A7 shares were purchased on December 1, 1996 based on the Budapest Treaty.
On 1st, FERMBP was sent to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture)
-5772.

The following shows that the YSA7 strain is a yeast having an increased ferulic acid decarboxylase activity.

Test Example 1 Test of Ferulic Acid Decarboxylase Activity Ability YSA7 strain was placed in a test tube containing 10 ml of YPD medium, and its parent strain K9H14 strain and K9H14 strain were YEp24.
The K9H14-C strain, which is a transformant into which Escherichia coli was introduced, was inoculated with one loop of each loop and cultured with shaking at 30 ° C. for 22 hours, and 0.9 ml of the obtained culture was added with 1 g / l ferulic acid solution in 0 ml. 0.1 ml was added, and the mixture was allowed to stand still at 25 ° C. for 22 hours.
After completion of the culture, the content of 4-vinylguaiacol in the culture supernatant was quantified by high performance liquid chromatography under the following conditions.

Column: Wakosil 5C 18-200 (id 4.6 × 250 mm) Column temperature: 50 ° C. Mobile phase: 10 mM phosphate buffer (pH 2.5) / methanol =
50/50 (v / v) Detector: Fluorescence detector (Ex 280 nm, Em 320 nm) Sample: Culture solution supernatant was diluted 10-fold with the mobile phase, and 10 μl was injected. The results are shown in Table 1.

[0056]

[Table 1]

The method for producing the distilled liquor of the present invention using the yeast having an increased ferulic acid decarboxylase activity produced by the above method is described below. As the carbon source, any carbohydrates and starches can be used, and preferably, grains such as rice, wheat, foam, corn, corn, fin, and cane,
Fruits such as potatoes, buckwheat, grapes, apples and the like or kojis thereof, and more preferably cereals or kojis thereof are used.

As the koji mold, filamentous fungi used for rice koji, barley koji, bran koji and the like, for example, Aspergillus ( Aspergillus)
s ) A microorganism belonging to the genus or Rhizopus is used. As saccharification enzymes, enzymes contained in malt,
Enzymes produced by Aspergillus or enzyme preparations such as α-amylase, glucoamylase, and protease are used.

As the alcohol fermentation, the following method is exemplified. In the case of using cereals, the starch, which is cereals, is first decomposed into saccharides by a saccharifying enzyme, and then yeast is added to perform fermentation in parallel. For example, in the production of shochu, koji is charged at the start of fermentation, and the remaining carbon source is added as the fermentation progresses. In the production of whiskey, a method is generally used in which malt is added to warm water to saccharify and ferment the resulting wort. When using sugars such as fruits, molasses, and glucose, simple fermentation is performed by directly adding yeast and fermenting.

The alcohol fermentation is usually carried out at pH 3.5 to pH 3.5.
5.0, temperature 5 ~ 25 ° C, shochu 7 ~ 1 after preparation
4 days, whiskey 3-4 days, brandy 7-1
Perform for 4 days. After completion of alcohol fermentation, the obtained moromi may be distilled as it is, but the moromi may be subjected to squeezing filtration, centrifugation, etc. to separate a fermentation residue, a liquid obtained by separating yeast cells, etc. . Ethanol is concentrated by distilling the moromi or a liquid obtained by subjecting the moromi to squeezing filtration, centrifugation, or the like, to prepare an original sake. The original sake may be used directly as distilled spirit, but the original sake is mixed, diluted,
Adjustment such as addition of alcohol may be performed, and this may be used as distilled spirits.

Examples will be described below.

[0062]

【Example】

Example 1 Cloning of Gene Encoding Ferulic Acid Decarboxylase (1) Assignment of ura3 Mutation to K9H14 Strain Saccharomyces cerevisiae Sake (Shochu) Yeast Haploid Strain No. 9 (Japan Brewing Association) And
As a marker for introducing a plasmid into the K9H14 strain, which is a strain having substantially no ferulic acid decarboxylase activity, the method of Boeke et al.
And General Genetics (Mol.Gen. Genet.), 1
97, 345-346 (1984)]. That is, one loopful of the K9H14 strain was inoculated into a YPD medium and cultured with shaking at 30 ° C. overnight. 100 μl of the obtained culture solution was applied to an FOA plate [0.67% yeast nitrogen-based W / O amino acid (manufactured by Difco),
0.1% 5-fluoroorotic acid, 0.005% uracil, 2% glucose, 2% agar] and cultured at 30 ° C for 3 days. After completion of the culture, of the resulting colonies,
One strain which is uracil auxotrophic and has no uracil auxotrophy and has no ferulic acid decarboxylation activity when transformed with plasmid YCp50 having URA3 as a marker was selected as K9H14-3u strain. The K9H14-3u strain exhibited properties equivalent to those of the K9H14 strain in terms of properties such as fermentation ability.

(2) Cloning The DNA fragment obtained by partially digesting the chromosomal DNA of the wine yeast W3 strain with BamHI was ligated into the BamH of plasmid YCp50.
A gene library was prepared by insertion at the I site. Using the gene library, the K9H14-3u strain was transformed, and transformants were selected without uracil auxotrophy. The obtained transformant is cultured in a YPD liquid medium with shaking at 30 ° C. overnight, and 0.1 ml of a 1 g / l ferulic acid solution is added to 0.9 ml of the obtained culture, and the mixture is allowed to stand at 25 ° C. for 22 hours. Cultured. After completion of the culture, the culture was smelled, a culture with a strong smoke-like scent was selected, and the culture supernatant was subjected to high-performance liquid chromatography to confirm the production of 4-vinylguaiacol.

A strain obtained from the culture solution was separated as a strain imparted with ferulic acid decarboxylation activity, and a recombinant plasmid pSA11 was extracted from the strain. Plasmid pS
A11 contains about 4 Kbp of Dam from BamHI to BamHI.
The NA fragment had been inserted. Plasmid pSA11 was cleaved with various restriction enzymes, and the obtained DNA fragments were separated by electrophoresis to measure the molecular weight, and a restriction enzyme map was prepared as shown in FIG.

(3) Determination of base sequence BamHI-B inserted into plasmid pSA11
The nucleotide sequence of a 4 Kbp DNA fragment of amHI was determined by the dideoxy method and a DNA sequencer (Pharmacia LKB).
ALF DNA Sequencer II). As a result, a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter referred to as FDC1 gene) was opened.
The nucleotide sequence represented by SEQ ID NO: 2, which was included as a reading frame, was determined. The protein encoded by the FDC1 gene predicted from the determined nucleotide sequence is 5
It consisted of 03 amino acid residues. This nucleotide sequence represented by SEQ ID NO: 1 can be obtained from the yeast genome project [for example, on the Internet http://genome-www.stanford.edu/
sacchdb /], the sequence I
It was the same as the nucleotide sequence located in the sequences 1512140 to 1513651 of chromosome V. The FDC1 gene and the Bacillus pumilus FDC gene [Appl. Envi
ron. Microbiol., 61, 4484-4486 (1995)] and a gene encoding phenylacrylic acid decarboxylase of Saccharomyces cerevisiae (hereinafter referred to as the PAD1 gene) [Gene, 142, 107-112 (1994)]. , DNASI
Simple homology analysis was performed using Sver3.0 (manufactured by Hitachi Software Engineering). As a result, 30% and 41% of the entire nucleotide sequence was obtained, and 11.25% of the total amino acid sequence obtained by translating the nucleotide sequence. 1
There was only 0.37% homology. Also, BLAS
As a result of performing homology search by T method [for example, using database of genome net, Kyoritsu Publishing (1996)],
No significant homology was found between the FDC1 gene and the Bacillus pumilus FDC and PAD1 genes.

Example 2 Preparation of Shochu Yeast Providing Ferulic Acid Decarboxylase Activity (1) Preparation of Plasmid for Expression of FDC1 Gene About 5 μg of plasmid pSA11 was added to an H buffer [50 mM Tris-HCl buffer (pH 7.5), 10 mM Magnesium chloride, 1 mM dithiothreitol, 100 mM sodium chloride] dissolved in 20 μl, and 10 units of the restriction enzyme Bam
HI and SalI were added and reacted at 37 ° C. overnight.
The reaction product was separated by 0.8% agarose electrophoresis, a DNA fragment of about 3.6 kb was cut out, and
Extraction and purification were performed using a LEAN II kit (manufactured by Bio-101). Further, a DNA fragment of about 7.7 kbp was extracted and purified using the same method as described above except that the plasmid pSA11 was replaced with the yeast-E. Coli shuttle vector YEp24. About 3.6 kbp of D from plasmid pSA11
1 μg of NA fragment and about 7.7 kbp of D from YEp24
A ligation reaction was performed at 16 ° C. overnight using 0.1 μg of the NA fragment using Ligation kit (Takara Shuzo). After completion of the reaction, competent cells E by using the reaction solution 5μl. co
li JM109 strain (Toyobo) was transformed. The obtained transformant was transformed into an ampicillin LB agar medium [1% bactotryptone (manufactured by Difco), 0.5% yeast extract, 1% sodium chloride, 1.5% agar, 50 µg / m2.
l ampicillin] and culturing was performed at 37 ° C for 20 hours. After completion of the culture, the resulting colony was isolated, and the plasmid DNA obtained by culturing the colony was extracted and purified. The obtained plasmid was digested with restriction enzymes BamHI and SalI, and a DNA fragment of about 3.6 Kbp was confirmed, which was used as an expression plasmid pSA8 (FIG. 2).

(2) Introduction and Expression of FDC1 Gene into Shochu Yeast K9H14-3u strain was inoculated into an Erlenmeyer flask containing 100 ml of YPD medium, and was incubated at 30 ° C. at 2 to 4 × 10 ⁷ cells / m 2.
The cells were shake-cultured until the total volume became 1. After completion of the culture, the cells were collected by centrifugation (2500 rpm, 5 minutes), and the obtained cells were contacted with the plasmid pSA8 by the lithium acetate method.
The K9H14-3u strain contacted with the plasmid pSA8 was transferred to an SD agar medium (0.67% yeast nitrogen
Base W / O amino acid, 2% glucose, 2% agar) and cultured at 30 ° C. for 2-5 days. After cultivation,
From one of the resulting colonies, YSA, a transformant in which the uracil auxotrophy of the K9H14-3u strain was complemented.
Seven shares were acquired.

The YSA7 strain, the K9H14 strain and the K9H1
Each 4-C strain was inoculated into a YPD medium, cultured overnight at 30 ° C., and 0.1 ml of a 1 g / l ferulic acid solution was added to 0.9 ml of the obtained culture, followed by stationary culture at 25 ° C. for 22 hours. did. After completion of the culture, the content of 4-vinyl guaiacol in the culture supernatant was quantified by high performance liquid chromatography. As a result, almost no production of 4-vinyl guaiacol was observed in the K9H14 strain and the K9H14-C strain.
In the SA7 strain, the production of 4-vinylguaiacol was confirmed.

Example 3 Production of Rice Shochu A small preparation of rice shochu was carried out for each of the YSA7 strain, K9H14 strain and K9H14-C strain using 840 g of total rice. Table 2 shows the charge ratios.

[0070]

[Table 2]

Alcohol fermentation at 20 ° C. after secondary charging 10
After a period of one day, the obtained moromi was distilled to produce rice shochu. The manufactured rice shochu was subjected to component analysis in accordance with the prescribed analysis method of the National Tax Agency. With respect to rice shochu produced using each yeast, the ethanol content and 4-vinyl guaiacol content were measured using high performance liquid chromatography, and sensory evaluation was performed by seven panelists.

The results are shown in Table 3. In addition, the evaluation score of the sensory evaluation was the average score of 7 persons.

[0073]

[Table 3]

As shown in Table 3, rice shochu produced using the YSA7 strain had significantly higher 4-vinyl guaiacol content than rice shochu produced using the parent strain K9H14, and was thus organoleptically sensitive. Also had a characteristic flavor.

[0075]

According to the present invention, it is possible to provide a ferulic acid decarboxylase useful for producing a distilled liquor having an excellent flavor.

[0076]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1512 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Saccharomyces ( Saccharomyces)
cerevisiae ) Strain name: YSA7 Sequence characteristics Symbol indicating characteristics: CDS Location: 1. 1512 Sequence determination method: E sequence ATG AGG AAG CTA AAT CCA GCT TTA GAA TTT AGA GAC TTT ATC CAG GTC 48 Met Arg Lys Leu Asn Pro Ala Leu Glu Phe Arg Asp Phe Ile Gln Val 1 5 10 15 TTA AAA GAT GAA GAT GAC TTA ATC GAA ATT ACC GAA GAG ATT GAT CCA 96 Leu Lys Asp Glu Asp Asp Leu Ile Glu Ile Thr Glu Glu Ile Asp Pro 20 25 30 AAT CTC GAA GTA GGT GCA ATT ATG AGG AAG GCC TAT GAA TCC CAC TTA 144 Asn Leu Glu Val Gly Ala Ile Met Arg Lys Ala Tyr Glu Ser His Leu 35 40 45 CCA GCC CCG TTA TTT AAA AAT CTC AAA GGT GCT TCG AAG GAT CTT TTC 192 Pro Ala Pro Leu Phe Lys Asn Leu Lys Gly Ala Ser Lys Asp Leu Phe 50 55 60 AGC ATT TTA GGT TGC CCA GCC GGT TTG AGA AGT AAG GAG AAA GGA GAT 240 Ser Ile Leu Gly Cys Pro Ala Gly Leu Arg Ser Lys Glu Lys Gly Asp 65 70 75 80 CAT GGT AGA ATT GCC CAT CAT CTG GGG CTC GAC CCA AAA ACA ACT ATC 288 His Gly Arg Ile Ala His His Leu Gly Leu Asp Pro Lys Thr Thr Ile 85 90 95 AAG GAA A TC ATA GAT TAT TTG CTG GAG TGT AAG GAG AAG GAA CCT CTC 336 Lys Glu Ile Ile Asp Tyr Leu Leu Glu Cys Lys Glu Lys Glu Pro Leu 100 105 110 CCC CCA ATC ACT GTT CCT GTG TCA TCT GCA CCT TGT AAA ACA CAT ATA 384 Pro Pro Ile Thr Val Pro Val Ser Ser Ala Pro Cys Lys Thr His Ile 115 120 125 CTT TCT GAA GAA AAA ATA CAT CTA CAA AGC CTG CCA ACA CCA TAT CTA 432 Leu Ser Glu Glu Lys Ile His Leu Gln Ser Leu Pro Thr Pro Tyr Leu 130 135 140 CAT GTT TCA GAC GGT GGC AAG TAC TTA CAA ACG TAC GGA ATG TGG ATT 480 His Val Ser Asp Gly Gly Lys Tyr Leu Gln Thr Tyr Gly Met Trp Ile 145 150 155 160 CTT CAA ACT CCA GAT AAA AAA TGG ACT AAT TGG TCA ATT GCT AGA GGT 528 Leu Gln Thr Pro Asp Lys Lys Trp Thr Asn Trp Ser Ile Ala Arg Gly 165 170 175 ATG GTT GTA GAT GAC AAG CAT ATC ACT GGT CTG GTA ATT AAA CCA CAA 576 Met Val Val Asp Asp Lys His Ile Thr Gly Leu Val Ile Lys Pro Gln 180 185 190 CAT ATT AGA CAA ATT GCT GAC TCT TGG GCA GCA ATT GGA AAA GCA AAT 624 His Ile Arg Gln Ile Ala Asp Ser Trp Ala Ala Ile Gly Lys Ala Asn 195 200 Two 05 GAA ATT CCT TTC GCG TTA TGT TTT GGC GTT CCC CCA GCA GCT ATT TTA 672 Glu Ile Pro Phe Ala Leu Cys Phe Gly Val Pro Pro Ala Ala Ile Leu 210 215 220 GTT AGT TCC ATG CCA ATT CCT GAA GGT GTT TCT GAA TCG GAT TAT GTT 720 Val Ser Ser Met Pro Ile Pro Glu Gly Val Ser Glu Ser Asp Tyr Val 225 230 235 240 GGC GCA ATC TTG GGT GAG TCG GTT CCA GTA GTA AAA TGT GAG ACC AAC 768 Gly Ala Ile Leu Gly Glu Ser Val Pro Val Val Lys Cys Glu Thr Asn 245 250 255 GAT TTA ATG GTT CCT GCA ACG AGT GAG ATG GTA TTT GAG GGT ACT TTG 816 Asp Leu Met Val Pro Ala Thr Ser Glu Met Val Phe Glu Gly Thr Leu 260 265 270 TCC TTA ACA GAT ACA CAT CTG GAA GGC CCA TTT GGT GAG ATG CAT GGA 864 Ser Leu Thr Asp Thr His Leu Glu Gly Pro Phe Gly Glu Met His Gly 275 280 285 TAT GTT TTC AAA AGC CAA GGT CAT CCT TGT CCA TTG TAC ACT GTC AAG 912 Tyr Val Phe Lys Ser Gln Gly His Pro Cys Pro Leu Tyr Thr Val Lys 290 295 300 GCT ATG AGT TAC AGA GAC AAT GCT ATT CTA CCT GTT TCG AAC CCC GGT 960 Ala Met Ser Tyr Arg Asp Asn Ala Ile Leu Pro Val Ser Asn Pro G ly 305 310 315 320 CTT TGT ACG GAT GAG ACA CAT ACC TTG ATT GGT TCA CTA GTG GCT ACT 1008 Leu Cys Thr Asp Glu Thr His Thr Leu Ile Gly Ser Leu Val Ala Thr 325 330 335 GAG GCC AAG GAG CTG GCT ATT GAA TCT GGC TTG CCA ATT CTG GAT GCC 1056 Glu Ala Lys Glu Leu Ala Ile Glu Ser Gly Leu Pro Ile Leu Asp Ala 340 345 350 TTT ATG CCT TAT GAG GCT CAG GCT CTT TGG CTT ATC TTA AAG GTG GAT 1104 Phe Met Pro Tyr Glu Ala Gln Ala Leu Trp Leu Ile Leu Lys Val Asp 355 360 365 TTG AAA GGG CTG CAA GCA TTG AAG ACA ACG CCT GAA GAA TTT TGT AAG 1152 Leu Lys Gly Leu Gln Ala Leu Lys Thr Thr Pro Glu Glu Phe Cys Lys AAG GTA GGT GAT ATT TAC TTT AGG ACA AAA GTT GGT TTT ATA GTC CAT 1200 Lys Val Gly Asp Ile Tyr Phe Arg Thr Lys Val Gly Phe Ile Val His 385 390 395 400 GAA ATA ATT TTG GTG GCA GAT GAT ATC GAC ATA TTT AAC TTC AAA GAA 1248 Glu Ile Ile Leu Val Ala Asp Asp Ile Asp Ile Phe Asn Phe Lys Glu 405 410 415 GTC ATC TGG GCC TAC GTT ACA AGA CAT ACA CCT GTT GCA GAT CAG ATG 1296 Val Ile Trp Ala Tyr Val Thr Arg His Thr Pro Val Al a Asp Gln Met 420 425 430 GCT TTT GAT GAT GTC ACT TCT TTT CCT TTG GCT CCC TTT GTT TCG CAG 1344 Ala Phe Asp Asp Val Thr Ser Phe Pro Leu Ala Pro Phe Val Ser Gln 435 440 445 445 TCA TCC AGA AGT AAG ACT ATG AAA GGT GGA AAG TGC GTT ACT AAT TGC 1392 Ser Ser Arg Ser Lys Thr Met Lys Gly Gly Lys Cys Val Thr Asn Cys 450 455 460 ATA TTT AGA CAG CAA TAT GAG CGC AGT TTT GAC TAC ATA ACT TGT AAT 1440 Ile Phe Arg Gln Gln Tyr Glu Arg Ser Phe Asp Tyr Ile Thr Cys Asn 465 470 475 480 TTT GAA AAG GGA TAT CCA AAA GGA TTA GTT GAC AAA GTA AAT GAA AAT 1488 Phe Glu Lys Gly Tyr Pro Lys Gly Leu Val Asp Lys Val Asn Glu Asn 485 490 495 TGG AAA AGG TAC GGA TAT AAA TAA 1512 Trp Lys Arg Tyr Gly Tyr Lys 500

SEQ ID NO: 2 Sequence length: 3930 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Saccharomyces ( Saccharomyces)
cerevisiae ) Strain name: YSA7 Characteristic of sequence Characteristic symbol: CDS Location: 532..2043 Method for determining characteristics: E Sequence characteristic Characteristic symbol: characteristic-site Location: 1..6, 3925. .3930 Other information: BamHI cleavage site Method used to determine the characteristics: S sequence characteristics Symbol indicating characteristics: cleavage-site Location: 656..661 Other information: StuI cleavage site Method used to determine the characteristics: S sequence Features Symbol indicating the feature: cleavage-site Location: 1527..1732 Other information: SpeI cleavage site Method for determining the feature: S sequence feature Symbol indicating the feature: cleavage-site Location: 3555..3600 other Information: SalI cleavage site Characterization method: S-sequence GGATCCTAGA CTGCTTTGGC ATCCACGCTG ACACTTTTCC TCGTTGGGAA GGAATAAAAA 60 GCAAGTAACA CTTTTTCTGA GCATTTTATT ACGTTACTCA ACTACTAATA GAGTTGATTT 120 GTTACTTCGATCGATCGATCTGATC 240 CTTACTCATT TTCAATGTCA GCTTAAATAT CGTCTAAAAC AATATTTTAC TAGATACGCA 300 GTTCAATCTT CGCGCATATT TTCACGAAAG TCCAAATTGC GTACGTAGTT TTATGTCAAA 360 GTGACCGCCG TTGTAGCGTA CTTTTTCCTA TAAGACAAGC TCGTGATATC AGGAATATAT 420 CAGGAATGTA AACGAATACC GCATATCTTT TTGATTTTTT TCCTCTGAGT TATTCTATTC 480 TTGACATTAT TACATCACCA ATTCAAAAGA ATTGTCAATT TATATATTTA AATGAGGAAG 540 CTAAATCCAG CTTTAGAATT TAGAGACTTT ATCCAGGTCT TAAAAGATGA AGATGACTTA 600 ATCGAAATTA CCGAAGAGAT TGATCCAAAT CTCGAAGTAG GTGCAATTAT GAGGAAGGCC 660 TATGAATCCC ACTTACCAGC CCCGTTATTT AAAAATCTCA AAGGTGCTTC GAAGGATCTT 720 TTCAGCATTT TAGGTTGCCC AGCCGGTTTG AGAAGTAAGG AGAAAGGAGA TCATGGTAGA 780 ATTGCCCATC ATCTGGGGCT CGACCCAAAA ACAACTATCA AGGAAATCAT AGATTATTTG 840 CTGGAGTGTA AGGAGAAGGA ACCTCTCCCC CCAATCACTG TTCCTGTGTC ATCTGCACCT 900 TGTAAAACAC ATATACTTTC TGAAGAAAAA ATACATCTAC AAAGCCTGCC AACACCATAT 960 CTACATGTTT CAGACGGTGG CAAGTACTTA CAAACGTACG GAATGTGGAT TCTTCAAACT 1020 CCAGATAAAA AATGGACTAA TTGGTCAATT GCTAGAGGTA TGGTTGTAGA TGACAAGCAT 1080 ATCACTGGTC TGG TAATTAA ACCACAACAT ATTAGACAAA TTGCTGACTC TTGGGCAGCA 1140 ATTGGAAAAG CAAATGAAAT TCCTTTCGCG TTATGTTTTG GCGTTCCCCC AGCAGCTATT 1200 TTAGTTAGTT CCATGCCAAT TCCTGAAGGT GTTTCTGAAT CGGATTATGT TGGCGCAATC 1260 TTGGGTGAGT CGGTTCCAGT AGTAAAATGT GAGACCAACG ATTTAATGGT TCCTGCAACG 1320 AGTGAGATGG TATTTGAGGG TACTTTGTCC TTAACAGATA CACATCTGGA AGGCCCATTT 1380 GGTGAGATGC ATGGATATGT TTTCAAAAGC CAAGGTCATC CTTGTCCATT GTACACTGTC 1440 AAGGCTATGA GTTACAGAGA CAATGCTATT CTACCTGTTT CGAACCCCGG TCTTTGTACG 1500 GATGAGACAC ATACCTTGAT TGGTTCACTA GTGGCTACTG AGGCCAAGGA GCTGGCTATT 1560 GAATCTGGCT TGCCAATTCT GGATGCCTTT ATGCCTTATG AGGCTCAGGC TCTTTGGCTT 1620 ATCTTAAAGG TGGATTTGAA AGGGCTGCAA GCATTGAAGA CAACGCCTGA AGAATTTTGT 1680 AAGAAGGTAG GTGATATTTA CTTTAGGACA AAAGTTGGTT TTATAGTCCA TGAAATAATT 1740 TTGGTGGCAG ATGATATCGA CATATTTAAC TTCAAAGAAG TCATCTGGGC CTACGTTACA 1800 AGACATACAC CTGTTGCAGA TCAGATGGCT TTTGATGATG TCACTTCTTT TCCTTTGGCT 1860 CCCTTTGTTT CGCAGTCATC CAGAAGTAAG ACTATGAAAG GTGGAAAGTG CGTTACTAAT 1920 TGCATATTTA GACAGCAAT A TGAGCGCAGT TTTGACTACA TAACTTGTAA TTTTGAAAAG 1980 GGATATCCAA AAGGATTAGT TGACAAAGTA AATGAAAATT GGAAAAGGTA CGGATATAAA 2040 TAATTGCCAT AGACTTTCTA CGGAAGAAAA ACCATATAAT CAGATTTTAA ATAAAATTTT 2100 CCGAACTTTT ATACTCCACG GTTTTGGAGT TGTTTGATTG CAGTGACAAG CAGTGCGCCA 2160 TTAACACTAT CCATCTTTCG TACAAAGTAA AGATAAAGTT ATTTTCCTGA GGTGAGAACC 2220 GTAAATCTTT ATAGACAAGG AGTATTTATA ACTAAACTAT TACCTTGTTA CTTATGGAAT 2280 TAATCTTGAC TAATAGGCAG ATGATCAACA GTTATTGATT TTGAGTGAAA GTCCATAAAG 2340 TTACAGTATG TAATTACAGT ACGTAATTAA GGAATGTCTG TAAATATATG CTCCTTTTTT 2400 TTTTTCCACT TACTATGATT TTAGTAAAGC ACCATGATGA TGTAGATGCG TAATACTCTA 2460 TAAATGTAAC ATCGTTAAAG CATTGGTTAT TTTAATTTCA TTCTATAAAC CAATATTTCT 2520 GACAGCACAT AAAAAATAAA TGGACTATAT TAACAGCAAA TATCGGTTAA TCTAGGGCAT 2580 AATTATTTAA CATCAAAAAG AAAGTTGCTA GTTGTTCTAG TATTGCTCGG AGTACCTCAA 2640 ACGGTAAAAA GATAATATTT GTTTCCGCTT TTATCATTGA ATAGCTTAGA AATTCTCTCC 2700 CTAGCGACCA TTTAAGGAAT GTAGCTAACA AAAATGATTC AAGTATGTTG CTCCTAAGCA 2760 GATATGTACT CTATAAGTTG AATC ACTATA TCATTGAAAT ATAGTGGCGA GGGCGTACAT 2820 AAAATCAAAG GAACTATGCA ATAGACTCAA TTAAATGCCA CATAGCTATT TAAGACTCCA 2880 AATCTCCAAT ACAATCATTC GTTAAAGATT TTTTGTATTC TGCTGATATC TTTTTCTACA 2940 GTTTCTTGAG TGTCTAGTGA TTGCATAAAA TGACCACAGT ATTTTTAGTA CTCATGGCCT 3000 GTATGCAATT GCAAGGAACG GTATTACTTT TACAAAAACC CTGCTTTCCG GCAAGTTCAG 3060 CTGTCATTTG GTAAGATTTT GAAAATAGTG GAAACAATGG ATTATCAACG AATAGTCTTT 3120 AAACATAAGT GCTAAAATTC AAAACATCAT TTGATGCGTG CGGTGCATTT TTTCGTGCTG 3180 AATGAACTTT TGGAGATATC TGCCTTCGCC AATGAATATG CATTACCATT AATTGCATAG 3240 TAACCGTATA CATGAAAATG GAAATAATGA ACTGATTATT AAAAATAGCT AATGCAGGTG 3300 GGATTTGAGA CATTGTGGTT ATCCTGTCAG CCTGTATTTT GCGCTTTAAG GTATTTCATA 3360 AAAGTTAGAA TAAAATTTAA AAGTTCCATT CTATGAAACT GTAATTATAG GTATATACTA 3420 TCATCTACCA ATCTTACCCA TGTATAGTTC TAATATTAAA GACAGAGTAG GTAAAAAAAA 3480 AAATGGTAAT CAAAACGTGA TCGCTTATAT TCGGTATGGA CAAGCTTTGA ATATTCCCTA 3540 GAAAATGCAA TAGTATGTCA TAATGAAAGA AGATTGTAAT ACAATGCTTG GAATGTCGAC 3600 CGGCAGAAAC CCGTGTCACA TGGCCTTATT CAACGTGACG TTGTGATATA TGTAGAACAT 3660 GCTTTAGATG AGGCGGTATT TGACTGTAGC ATCTTCTAAA ATGTGCTGAT ATTGTTAAAT 3720 CTCAATCAAA CTGAGAGAGT ATGAGAGACT GAAAAAGTGG GATTCTGCCT GTGGTGCTAA 3780 TATCCTTAAA ATGCTAAACT GAAAGAAGTA ATATAATCAT ATATATTGAT CATGTATCAT 3840 ACAAAAGATG CATGTATTTT AGTAATATTA ACTGCTACTA TGATGTAGTA GACGATCGAT 3900 AATCGAATCT TGCGGTATAT TCTAGGATCC 3930

[Brief description of the drawings]

FIG. 1 shows a restriction map of a DNA fragment containing the FDC1 gene, the results of subcloning performed to identify the FDC1 gene, and the FD of the obtained DNA fragment.
It is a figure which shows the presence or absence of C activity.

FIG. 2 is a view showing a step of preparing a plasmid for expressing FDC1 gene.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 7/22 C12P 7/22 7/24 7/24 7/42 7/42 // (C12N 1/19 C12R 1: 865) (C12N 1 / 21 C12R 1:19) (C12N 9/02 C12R 1: 865)

Claims (14)

[Claims] 1. A protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid in the amino acid sequence (a) with deletion, substitution or addition Consisting of the amino acid sequence
And a protein having ferulic acid decarboxylase activity. 2. A gene encoding the protein according to claim 1. 3. A recombinant vector containing the gene according to claim 2. A transformant comprising the recombinant vector according to claim 3. A yeast comprising the recombinant vector according to claim 3. 6. A 4-vinyl formed in an aqueous medium by reacting ferulic acid in an aqueous medium in the presence of an enzyme source derived from the transformant according to claim 4 and having ferulic acid decarboxylase activity. 4-vinyl guaiacol, characterized by collecting guaiacol, vanillin or vanillic acid,
A method for producing vanillin or vanillic acid. 7. The method according to claim 6, wherein the enzyme source is a culture, a cell, or a treated product of the protein according to claim 1 or the transformant according to claim 4. 8. A method for producing a distilled liquor, comprising adding an enzyme source derived from the transformant according to claim 4 and having ferulic acid decarboxylase activity to moromi. 9. The method according to claim 8, wherein the enzyme source is a culture, a cell, or a processed product of the protein according to claim 1 or the transformant according to claim 4. 10. A method for producing a distilled liquor, comprising using a yeast having an increased ferulic acid decarboxylase activity. 11. The method according to claim 10, wherein the yeast having an increased ferulic acid decarboxylase activity is a yeast containing a recombinant vector containing a gene encoding ferulic acid decarboxylase. 12. The method according to claim 10, wherein the yeast having an increased ferulic acid decarboxylase activity is the yeast according to claim 5. 13. A distilled liquor obtained by the method according to claim 8. 14. A distilled liquor obtained by the method according to claim 10.