JP6064183B2 - Reductase gene amplification primer set and reductase gene acquisition method - Google Patents

Description

  The present invention relates to a method for obtaining a nucleic acid encoding a reductase, a nucleic acid encoding a reductase obtained by the method, and the like.

Reductase has the ability to reduce a substrate, and has recently been used in organic synthesis reactions for producing, for example, compounds that are active ingredients of medicines and agricultural chemicals and intermediates thereof. For example, Patent Document 1 discloses a method for producing an optically active alcohol by cloning a nucleic acid encoding a reductase from a microorganism belonging to the genus Leifsonia sp. And using a transformant expressing the reductase. Has been.
Such industrially utilized reductase desirably has high stability with respect to temperature, pH, solvent, pressure and the like. For example, when the stability to an organic solvent is high, shortening of the reaction time and improvement of the reaction efficiency can be expected. Moreover, when the stability with respect to temperature (that is, thermal stability) is high, it becomes possible to increase the reaction temperature, not only increase the reaction rate, but also reduce the deactivation of the reductase during the reaction.

Japanese Patent Laid-Open No. 2004-261042

  In order to find a target reductase from microorganisms by a conventional screening method, it is necessary to culture microorganisms that can be purely cultured and to screen microorganisms that degrade or convert a specific target substrate. Since purely culturable microorganisms are said to be less than 1% of all microorganisms present in the environment such as soil, in the conventional screening method, the screening source of reductase is about 1 of the microorganisms in the environment. Only%. Further, in the conventional screening method described above, it is necessary to culture a large number of different types of microorganisms under conditions suitable for each, which may require considerable labor and time.

The present invention provides a method for efficiently obtaining a nucleic acid encoding a reductase, a nucleic acid encoding a reductase that can be obtained by the method, and the like.
That is, the present invention
1. A method for obtaining a nucleic acid encoding a reductase, comprising:
(1) An oligonucleotide having a base sequence represented by SEQ ID NO: 1 (5'-gaccgggtccgcgatgtgtacmggmgsmg-3 ') and an oligonucleotide having a base sequence represented by SEQ ID NO: 2 (5'-cggcactggggcggtrtabccrcccrtcb-3') are used as primers. Performing a nucleic acid amplification reaction using a DNA sample as a template to obtain an amplification product;
(2) The amplification product and a double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized are fused in the presence of a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity. , A step of obtaining a circular recombinant expression vector containing the amplification product, wherein one of the two strands constituting the double-stranded linear vector DNA is a base sequence represented by SEQ ID NO: 6 at its 5 ′ end (5'-accgcccagtga-3 '), having a base sequence represented by SEQ ID NO: 7 at its 3' end (5'-atggctcagtacgacgtcgccgaccgggtccgcgactgt-3 ') and encoded by the base sequence represented by SEQ ID NO: 7. A nucleotide sequence that enables expression of the amino acid sequence in the host cell, a replication origin that can function in the host cell, And its complementary sequence is single-stranded DNA having a 5 'upstream of the nucleotide sequence represented by SEQ ID NO: 7;
(3) introducing the circular recombinant expression vector into a host cell to obtain a transformant;
(4) a step of measuring the activity of the transformant or its treatment product to reduce a substrate and selecting a transformant having the activity; and (5) from the transformant selected in step (4). Obtaining a nucleic acid encoding the reductase having the activity;
(Hereinafter sometimes referred to as the nucleic acid obtaining method of the present invention);
2. The single-stranded DNA has, at its 3 ′ end, a base sequence represented by SEQ ID NO: 7 (3′-aggagagatacat-3 ′) continuously downstream of the base sequence represented by SEQ ID NO: 8 (5′-aggagatacatat-3 ′). 2. The method according to item 1 above, which is a single-stranded DNA having atggctcagtacgacgtcgccgaccgggtccgcgactcgt-3 ′);
3. 3. The method according to item 1 or 2 above, wherein the single-stranded DNA is a single-stranded DNA having the base sequence represented by SEQ ID NO: 3;
4). 4. The method according to any one of items 1 to 3, wherein the DNA sample is environmental DNA derived from an environmental sample;
5. 5. The method according to any one of items 1 to 4 above, wherein the activity of reducing the substrate is an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol;
6). 6. The method according to any one of 1 to 5 above, wherein the host cell is Escherichia coli;
7). A primer set consisting of an oligonucleotide having the base sequence represented by SEQ ID NO: 1 (5′-gaccgggtccgcgactgvaccmmgmgsmg-3 ′) and an oligonucleotide having the base sequence represented by SEQ ID NO: 2 (5′-cgtcactggggcggtrtabccrcccrtcb-3 ′) May be referred to as the primer set of the present invention.);
8). A double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized, and one of the two strands constituting the DNA has a nucleotide sequence (5) An amino acid encoded by the base sequence represented by SEQ ID NO: 7 and having the base sequence represented by SEQ ID NO: 7 at its 3 'end (5'-atggctcagtacgacgtcgccgaccgggtccgcgactgt-3') Two single-stranded DNAs that have a base sequence that allows expression of the sequence in a host cell and a replication origin that can function in the host cell or its complementary sequence 5 ′ upstream of the base sequence represented by SEQ ID NO: 7 Chain linear vector DNA;
9. A protein selected from the following group:
(A) a protein having an amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28;
(B) In the amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, one or several amino acids are deleted, inserted, substituted or added. A protein having an amino acid sequence and having an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol; and (c) SEQ ID NOs: 10, 12, 14, It has an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of 16, 18, 20, 22, 24, 26, or 28, and 2,2,2-trifluoroacetophenone is 2,2 , A protein having activity of reducing to 2-trifluoro-1-phenylethanol;
10. A nucleic acid encoding the protein according to item 9;
11. The nucleic acid according to item 10 above, which has a base sequence represented by any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27;
12 A recombinant vector containing the nucleic acid according to item 10 or 11;
13. 13. The recombinant vector according to item 12, wherein a promoter capable of functioning in a host cell is operably linked upstream of the nucleic acid according to item 10 or 11 above;
14 A method for producing a vector, wherein the nucleic acid according to item 10 or 11 is incorporated into a vector capable of replicating in a host cell;
15. A transformant obtained by introducing the nucleic acid according to the above item 10 or 11 or the vector according to the above item 12 or 13 into a host cell (hereinafter also referred to as the transformant of the present invention);
16. 16. The transformant according to 15 above, wherein the host cell is E. coli; A method for producing a transformant, which comprises introducing the nucleic acid according to item 10 or 11 or the vector according to item 12 or 13 into a host cell;
Etc.

  The present invention makes it possible to provide an efficient method for obtaining a nucleic acid encoding a reductase. For example, it is obtained from an environmental DNA derived from a microorganism present in the environment, and is excellent in stability to organic solvents and thermal stability. Nucleic acids encoding reductases can be provided.

Hereinafter, the present invention will be described in detail.

The primer set of the present invention will be described.
The primer set of the present invention comprises an oligonucleotide 1 having the base sequence represented by SEQ ID NO: 1 and an oligonucleotide 2 having the base sequence represented by SEQ ID NO: 2.
Sequence number 1: 5'-GACCGGTCCGCGATCGTVACMGGGMGSMMG-3 '
SEQ ID NO: 2: 5′-CGGTCACTGGGCGGTRTABCCRCCRTCB-3 ′
Here, R represents adenine or guanine, M represents adenine or cytosine, S represents cytosine or guanine, V represents adenine or cytosine or guanine, and B represents cytosine, guanine or thymine.

  Each of the oligonucleotide 1 and the oligonucleotide 2 is preferably a mixture (mixed oligonucleotide, degenerate oligonucleotide) composed of all combinations of oligonucleotides having the base sequence represented by SEQ ID NO: 1 or 2.

  Examples of the oligonucleotide 1 include an oligonucleotide having a base sequence represented by SEQ ID NO: 1, and examples of the oligonucleotide 2 include an oligonucleotide having a base sequence represented by SEQ ID NO: 2. Further, the oligonucleotide 1 and the oligonucleotide 2 may be oligonucleotides in which a base sequence including a restriction enzyme site or the like is added to the 5 ′ end side of the base sequence represented by SEQ ID NO: 1 or 2. As the restriction enzyme site (restriction enzyme recognition sequence), a sequence known to those skilled in the art can be appropriately selected. Further, several bases may be further included on the 5 'end side of the restriction enzyme site. In general, the number of bases added to the 5 ′ end of the restriction enzyme site is 3 or more bases so that the restriction enzyme site can be efficiently cleaved. The number of bases that can be efficiently cleaved can be selected. As a base sequence added so as to include a cleavage sequence by a restriction enzyme, it is desirable to select a sequence that is specific for the reductase gene and has little homology with other enzyme genes. Moreover, it is preferable that a terminal sequence is added within the range which maintains the hybridization ability with respect to a reductase gene so that each primer of a primer set may not mutually hybridize. The oligonucleotide 1 and the oligonucleotide 2 are single-stranded oligonucleotides having a sequence length of 15 to 50 bases. The oligonucleotide 1 and the oligonucleotide 2 can be used as a mix primer or a degenerate primer.

Specifically, as the primer set of the present invention,
A primer set consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 can be mentioned.

As the primer set of the present invention,
An oligonucleotide having a base sequence containing a restriction enzyme site added to the 5 ′ end side of the base sequence shown in SEQ ID NO: 1 and a restriction enzyme site on the 5 ′ end side of the base sequence shown in SEQ ID NO: 2 A primer set consisting of an oligonucleotide to which a base sequence is added can also be mentioned.

  According to the oligonucleotide and primer set of the present invention, a nucleic acid encoding the amino acid sequence of a reductase can be efficiently obtained. For example, a nucleic acid that encodes the amino acid sequence of a reductase derived from a microorganism that exists in the environment and is difficult to isolate and culture can be efficiently obtained. When a nucleotide sequence containing a restriction enzyme site is added to the 5 ′ end of each oligonucleotide of the primer set of the present invention, it is convenient for cloning of the obtained nucleic acid or construction of a recombinant vector containing a nucleic acid encoding a reductase. It is.

The nucleic acid acquisition method of the present invention will be described.
The nucleic acid obtaining method of the present invention is a method for obtaining a nucleic acid encoding a reductase,
(1) An oligonucleotide having a base sequence represented by SEQ ID NO: 1 (5'-gaccgggtccgcgatgtgtacmgggmgsmg-3 ') and an oligonucleotide having a base sequence represented by SEQ ID NO: 2 (5'-cggcactggggcggtrtabccrcccrtcb-3') are used as primers. Performing a nucleic acid amplification reaction using a DNA sample as a template to obtain an amplification product;
(2) The amplification product and a double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized are fused in the presence of a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity. Obtaining a circular recombinant expression vector containing the amplification product,
Here, one of the two strands constituting the double-stranded linear vector DNA has the base sequence (5′-accgccccgtga-3 ′) represented by SEQ ID NO: 6 at its 5 ′ end, and the 3 ′ A base sequence having a base sequence represented by SEQ ID NO: 7 at its terminal (5'-atggctcagtacgacgtcgccgaccgggtccgcgactcgt-3 ') and an amino acid sequence encoded by the base sequence represented by SEQ ID NO: 7 can be expressed in a host cell; A single-stranded DNA having a replication origin that can function in a cell or its complementary sequence 5 ′ upstream of the base sequence represented by SEQ ID NO: 7;
(3) introducing the circular recombinant expression vector into a host cell to obtain a transformant;
(4) a step of measuring the activity of the transformant or its treatment product to reduce a substrate and selecting a transformant having the activity; and (5) from the transformant selected in step (4). Obtaining a nucleic acid encoding the reductase having the activity;
It is a method including.

  The step (1) is a step of performing a nucleic acid amplification reaction using the primer set of the present invention and a DNA sample as a template to obtain an amplification product. Examples of the DNA sample include environmental DNA derived from environmental samples.

  In the present invention, the “environment” refers to the living environment of microorganisms such as soil, compost, sludge, sewage, rivers, lakes, seawater, seabed, insects, animals and plants solid tissues and surfaces, and excreta. means. The “environmental sample” is a sample collected from the environment, and specifically includes a sample collected from soil, compost or the like. “Environmental DNA” means DNA obtained from an environmental sample. As a method for extracting environmental DNA, for example, 5 g of a soil sample is treated with 100 mM Tris-HCl (pH 8), 100 mM sodium EDTA (pH 8), 100 mM sodium phosphate (pH 8), 1.5 M NaCl, and 1% hexadethyryl ammonium bromide (CTAB). Suspended in a solution containing 13.5 ml of extraction solution containing 100 μl of proteinase K (10 mg / ml), shaken at 37 ° C. for 30 minutes, and then added with 1.5 ml of 20% SDS aqueous solution, at 65 ° C. for 2 hours. After extraction, a method for obtaining DNA from a supernatant obtained by treating with chloroform / isoamyl alcohol (Appl. Environ. Microbiol., 62 (2): 316, 1996) or a soil DNA extraction kit (ISOIL for Be ds Beating, include a method of preparing according to the manual using a Nippon Gene), may use any means if it is possible to extract the DNA in microorganisms present in environmental samples.

  As the nucleic acid amplification reaction, a method known to those skilled in the art can be used, and examples thereof include a polymerase chain reaction (PCR). For example, PCR is performed using an environmental DNA as a template and a primer set consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 to obtain an amplification product. As PCR conditions, for example, 200 μM each of 4 types of dNTPs, 80 pmol of each of 2 types of oligonucleotide primers, 0.5 U of Taq polymerase, and a DNA sample serving as a template were incubated at 94 ° C. for 2 minutes. Then, the temperature is kept at 94 ° C. for 0.5 minutes, then at 69 ° C. for 1 minute, then at 72 ° C. for 1 minute, 40 times, and further at 72 ° C. for 10 minutes. Examples of a method for confirming that an amplification product has been obtained include agarose gel electrophoresis.

  The amplification product obtained in the step (1) can be expected to contain a nucleic acid encoding the amino acid sequence of the desired reductase. Isolation and identification of the nucleic acid encoding the amino acid sequence of such a reductase can be performed as follows. First, the amplification product is fused with a double-stranded linear vector DNA that can function as an expression vector in a host cell when it is circularized to construct a recombinant expression vector (step (2)). Next, the obtained recombinant expression vector is introduced into a host cell to produce a transformant (step (3)). The activity of reducing the substrate is measured using the obtained transformant or its treated product, and a transformant having the activity is selected (step (4)). From the selected transformant, a nucleic acid encoding the reductase having the activity is obtained (step (5)).

Below, process (2)-(5) is demonstrated.
In the step (2), the amplification product and a double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized are obtained in the presence of a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity. In order to obtain a circular recombinant expression vector containing the amplification product.

The above “double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized” used in the nucleic acid obtaining method of the present invention is a double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized. One of the two strands constituting the DNA has the base sequence represented by SEQ ID NO: 6 (5'-accgccccgtga-3 ') at its 5' end, and SEQ ID NO: 7 at its 3 'end. And a base sequence capable of expressing in the host cell the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 7, which has the base sequence represented by (5'-atggctcagtacgacgtcgccgaccgggtccgcgactgt-3 ') An origin of replication or its complementary sequence is located 5 ′ upstream of the base sequence shown in SEQ ID NO: 7. Two chain linear vector DNA is a single-stranded DNA that is a (hereinafter, the present invention is sometimes referred to as a two-chain linear vector DNA.).
The nucleotide sequence represented by SEQ ID NO: 6 (5′-accgccccaggtga-3 ′) and the nucleotide sequence represented by SEQ ID NO: 7 (5′-atggctcagtacgagtcgccgaccggtccgccatcgt-3 ′) are each of the primer set of the present invention used in step (1). It contains a sequence that overlaps the base sequence of the oligonucleotide. Therefore, the amplification product obtained in step (1) and the double-stranded linear vector DNA of the present invention have the same base sequence at each end. Incubating such an amplification product and the double-stranded linear vector DNA of the present invention in the presence of a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity under conditions that allow the DNA polymerase to exhibit activity, By the 3′-5 ′ exonuclease activity of the DNA polymerase, nucleotides are removed from the amplification product and the 3 ′ end of the double-stranded linear vector DNA of the present invention. As a result, single-stranded regions complementary to each other are generated on the amplification product and the double-stranded linear vector DNA of the present invention, and the amplification product and the two double-stranded DNAs of the present invention are formed by base pairing between these complementary single-stranded regions. The strand linear vector DNA anneals. As a result, a circular recombinant expression vector containing the amplification product is obtained.
Examples of such a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity used in a reaction for fusing a homologous base sequence existing at the end of the vector DNA and the inserted DNA include poxvirus DNA polymerase, etc. A commercially available kit (for example, In Fusion HD Cloning Kit (manufactured by Clontech)) or the like can also be used.

In the double-stranded linear vector DNA of the present invention, the “base sequence enabling expression of the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 7 in the host cell” refers to the base sequence represented by SEQ ID NO: 7 as the host cell. And a ribosome binding sequence that enables the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 7 to be translated in a host cell.
Examples of promoters that can function in E. coli include lac promoter, trc promoter, and tac promoter.
Examples of ribosome binding sequences that can function in prokaryotes such as E. coli include SD sequences. In order to improve the expression level, the distance between the SD sequence and the start codon, or the distance between the promoter and the start codon can be devised. For example, the distance between the SD sequence and the start codon may be 5 to 13 nucleotides. Examples of the base sequence from the SD sequence to the start codon include the base sequence represented by SEQ ID NO: 8 (5′-aggagatacat-3 ′).
Examples of the origin of replication that can function in the host cell include the origin of replication of ordinary vectors that can be used in the host cell. For example, replication origins that can function in E. coli include pUC119 (Takara Bio), pTV118N (Takara Bio), pBluescript II (Toyobo), pCR2.1-TOPO (Invitrogen), pTrc99A (GE). The origin of replication contained in commercially available vectors, such as Healthcare Co., Ltd. and pKK223-3 (GE Healthcare Co., Ltd.), can be mentioned.
Of the two strands constituting the double-stranded linear vector DNA of the present invention, in the single-stranded DNA having the base sequence represented by SEQ ID NO: 7 at the 3 ′ end, the above-mentioned “base sequence represented by SEQ ID NO: 7” The nucleotide sequence enabling the expression of the amino acid sequence encoded by the host cell in the host cell and the replication origin that can function in the host cell or its complementary sequence are 5 ′ upstream of the nucleotide sequence represented by SEQ ID NO: 7, respectively. It exists in a functionable position.
Specifically, as the double-stranded linear vector DNA of the present invention, one of two strands constituting the double-stranded linear vector DNA has a base sequence represented by SEQ ID NO: 8 at its 3 ′ end (5 A double-stranded linear vector DNA which is a single-stranded DNA having the base sequence (5′-atggctcagtacgacgtcgccgaccgggtccgcgactgt-3 ′) continuously represented 3 ′ downstream of “-aggagatacatat-3”) it can. That is, the single-stranded DNA has a base sequence represented by SEQ ID NO: 29 (5′-aggagataatacatgggctcagtacgacgtcgccgaccgggtccgcgactgt-3 ′) at its 3 ′ end.
As the double-stranded linear vector DNA of the present invention, more specifically, one of the two strands constituting the double-stranded linear vector DNA is a single-stranded DNA having the base sequence represented by SEQ ID NO: 3. A double-stranded linear vector DNA can be mentioned.
The double-stranded linear vector DNA of the present invention can be prepared by the method described in Examples described later.

  The circular recombinant expression vector obtained in step (2) includes, for example, the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 7 and the desired reductase encoded by the amplification product obtained in step (1). A reductase obtained by fusing with an amino acid sequence can be expressed in a host cell.

In step (3), the circular recombinant expression vector obtained in step (2) (hereinafter sometimes referred to as the present circular recombinant expression vector) is introduced into a host cell to produce a transformant. It is a process.
Examples of the host cell include microorganisms belonging to the genus Escherichia, the genus Bacillus, and the genus Corynebacterium. The host cell is preferably Escherichia coli. E. coli is preferably a K-12-derived strain, specifically, JM109 strain, BL21 (DE3) strain, BL21 strain, XL1 Blue MRF 'strain, DH5α strain, HB101 strain, DH1 strain, MV1184 strain, JM105 strain, MC1061 Stock, C600 stock, etc. are exemplified.

  The method for introducing the circular recombinant expression vector of the present invention into a host cell may be any method commonly used depending on the host cell used. For example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Molecular Biology” (1987), John Wiley & Sons, Inc. The calcium chloride method described in ISBNO-471-50338-X and the like, and the electroporation method described in “METHODS in Electroporation: Gene Pulser / E. Coli Pulser System”, Bio-Rad Laboratories, (1993), etc. Can be mentioned.

  A transformant introduced with the circular recombinant expression vector of the present invention can be selected using, for example, the phenotype of the selection marker gene contained in the vector as described above as an index.

The step (4) is a step in which the transformant obtained in step (3) or a processed product thereof measures the activity of reducing the substrate, and a transformant having the activity is selected.
The fact that the transformant has a nucleic acid encoding a reductase is, for example, in accordance with a normal method described in “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, etc. It can also be confirmed by analyzing the base sequence, Southern hybridization and the like.

  As a medium for culturing the transformant, for example, various media appropriately containing a carbon source, a nitrogen source, an organic salt, an inorganic salt, or the like that are usually used for culturing host cells such as microorganisms can be used.

  Examples of the carbon source include sugars such as glucose, dextrin and sucrose, sugar alcohols such as glycerol, organic acids such as fumaric acid, citric acid and pyruvic acid, animal oils, vegetable oils and molasses. The amount of these carbon sources added to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.

  Examples of the nitrogen source include meat extract, peptone, yeast extract, malt extract, soy flour, corn steep liquor, cottonseed flour, dry yeast, casamino acid and other natural organic nitrogen sources, amino acids And sodium salts of inorganic acids such as sodium nitrate, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, and urea. Of these, ammonium salts of organic acids, natural organic nitrogen sources, amino acids and the like can be used as carbon sources in many cases. The amount of these nitrogen sources added to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.

  Examples of organic salts and inorganic salts include chlorides such as potassium, sodium, magnesium, iron, manganese, cobalt, and zinc, sulfates, acetates, carbonates, and phosphates. Specifically, for example, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, monopotassium hydrogen phosphate and dipotassium hydrogen phosphate Is mentioned. The amount of these organic salts and / or inorganic salts added to the medium is usually about 0.0001 to 5% (w / v) with respect to the culture solution.

  Furthermore, a transformant in which the circular recombinant expression vector of the present invention in which a lac promoter, trc promoter, tac promoter, etc. of a type induced by allolactose and a reductase-encoding nucleic acid are functionally connected is introduced In this case, as an inducer for inducing the production of reductase, for example, isopropyl thio-β-D-galactoside (IPTG) can be added in a small amount to the medium.

  The transformant carrying the circular recombinant expression vector of the present invention can be cultured according to a method usually used for culturing host cells such as microorganisms. For example, test tube shaking culture, reciprocating shaking culture , Jar Fermenter culture, liquid culture such as tank culture, and solid culture.

  The culture temperature can be appropriately changed within the range in which the transformant can grow, but is usually about 15 to about 40 ° C. The pH of the medium is preferably in the range of about 6 to about 8. Although the culture time varies depending on the culture conditions, it is usually preferably about 1 day to about 5 days.

  Examples of the transformant used for detecting the activity or a processed product thereof include, for example, a culture of the transformant, a live transformant, a crushed material, a cell-free extract, a crude protein, a purified protein, and these An immobilization thing is mentioned. Here, the processed product of the transformant is, for example, a freeze-dried transformant, an organic solvent-treated transformant, a dried transformant, a transformant grind, an autolysate of the transformant, or a transformant. Examples include ultrasonically treated products, transformant extracts, and alkaline processed products of transformants. Examples of a method for obtaining an immobilized product include a carrier binding method (a method of adsorbing a reductase or the like on an inorganic carrier such as silica gel or ceramic, cellulose, an ion exchange resin, etc.) and a comprehensive method (polyacrylamide, sulfur-containing polysaccharide gel). (For example, a method of confining a reductase or the like in a polymer network such as carrageenan gel, alginic acid gel, or agar gel).

  The activity of reducing the substrate can be detected by the following method. For example, the above transformant or its treated product is mixed with a substrate and NADH, and kept at 30 ° C., and the reducing activity is measured by quantifying the amount of NADH consumed using the absorbance at 340 nm of the reaction solution as an index. . As substrates, acetophenone, 2,2,2-trifluoroacetophenone, 3′-chloroacetophenone, n-valeraldehyde, ethyl 4-chloro-3-oxobutyrate, 4′-chloroacetophenone, 3-methyl-2-oxo Examples include butyric acid ethyl ester, 2-heptanone, 4-hydroxy-2-butanone, n-heptylaldehyde, ethyl benzoylformate, propionaldehyde, 1-phenyl-2-butanone, n-butyraldehyde, ethyl 3-oxobutyrate, and the like. . For example, a transformant having a reducing activity is selected using as an index the ability to reduce 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol.

Step (5) is a step of obtaining a nucleic acid encoding a reductase having the activity from the transformant selected in step (4).
The base sequence of the nucleic acid encoding the reductase is F.I. Sanger er al. , Proc. Natl. Acad. Sci. U. S. A. (1977) 74: 5463-5467, etc. and can be analyzed by the dideoxy terminator method. For sample preparation for base sequence analysis, for example, a commercially available reagent such as ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit from Life Technologies may be used.

  By using the nucleic acid obtaining method of the present invention, a nucleic acid encoding a reductase can be efficiently obtained. In addition, the present invention can provide a nucleic acid encoding a reductase obtained by the nucleic acid obtaining method of the present invention, a reductase, a recombinant vector containing a nucleic acid encoding a reductase, a transformant containing the same, and the like. .

  The reductase, the nucleic acid encoding the reductase, the recombinant vector containing the nucleic acid encoding the reductase, and the transformant containing the same will be described.

The reductase of the present invention is a protein selected from the following group:
(A) a protein having an amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28;
(B) In the amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, one or several amino acids are deleted, inserted, substituted or added. A protein having an amino acid sequence and having an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol; and (c) SEQ ID NOs: 10, 12, 14, It has an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of 16, 18, 20, 22, 24, 26, or 28, and 2,2,2-trifluoroacetophenone is 2,2 , A protein having activity of reducing to 2-trifluoro-1-phenylethanol;
It is.

In the amino acid sequence of the protein shown in the above (b) and (c), the difference that may be observed between the amino acid sequence of the protein shown in (a) is the deletion, insertion of some amino acids, Substitution, addition, etc. These include, for example, deletion due to processing that the protein shown in (a) above undergoes in the cell. In addition, gene mutations that occur naturally due to species differences or individual differences in the organism from which the protein is derived, or gene mutations that are artificially introduced by site-specific mutagenesis, random mutagenesis, mutation treatment, etc. Amino acid deletions, substitutions, additions and the like are included.
The number of amino acids that undergo such deletions, insertions, substitutions, additions, etc. exerts the activity of the protein to reduce 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol Any number within the possible range is acceptable. Examples of the “several amino acids” in the above (b) include 2, 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids.
Examples of amino acid substitutions include conservative substitutions with similar amino acids such as hydrophobicity, charge, pK, and steric features. Specific examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; ) Lysine, arginine; (6) substitution within groups such as phenylalanine, tyrosine and the like.
As a technique for artificially performing such amino acid deletion, insertion, addition or substitution (hereinafter sometimes referred to as amino acid modification as a whole), for example, the amino acid sequence of the protein shown in (a) above is encoded. An example is a technique in which site-directed mutagenesis is performed on a polynucleotide, and then the polynucleotide is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method utilizing amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc.
As a technique for artificially modifying amino acids, for example, mutation is randomly introduced into a polynucleotide encoding the amino acid sequence of the protein shown in (a) above, and then this polynucleotide is expressed by a conventional method. A method is also mentioned. Here, as a method for randomly introducing mutations, for example, a polynucleotide encoding one of the above amino acid sequences is used as a template, and a primer pair capable of amplifying the full length of each polynucleotide is used, and dATP used as a substrate is used. , DTTP, dGTP, dCTP, the method of performing PCR under the reaction conditions in which the addition concentrations of each are changed from normal, or the reaction conditions in which the concentration of Mg ²⁺ that promotes the polymerase reaction is increased than usual. It is done. Examples of such a PCR method include the method described in Method in Molecular Biology, (31), 1994, 97-112. Moreover, the method described in WO0009682 can also be mentioned.

“Sequence identity” refers to identity between two base sequences or two amino acid sequences. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, in the optimal alignment of the base sequence or amino acid sequence to be compared, addition or deletion (for example, a gap or the like) may be allowed. Such sequence identity can be determined, for example, by FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403- 410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] and the like. The above program is, for example, the DNA Data Bank of Japan [International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)]. It is generally available on the website of Sequence identity can also be determined using commercially available sequence analysis software. Specifically, for example, GENETYX-WIN Ver.5 (manufactured by Software Development Co., Ltd.) is used, and the homology is obtained by the Lipman-Pearson method [Lipman, DJ and Pearson, WR, Science, 227, 1435-1441, (1985)] It can be calculated by creating an alignment by performing sex analysis.
Examples of “90% or more sequence identity” described in (c) above include 90, 95, 98, or 99% or more sequence identity.

The nucleic acid encoding the reductase of the present invention is a nucleic acid encoding the protein shown in the above (a) to (c) (hereinafter sometimes referred to as the present nucleic acid).
Specifically, the nucleic acid encoding the reductase of the present invention is a nucleic acid having a base sequence represented by any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Can be mentioned.

The nucleic acid encoding the reductase of the present invention is described, for example, from environmental DNA derived from environmental samples in Sambrook, J. et al., Molecular Cloning: A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc. Can be obtained according to genetic engineering methods.
For example, an environmental DNA library is obtained by ligating environmental DNA derived from an environmental sample using a vector such as λgt10 and ligase.
From the obtained environmental DNA library, a partial base sequence of the base sequence represented by any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 or complementary to the partial base sequence PCR reaction using an oligonucleotide having a simple base sequence as a primer, or the base shown by any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 from the environmental DNA library A nucleic acid encoding the reductase of the present invention can be obtained by a hybridization method using a polynucleotide having a base sequence complementary to the sequence or a polynucleotide having a partial base sequence of the base sequence as a probe.
As a primer used for PCR, for example, an oligonucleotide having a length of about 15 bases to about 50 bases, comprising SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 An oligonucleotide having a nucleotide sequence starting from the translation start point on the 5 ′ side of the nucleotide sequence represented by any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 And an oligonucleotide having a base sequence complementary to the base sequence starting from the translation end point on the 3 ′ side of the base sequence shown in FIG. PCR is performed, for example, using KOD-Plus-Neo (manufactured by Toyobo Co., Ltd.) at 95 ° C. for 30 seconds, then at 60 ° C. for 30 seconds, and then at 68 ° C. for 2 minutes for 1 cycle. The DNA fragment amplified by such PCR can be purified and collected by ethanol precipitation after being subjected to purification by low melting point agarose electrophoresis or the like, if necessary.
As a probe used for the hybridization method, for example, it has a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Examples include a polynucleotide or a polynucleotide having a partial base sequence of the polynucleotide. As hybridization conditions, for example, 6 × SSC (0.9 M sodium chloride, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% (w / v BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA, incubated at 65 ° C., then 1 × SSC (0.15 M sodium chloride, 0.015 M citric acid (Sodium) and 0.5% (w / v) SDS in the presence of SDS for 15 minutes at room temperature, then 0.1 × SSC (0.015M sodium chloride, 0.0015M sodium citrate) and 0.5% (w / v) The conditions etc. which hold | maintain for 30 minutes at 68 degreeC in SDS presence can be mentioned. For example, incubate at 65 ° C. in the presence of 5 × SSC, 50 mM HEPES pH 7.0, 10 × Denhardt's solution and 20 μg / ml denatured salmon sperm DNA, and then incubate at 2 × SSC for 30 minutes at room temperature. In addition, there may be mentioned conditions in which the incubation is performed twice at 65 ° C. for 40 minutes in 0.1 × SSC.

  The nucleic acid encoding the reductase of the present invention is, for example, based on the base sequence represented by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, for example, the phosphite triester method. (Hunkapiller, M. et al., Nature, 310, 105, 1984) and the like, and can also be prepared by chemically synthesizing a nucleic acid having the target base sequence.

The nucleic acid encoding the reductase of the present invention is a cell engineering separate volume, Plant Cell Engineering Series 7 “Plant PCR Experiment Protocol”, pages 95-100, Shimamoto, Takuji Sasaki, Shujunsha, July 1, 1997 It can also be synthesized with reference to the method described in the publication. In this method, DNA is synthesized using only long synthetic oligonucleotide primers. The primer pair is synthesized so as to have a complementary strand or overlap of about 10 bp to about 12 bp at the 3 ′ end of each primer, and DNA synthesis is performed using each primer as a template. The total length of the primer is about 60 mer to about 100 mer, preferably about 80 mer to about 100 mer.
Specifically, first, based on the designed base sequence, a DNA oligomer is designed and synthesized, for example, every about 90 bases. The synthesis of the DNA oligomer can be performed using a DNA synthesizer by the β-cyanoethyl phosphoramidide method.
A first DNA oligomer is designed and synthesized using a sequence from the vicinity of the center of the designed base sequence to about 90 residues upstream of the 5 ′ side. Next, a complementary strand oligomer having a length of about 90 residues is synthesized from the portion 3 ′ downstream of the polynucleotide, including the sequence of 12 residues on the 3 ′ side of the first DNA oligomer. DNA oligomer. In addition, it comprises 12 residues on the 5 ′ side of the first DNA oligomer, and a complementary strand oligomer having a length of about 90 residues is synthesized 5 ′ upstream of the polynucleotide from this portion, and this is synthesized into the third DNA oligomer. And Furthermore, it contains a 12-residue sequence on the 5 ′ side (3 ′ side when viewed from the gene side) of the second DNA oligomer, and the complementary strand of the second DNA oligomer is placed 3 ′ downstream of the polynucleotide from this part. This is synthesized and used as a fourth DNA oligomer. Similarly, the fifth, sixth and DNA oligomers are synthesized, and further oligomers are synthesized until the entire region can be covered.
Next, these oligomers are sequentially bound by a PCR reaction. First, a PCR reaction is performed using both the first DNA oligomer and the second DNA oligomer as primers. Next, PCR reaction is performed using the PCR product as a template and both the third DNA oligomer and the fourth DNA oligomer as primers. For example, the PCR reaction is performed by 5 cycles of a denaturation temperature of 94 ° C. for 1 minute, an annealing temperature of 51 ° C. for 1 minute, and an extension temperature of 72 ° C. for 2 minutes, followed by a denaturation temperature of 94 ° C. for 1 minute and an annealing temperature of 60 ° C. for 1 minute. The reaction is carried out for 20 cycles with one set of extension temperature 72 ° C. for 2 minutes. The DNA polymerase used in the reaction is preferably an enzyme with a low base incorporation error rate. Thereafter, this operation is repeated to extend the base sequence to obtain the target base sequence. Restriction enzyme sites are provided at both ends of the target base sequence, if necessary, introduced into a cloning vector according to a conventional method, and subcloned or a plasmid is constructed by self-ligation. The base sequence of the obtained polynucleotide is confirmed with a DNA sequencer to confirm that the target base sequence has been obtained.

  The base sequence of the nucleic acid encoding the reductase of the present invention is the dideoxy described in F. Sanger, S. Nicklen, ARCoulson, Proceeding of Natural Academy of Science USA (1977) 74: 5463-5467, etc. It can be analyzed by a terminator method or the like. For sample preparation for base sequence analysis, for example, a commercially available reagent such as ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit manufactured by Life Technologies may be used.

The nucleic acid obtained as described above encodes an amino acid sequence of a protein having an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol. The confirmation can be performed as follows, for example.
First, as described later, the nucleic acid obtained as described above is inserted into a vector so as to be connected downstream of a promoter that can function in the host cell, and this vector is introduced into the host cell to obtain a transformant. . Subsequently, the culture of the transformant was mixed with 2,2,2-trifluoroacetophenone and NADH, incubated at 30 ° C., and reduced by quantifying the amount of NADH consumed using the absorbance at 340 nm of the reaction solution as an index. Measure activity. In this way, it can be confirmed that the obtained nucleic acid encodes an amino acid sequence of a protein having such activity.

  The recombinant vector of the present invention contains the nucleic acid of the present invention or a nucleic acid in which a promoter capable of functioning in a host cell and the nucleic acid of the present invention are operably linked.

  The recombinant vector of the present invention is prepared by preparing a nucleic acid or the like in which the nucleic acid of the present invention or a promoter capable of functioning in the host cell and the nucleic acid of the present invention are operably linked to the host cell into which the nucleic acid of the present invention is introduced. For example, vectors containing genetic information that can be replicated in a host cell, can be propagated autonomously, can be isolated and purified from the host cell, and have a detectable marker (hereinafter, basic In some cases, it may be described as a vector).

  Here, examples of the “basic vector” include a vector pUC119 (manufactured by Takara Bio Inc.), a phagemid pBluescript II (manufactured by Stratagene) and the like when Escherichia coli is used as a host cell. Moreover, when using budding yeast as a host cell, vectors pGBT9, pGAD424, pACT2 (manufactured by Clontech) and the like can be exemplified. When mammalian cells are used as host cells, for example, vectors such as pRc / RSV and pRc / CMV (manufactured by Invitrogen), bovine papilloma virus vector pBPV (manufactured by GE Healthcare), EB virus vector pCEP4 ( Invitrogen) and other virus-derived vectors containing autonomous origins of replication, viruses such as vaccinia virus, and the like. In addition, when insect animal cells are used as host cells, for example, insect viruses such as baculovirus can be mentioned.

  When the recombinant vector of the present invention is constructed using a vector containing an autonomous replication origin (specifically, for example, the yeast vector pACT2, the bovine papilloma virus vector pBPV, the EB virus vector pCEP4, etc.), the vector is transferred to the host cell. When introduced, it is retained in the cell as an episome.

  In order to express the nucleic acid of the present invention in a host cell, for example, a nucleic acid in which a promoter that can function in the host cell and the nucleic acid of the present invention are connected in a functional manner may be introduced into the host cell.

  Here, “in a functional form” means that the promoter and the nucleic acid of the present invention are connected so that the nucleic acid of the present invention is expressed under the control of the promoter in the host cell into which the nucleic acid of the present invention is introduced. Means. Examples of a promoter that can function in a host cell include DNA that exhibits promoter activity in the host cell into which it is introduced. For example, when the host cell is E. coli, the lactose operon promoter (lacP), tryptophan operon promoter (trpP), arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T7 Examples include promoters, T3 promoters, λ phage promoters (λ-pL, λ-pR), and the like. When the host cell is an animal cell or fission yeast, for example, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) early or late promoter, mouse papilloma virus ( MMTV) promoter and the like. Moreover, when a host cell is budding yeast, ADH1 promoter etc. can be mentioned, for example.

  In addition, when using a basic vector having a promoter that functions in a host cell in advance, the nucleic acid of the present invention is inserted downstream of the above promoter so that the promoter and the nucleic acid of the present invention are connected in a functional manner. do it. For example, in the case of pRc / RSV, pRc / CMV, etc., a cloning site is provided downstream of a promoter that can function in animal cells. By introducing a vector obtained by inserting the nucleic acid of the present invention into the cloning site into an animal cell, the nucleic acid of the present invention can be expressed in the animal cell. Since these vectors have SV40 autonomous replication origin (ori) incorporated in advance, when the vector is introduced into cultured cells transformed with the SV40 genome lacking ori, such as COS cells, As a result, the copy number of the vector is greatly increased, and as a result, the nucleic acid of the present invention incorporated in the vector can be expressed in a large amount. The yeast vector pACT2 has an ADH1 promoter, and if the nucleic acid of the present invention is inserted downstream of the ADH1 promoter of the vector or its derivative, the nucleic acid of the present invention can be converted into, for example, CG1945 (manufactured by Clontech). Vectors that can be expressed in large quantities in budding yeast can be constructed.

  The transformant of the present invention is prepared by introducing the nucleic acid of the present invention or the recombinant vector of the present invention into a host cell. Next, by culturing the produced transformant of the present invention according to a normal cell culture method, the reductase of the present invention can be produced and obtained in large quantities.

  Examples of host cells include eukaryotes and prokaryotes in the case of microorganisms. Preferable examples include E. coli. A host cell can be transformed by introducing the vector as described above into the host cell by an ordinary genetic engineering method.

  As a method for introducing the recombinant vector of the present invention into a host cell, a normal introduction method corresponding to the host cell may be applied. When Escherichia coli is used as a host cell, for example, J. et al. Sambrook, E .; F. Frisch, T .; Maniatis; “Normal Cloning 2nd Edition”, Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory, published in 1989), etc. In addition, when mammalian cells or insect animal cells are used as host cells, for example, normal gene transfer methods such as calcium phosphate method, DEAE dextran method, electroporation method, lipofection method, etc. In addition, when yeast is used as a host cell, the lithium method used in, for example, Yeast transformation kit (Clontech) and the like In addition, when a virus is used as a vector, the virus genome can be introduced into a host cell by a general gene transfer method as described above, and a virus into which the gene of the present invention is inserted It is also possible to introduce the genome of the virus into the host cell by infecting the host cell with a virus particle containing the genome.

When the vector of the present invention is constructed using a vector containing an autonomous origin of replication, such as the above-described yeast vector pACT2, bovine papilloma virus vector pBPV, EB virus vector pCEP4, etc., the vector is episomal when introduced into a host cell. As retained in the cell.
When a vector containing a selection marker gene (for example, an antibiotic resistance-conferring gene such as a kanamycin resistance gene or a neomycin resistance gene) is used as a basic vector, a transformant into which the vector has been introduced is used as a phenotype of the selection marker gene, etc. Can be selected as an index.

  Examples of the host cell into which the nucleic acid of the present invention or the recombinant vector of the present invention is introduced into the host cell include, for example, microorganisms belonging to the genus Escherichia, Bacillus, Corynebacterium, Staphylococcus, Streptomyces, Saccharomyces, Kluyveromyces and Aspergillus Etc.

The method of introducing the nucleic acid of the present invention or the recombinant vector of the present invention, which is operably connected to a promoter that can function in the host cell, into the host cell is a method commonly used depending on the host cell used. sufficient if, for example, ^{"Molecular Cloning: A Laboratory Manual 2 nd} edition " (1989), Cold Spring Harbor Laboratory Press, "Current Protocols in Molecular Biology" (1987), John Wiley & Sons , Inc. ISBNO-471-50338 Calcium chloride method described in -X and the like, and "Methods in Electroporation: Gene Pulser / E. Coli Pulser System" Bio-Rad Laboratories, (1993) and the like.

  In order to select a transformant introduced with the nucleic acid of the present invention or the recombinant vector of the present invention operably connected to a promoter capable of functioning in a host cell, for example, it is contained in the vector as described above. Selection may be made using the phenotype of the selected marker gene as an index.

  By using the nucleic acid of the present invention, the reductase, the recombinant vector containing the nucleic acid of the present invention, and the transformant thereof, for example, a useful optically active alcohol can be industrially advantageously obtained from a carbonyl compound. In such a method for producing an optically active alcohol, a reductase, a transformant that produces it, or a processed product thereof is allowed to act on a carbonyl compound that is a substrate.

  As a method for culturing a transformant, a method for preparing a processed product of the transformant, and the like, the method described in the above-described step (4) of the nucleic acid obtaining method of the present invention can be used. In addition, as a method for purifying a reductase from a culture of a transformant, a method commonly used in protein purification can be applied, and examples thereof include the following methods.

  First, cells are collected from the transformant culture by centrifugation, etc., and then subjected to physical disruption methods such as ultrasonic treatment, dynomill treatment, French press treatment, or chemistry using a lytic enzyme such as a surfactant or lysozyme. Crush by mechanical crushing method. Cell-free extracts are prepared by removing impurities from the crushed liquid by centrifugation, membrane filter filtration, etc., and this is prepared by cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel chromatography, etc. The reductase can be purified by fractionation using the separation and purification method as appropriate.

  Examples of the carrier used for the chromatography include insoluble polymer carriers such as cellulose, dextrin, or agarose into which carboxymethyl (CM) group, diethylaminoethyl (DEAE) group, phenyl group or butyl group is introduced. Commercially available carrier-filled columns can also be used. Examples of such commercially available carrier-filled columns include Q-Sepharose FF, Phenyl-Sepharose HP (GE Healthcare), TSK-gel G3000SW (Tosoh) Etc.

  In order to select a fraction containing reductase, for example, it is possible to select based on the ability to reduce 2,2,2-trifluoroacetophenone to produce 2,2,2-trifluoro-1-phenylethanol. That's fine.

  Examples of the substrate ketone compound or aldehyde compound include propionaldehyde, n-butyraldehyde, n-valeraldehyde, n-hexylaldehyde, n-peptylaldehyde, n-decylaldehyde, 2-oxopropionaldehyde, trans- Cinnamaldehyde, 4-bromobenzaldehyde, 2-nitrobenzaldehyde, 3-nitrobenzaldehyde, phenylacetaldehyde, 4-chlorobenzaldehyde, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone, 2-nonanone, 3-pentanone, 3-chloro-2-butanone, tert-butyl acetoacetate, 4-hydroxy-2-butanone, hydroxyacetone, 1,1-dichloroacetone, chloroacetone, dihydroxyacetone, Ethyl binate, methyl 3-oxobutanoate, ethyl 3-oxabutanoate, ethyl 4-chloroacetoacetate, methyl 4-bromo-3-oxabutanoate, ethyl 4-bromo-3-oxabutanoate N-tert-butoxycarbonyl-3-pyrrolidinone, isopropyl 4-cyano-3-oxobutanoate, ethyl 4-cyano-3-oxobutanoate, methyl 4-cyano-3-oxobutanoate, methyl 3 -Oxopentanoate, 2,2,2-trifluoroacetophenone, acetophenone, 2'-bromoacetophenone, 3'-bromoacetophenone, 4'-bromoacetophenone, 2-chloroacetophenone, 3'-chloroacetophenone, 4'- Chloroacetophenone, benzylacetone, 1- Enyl-2-butanone, m-methoxyacetophenone, 3,4-dimethoxyacetophenone, 4′-methoxyacetophenone, 2,3′-dichloroacetophenone, 3,4-dimethoxyphenylacetone, cyclopentanone, 4-acetylbenzoic acid, D-(+)-glucose, ethyl 4-chloro-3-oxobutyrate, ethyl 3-methyl-2-oxobutyrate, 4-hydroxy-2-butanone, ethyl benzoylformate, 1-phenyl-2-butanone, 3- And ethyl oxobutyrate.

  The above method is usually performed in the presence of water and reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH). The water used at this time may be a buffered aqueous solution. Examples of the buffer used in the buffered aqueous solution include alkali metal phosphates such as sodium phosphate and potassium phosphate, alkali metal acetates such as sodium acetate aqueous solution and potassium acetate, and mixtures thereof.

  In the above method, an organic solvent can be allowed to coexist in addition to water. Examples of the organic solvent that can coexist include ethers such as t-butyl methyl ether, diisopropyl ether, and tetrahydrofuran; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, ethyl propionate, and butyl propionate Hydrocarbons such as toluene, hexane, cyclohexane, heptane and isooctane; alcohols such as methanol, ethanol, 2-propanol, butanol and t-butyl alcohol; organic sulfur compounds such as dimethyl sulfoxide; ketones such as acetone; Nitriles such as acetonitrile, and mixtures thereof.

  The reaction in the above method is, for example, stirring water, NADH, and a ketone compound or an aldehyde compound, together with a reductase, a transformant that produces the compound, or a processed product thereof, if necessary, further containing an organic solvent or the like. It is performed by mixing by shaking or the like.

  The pH during the reaction in the above method can be selected as appropriate, but is usually in the range of pH 3-10. Moreover, although reaction temperature can be selected suitably, it is the range of 0-60 degreeC normally from the point of stability of a raw material and a product, and the point of reaction rate. Further, the reaction may be carried out while blowing nitrogen or air. The air flow rate is usually in the range of 0.1 to 5 L / min.

  The end point of the reaction can be determined, for example, by following the amount of the carbonyl compound in the reaction solution by liquid chromatography or the like. The reaction time can be appropriately selected and is usually in the range of 0.5 hours to 10 days.

  Recovery of alcohol from the reaction solution may be performed by any generally known method. For example, a method of purifying by performing post-treatment such as organic solvent extraction operation and concentration operation of the reaction solution in combination with column chromatography, distillation or the like, if necessary.

  As the transformant or the processed product thereof, the above-mentioned ones can be used. However, in consideration of industrial production using the transformant of the present invention, transformation is performed rather than using live transformants. A method using a processed product whose body has been killed is preferable in that there are few restrictions on production facilities. As a killing treatment method for that purpose, for example, physical sterilization methods (heating, drying, freezing, light, ultrasonic waves, filtration, energization) and sterilization methods using chemicals (alkali, acid, halogen, oxidizing agent, Sulfur, boron, arsenic, metal, alcohol, phenol, amine, sulfide, ether, aldehyde, ketone, cyan and antibiotics). In general, it is desirable to select a treatment method among these sterilization methods that does not deactivate the enzyme activity of the reductase as much as possible and that has less influence on the reaction system such as residue and contamination.

  The method for producing alcohol using the reductase of the present invention is carried out in the presence of NADH, and NADH is converted to oxidized β-nicotinamide adenine dinucleotide (hereinafter referred to as NAD +) as the reduction reaction of the ketone compound or aldehyde compound proceeds. Is written). When the reductase has the ability to convert NAD + generated by the conversion into, for example, a reduced form (NADH) accompanying the oxidation reaction of 2-propanol, in the reaction system of the above method, As a substrate that enables conversion of NAD + to NADH, 2-propanol or the like can be present together.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention further in detail, this invention is not limited at all by these Examples.

Example 1 (Acquisition of environmental DNA)
Using the following environmental samples (A) to (E) stored at 4 ° C. and a soil DNA extraction kit (ISOIL for Beads Beating, manufactured by Nippon Gene), DNA was extracted according to the manual of the kit, and the resulting DNA Were designated as environmental DNAs (A) to (E), respectively. The extracted DNA was confirmed by 1.5% agarose gel electrophoresis.
(A) Compost from Kataguchi, Imizu City, Toyama Prefecture (grass and branches 79 ° C)
(B) Tomi, Izumi City, Kataguchi's compost (wood bark 60 ° C)
(C) Compost (grass and branch surface) at Kataguchi, Imizu City, Toyama Prefecture
(D) Tomi, Izumi City, Kataguchi compost (wood bark 59 ° C)
(E) Compost from Kataguchi, Imizu City, Toyama Prefecture (grass and branches below 79 ° C)

Example 2 (Preparation of oligonucleotide primers)
The following four types of oligonucleotide primers were synthesized.
Primer 1: 5′-GACCGGTCCGCGATCGTVACMGGGSMMG-3 ′ (SEQ ID NO: 1)
Primer 2: 5′-CGGTCACTGGGGCGTRTABCCRCCRTCB-3 ′ (SEQ ID NO: 2)
Primer 3: 5′-ACGATCCGCGGACGGGTCGGCGACGT-3 ′ (SEQ ID NO: 4)
Primer 4: 5'-ACCGCCCCAGTGACCGGGGCTGCAGGT-3 '(SEQ ID NO: 5)
R represents adenine or guanine, V represents adenine or cytosine or guanine, M represents adenine or cytosine, S represents cytosine or guanine, and B represents cytosine, guanine or thymine, respectively.

Example 3 (Production of double-stranded linear vector DNA)
The plasmid pKELA described in JP2008-017773 contains a nucleic acid encoding a protein having the ability to reduce 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol. Recombinant vector.
Plasmid pKELA was digested with restriction enzyme XhoI. The digested DNA was subjected to 1.5% agarose gel electrophoresis, and then 5.3 kb DNA was recovered from the gel and purified using Wizard Plus SV Gel and PCR Clean-up System (Promega). DNA was ethanol precipitated from the obtained purified solution, and the resulting precipitate was dried and then dissolved in 5 μl of sterilized water. 10 μl of Ligation Mix (manufactured by Takara Bio Inc.) was added to the obtained enzyme digested DNA aqueous solution and left overnight at 16 ° C., then DNA was ethanol precipitated, the resulting precipitate was dried, and 5 μl of sterile water Dissolved in. The resulting DNA solution was used to transform E. coli by the transformation method. E. coli JM109 Competent cell (Toyobo Co., Ltd.) was transformed. The obtained transformant was inoculated into an LB (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% sodium chloride) medium containing 100 μg / ml ampicillin and cultured. Plasmid DNA was prepared from the cultured cells using Wizard Plus SV Minipreps DNA Purification System (Promega). The resulting plasmid is hereinafter referred to as pKELAs.

  Using the oligonucleotide primer 3 consisting of the base sequence shown in SEQ ID NO: 4 and the oligonucleotide primer 4 consisting of the base sequence shown in SEQ ID NO: 5, using the plasmid pKELAs as a template, PCR was performed with the following reaction solution composition and reaction conditions. went.

[Primer]
Primer 3: 5′-ACGATCCGCGGACGGGTCGGCGACGT-3 ′ (SEQ ID NO: 4)
Primer 4: 5'-ACCGCCCCAGTGACCGGGGCTGCAGGT-3 '(SEQ ID NO: 5)
[Reaction solution composition]
pKELAs solution (100 μg / ml) 1 μl
dNTPs (mixture of 2 mM each) 4 μl
Primer (10 μM) 0.8 μl each
2 × Buffer for KOD FX Neo 10 μl
KOD FX Neo (manufactured by Toyobo Co., Ltd.) 0.4 μl
Sterile water 3μl
[Reaction conditions]
A container containing the reaction solution of the above composition is set in Bio-RAD DNA Engine, and kept at 94 ° C. for 2 minutes, followed by a heating cycle of 98 ° C. for 10 seconds, 60 ° C. for 30 seconds and then 68 ° C. for 3 minutes. This was performed 30 times, and further kept at 68 ° C. for 5 minutes. The amplified product was purified using Wizard Plus SV Minipreps DNA Purification System (Promega), which was used as a double-stranded linear vector DNA.

Example 4 (Amplification of nucleic acid from DNA sample, preparation of circular recombinant expression vector and acquisition of transformant)
(4-1) Nucleic acid amplification from environmental DNA
Using environmental primer obtained from environmental samples (A) to (E) as a template, using oligonucleotide primer 1 consisting of the base sequence shown in SEQ ID NO: 1 and oligonucleotide primer 2 consisting of the base sequence shown in SEQ ID NO: 2 Then, PCR was performed with the following reaction solution composition and reaction conditions.

Primer 1: 5′-GACCGGTCCGCGATCGTVACMGGGSMMG-3 ′ (SEQ ID NO: 1)
Primer 2: 5′-CGGTCACTGGGCGGTRTABCCRCCRTCB-3 ′ (SEQ ID NO: 2)
R represents adenine or guanine, V represents adenine or cytosine or guanine, M represents adenine or cytosine, S represents cytosine or guanine, and B represents cytosine, guanine or thymine, respectively.
[Reaction solution composition]
Environmental DNA solution (10-50 ng) 1 μl
dNTPs (mixture of 2 mM each) 4 μl
Primer 1 (10 μM) 0.8 μl
Primer 2 (10 μM) 0.8 μl
5x Phusion GC Buffer 4μl
Phusion Hot Start Flex DNA Polymerase (New England BioLabs) 0.4 μl
DMSO 1 μl
Sterile water 8μl
[Reaction conditions]
A container containing the reaction solution of the above composition is set in Bio-RAD DNA Engine, kept at 94 ° C. for 2 minutes, then subjected to 5 incubation cycles of 98 ° C. for 10 seconds and then at 74 ° C. for 1 minute, Incubation cycle of 98 ° C. for 10 seconds and then 72 ° C. for 1 minute 5 times, followed by 5 incubation cycles of 98 ° C. for 10 seconds and then 70 ° C. for 1 minute, then 98 ° C. for 10 seconds Next, a heat insulation cycle of 68 ° C. for 1 minute was performed 20 times, and the mixture was further kept at 68 ° C. for 7 minutes. The amplification product was purified using Wizard Plus SV Minipreps DNA Purification System (Promega).

(4-2) Preparation of circular recombinant expression vector and acquisition of transformant The double-stranded linear vector DNA obtained in Example 3 was added to 2 μl of the solution after purification of the amplification product obtained in (4-1). 6 μl of the purified solution was added, 2 μl of 5 × In-Fusion HD Enzyme Premix (Clontech) was added, and the mixture was incubated at 50 ° C. for 15 minutes, and then allowed to stand on ice. Using this, E. E. coli strain JM109 was transformed.

  334 colonies of E. coli recombinants cloned with DNA amplified from environmental DNA (A) were obtained. 40 colonies of E. coli recombinants cloned with DNA amplified from environmental DNA (B) were obtained. 286 colonies of E. coli recombinants cloned with DNA amplified from environmental DNA (C) were obtained. 39 colonies of E. coli recombinants in which DNA amplified from environmental DNA (D) was cloned were obtained. 422 colonies of E. coli recombinants cloned with DNA amplified from environmental DNA (E) were obtained.

Example 5 (Measurement of acetophenone reduction activity of transformant and acquisition of nucleic acid encoding reductase)
(5-1) Preparation of crude enzyme solution
The transformant obtained in Example 4 was transformed into E. coli using plasmid pKELAs. A transformant obtained by transforming E. coli JM109 strain or a plasmid pKELA described in Japanese Patent Application Laid-Open No. 2008-017773 is used for E. coli. The transformant obtained by transforming E. coli strain JM109 was cultured with shaking at 37 ° C. for 22 hours in 4 ml of LB medium containing 100 μg / ml ampicillin and 0.4 mM IPTG. 1 ml of the obtained culture broth was placed in a 2 ml centrifuge tube and centrifuged at 15000 rpm for 30 seconds at 4 ° C. to precipitate wet cells. 500 mg beads (Φ0.1 mm) and 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) are added to the wet cells excluding the centrifugal supernatant, and Multi-Beads Shocker (manufactured by Yasui Kikai Co., Ltd.) is added. The crushing treatment was performed under the conditions of 2500 rpm, 150 seconds: ON (30 seconds × 3), OFF (30 seconds × 2). After the crushing liquid was centrifuged for a short time, a hole was made in the bottom of the tube containing the crushing liquid, and the tube was pushed into a new centrifuge tube having a volume of 1.5 ml. After centrifuging at 3000 rpm for 5 minutes at 4 ° C., the 2 ml centrifuge tube is removed, and the sample in the 1.5 ml centrifuge tube is further centrifuged at 15000 rpm for 5 minutes at 4 ° C., and the resulting centrifugal supernatant is obtained. The solution was used as a crude enzyme solution.

(5-2) Measurement of reducing activity and acquisition of nucleic acid encoding reductase (part 1)
990 μl of a reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 0.3 mM NADH, and 10 mM 2,2,2-trifluoroacetophenone was kept at 25 ° C. for 1.5 minutes, and (5-1) After 10 μl of the obtained crude enzyme solution was added, the decrease in NADH absorption at 340 nm was measured. In addition, 990 μl of a reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 0.3 mM NADH, 15% 1-propanol, and 10 mM 2,2,2-trifluoroacetophenone was incubated at 25 ° C. for 1.5 minutes. Then, 10 μl of the crude enzyme solution obtained in (5-1) was added, and then the decrease in NADH absorption at 340 nm was measured. In plasmid pKELAs In the crude enzyme solution obtained from the transformant obtained by transforming E. coli JM109 strain, a decrease in absorption at 340 nm was not detected, and this was used as a blank. The NADH molar extinction coefficient (ε) was 6.22 × 1000 M ⁻¹ cm ⁻¹ , the amount of enzyme converting 1 μmol of NADH per minute at 25 ° C. was 1 U, and the enzyme activity value per 1 ml of the culture solution was calculated. . The relative value of 2,2,2-trifluoroacetophenone reduction activity value in the presence of 15% 1-propanol, with the value of 2,2,2-trifluoroacetophenone reduction activity value in the absence of 15% 1-propanol being 100 Values were calculated (Table 1).

  In a crude enzyme solution of a transformant containing plasmid pKELA, 2,2 in the presence of 15% 1-propanol with respect to the 2,2,2-trifluoroacetophenone reduction activity value in the absence of 15% 1-propanol. , 2-trifluoroacetophenone reduction activity value compared with the circular recombinant expression vector # 0012, # 0013, # 0014, # 0015, # 0016, # 0017, # 0018 containing DNA amplified from environmental DNA , # 0021, and # 0022 respectively, the crude enzyme solution of the transformant has a small decrease rate of 2,2,2-trifluoroacetophenone reduction activity value due to the presence of 15% 1-propanol, and these transformants Was found to produce a reductase with better resistance to organic solvents than the reductase produced by transformants containing plasmid pKELA (Table 1).

  A recombinant expression vector (hereinafter referred to as plasmid # 0012) containing DNA amplified from environmental DNA using QIAprep Spin Miniprep Kit (manufactured by Qiagen) from the above-mentioned nine types of transformants in which the reducing activity of the crude enzyme solution was confirmed. , # 0013, # 0014, # 0015, # 0016, # 0017, # 0018, # 0021, and # 0022).

(5-3) Reducing activity measurement and acquisition of nucleic acid encoding reductase (part 2)
The crude enzyme solution obtained in (5-1) was kept at each temperature shown in Table 2 for 30 minutes. 990 μl of a reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 0.3 mM NADH, and 10 mM 2,2,2-trifluoroacetophenone was kept at 25 ° C. for 1.5 minutes, and then kept at the above temperature. 10 μl of the crude enzyme solution was added, and the decrease in NADH absorption at 340 nm was measured. The relative activity value of the crude enzyme solution after each temperature treatment was calculated with the activity value in the crude enzyme solution kept at 4 ° C. being 100 ° C. instead of each temperature shown in Table 2 (Table 2).
A circular recombinant expression vector comprising DNA amplified from environmental DNA as compared to the rate of decrease in 2,2,2-trifluoroacetophenone reduction activity value due to temperature treatment in the crude enzyme solution of a transformant containing plasmid pKELA In the crude enzyme solution of the transformant containing # 0024, the rate of decrease in the 2,2,2-trifluoroacetophenone reduction activity value due to the temperature treatment is small, and the transformant contains the transformant containing the plasmid pKELA. It was found that a reductase having better thermal stability than the reductase produced was produced (Table 2).

  A recombinant expression vector (hereinafter referred to as plasmid # 0024) containing DNA amplified from environmental DNA using QIAprep Spin Miniprep Kit (manufactured by Qiagen) from the above transformant in which the reducing activity of the crude enzyme solution was confirmed. There is also.)

Example 6 (Determining the base sequence of a nucleic acid encoding a reductase)
Plasmids # 0012, # 0013, # 0014, # 0015, # 0016, # 0017, # 0017 obtained from the transformants in which the 2,2,2-trifluoroacetophenone reducing activity of the crude enzyme solution was confirmed in Example 5 The nucleotide sequences of the DNAs cloned into 0018, # 0021, # 0022, and # 0024 were determined. A sequence reaction was carried out using each plasmid as a template using Dye Terminator Cycle sequencing FS ready Reaction Kit (manufactured by PerkinElmer), and the base sequence of the obtained DNA was analyzed by DNA sequencer 373A (manufactured by PerkinElmer). The nucleotide sequences obtained from the plasmids # 0012, # 0013, # 0014, # 0015, # 0016, # 0017, # 0018, # 0021, # 0022 and # 0024, respectively, are shown in SEQ ID NOs: 11, 13, 15, 17, 19, The amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, and 10, respectively. Each amino acid sequence represented by SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28 and 10 and 2,2,2-trifluoroacetophenone encoded by the plasmid pKELA are 2,2,2 -Sequence identity with the amino acid sequence of a protein having the ability to reduce to trifluoro-1-phenylethanol is about 50-75%.

The present invention makes it possible to provide an efficient method for obtaining a nucleic acid encoding a reductase used in an organic synthesis reaction for producing a compound or an intermediate thereof as an active ingredient of medical and agricultural chemicals. For example, in the environment It is possible to provide a nucleic acid encoding a reductase excellent in stability to organic solvents and heat stability, which is obtained from environmental DNA derived from microorganisms present in
[Sequence table free text]
SEQ ID NO: 1
Oligonucleotide primer SEQ ID NO: 2
Oligonucleotide primer SEQ ID NO: 3
Vector sequence number 4
Oligonucleotide primer SEQ ID NO: 5
Oligonucleotide primer SEQ ID NO: 6
Vector base sequence SEQ ID NO: 7
Vector base sequence SEQ ID NO: 8
Vector base sequence SEQ ID NO: 9
Sequence number # 10 of # 0024
Amino acid sequence of # 0024 SEQ ID NO: 11
Base sequence of # 0012 SEQ ID NO: 12
Amino acid sequence of # 0012 SEQ ID NO: 13
Base sequence of # 0013 SEQ ID NO: 14
Amino acid sequence of # 0013 SEQ ID NO: 15
SEQ ID NO: 16 of the nucleotide sequence of # 0014
Amino acid sequence of # 0014 SEQ ID NO: 17
SEQ ID NO: 18 of the nucleotide sequence of # 0015
Amino acid sequence of # 0015 SEQ ID NO: 19
SEQ ID NO: 20 of the nucleotide sequence of # 0016
Amino acid sequence of # 0016 SEQ ID NO: 21
SEQ ID NO: 22 of the nucleotide sequence of # 0017
Amino acid sequence of # 0017 SEQ ID NO: 23
Base sequence of # 0018 SEQ ID NO: 24
Amino acid sequence of # 0018 SEQ ID NO: 25
SEQ ID NO: 26 of nucleotide sequence of # 0021
Amino acid sequence of # 0021 SEQ ID NO: 27
Base sequence of SEQ ID NO: 28
Amino acid sequence of SEQ ID NO: 29
Vector sequence

Claims (17)

A method for obtaining a nucleic acid encoding a reductase, comprising:
(1) A step of performing a nucleic acid amplification reaction using a DNA sample as a template by using an oligonucleotide having the base sequence shown by SEQ ID NO: 1 and an oligonucleotide having the base sequence shown by SEQ ID NO: 2 as a primer to obtain an amplification product;
(2) The amplification product and a double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized are fused in the presence of a DNA polymerase having an endogenous 3′-5 ′ exonuclease activity. Obtaining a circular recombinant expression vector containing the amplification product,
Here, one of the two strands constituting the double-stranded linear vector DNA has a base sequence represented by SEQ ID NO: 6 at its 5 ′ end and a base represented by SEQ ID NO: 7 at its 3 ′ end. A nucleotide sequence having the sequence and capable of expressing the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 7 in a host cell, and an origin of replication that can function in the host cell or a complementary sequence thereof, represented by SEQ ID NO: 7 A single-stranded DNA 5 ′ upstream of the base sequence shown;
(3) introducing the circular recombinant expression vector into a host cell to obtain a transformant;
(4) a step of measuring the activity of the transformant or its treatment product to reduce a substrate and selecting a transformant having the activity; and (5) from the transformant selected in step (4). Obtaining a nucleic acid encoding the reductase having the activity;
Including methods.   The single-stranded DNA is a single-stranded DNA having a base sequence represented by SEQ ID NO: 7 continuously 3 'downstream of the base sequence represented by SEQ ID NO: 8 at its 3' end. the method of.   The method according to claim 1 or 2, wherein the single-stranded DNA is a single-stranded DNA having a base sequence represented by SEQ ID NO: 3.   The method according to any one of claims 1 to 3, wherein the DNA sample is environmental DNA derived from an environmental sample.   The method according to any one of claims 1 to 4, wherein the activity of reducing the substrate is an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol. .   The method according to any one of claims 1 to 5, wherein the host cell is Escherichia coli.   A primer set comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 1 and an oligonucleotide having a base sequence represented by SEQ ID NO: 2.   A double-stranded linear vector DNA that can function as an expression vector in a host cell when circularized, and one of the two strands constituting the DNA has a base sequence represented by SEQ ID NO: 6 at its 5 ′ end. A nucleotide sequence having the nucleotide sequence represented by SEQ ID NO: 7 at its 3 ′ end and capable of expressing in the host cell the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 7; A double-stranded linear vector DNA, which is a single-stranded DNA having a simple replication origin or its complementary sequence 5 ′ upstream of the base sequence represented by SEQ ID NO: 7. A protein selected from the following group:
(A) a protein having an amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28;
(B) In the amino acid sequence represented by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, one or several amino acids are deleted, inserted, substituted or added. A protein having an amino acid sequence and having an activity of reducing 2,2,2-trifluoroacetophenone to 2,2,2-trifluoro-1-phenylethanol; and (c) SEQ ID NOs: 10, 12, 14, It has an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of 16, 18, 20, 22, 24, 26, or 28, and 2,2,2-trifluoroacetophenone is 2,2 , A protein having activity of reducing to 2-trifluoro-1-phenylethanol.   A nucleic acid encoding the protein according to claim 9.   The nucleic acid according to claim 10, which has a base sequence represented by any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27.   A recombinant vector containing the nucleic acid according to claim 10 or 11.   The recombinant vector according to claim 12, wherein a promoter capable of functioning in a host cell is operably linked upstream of the nucleic acid according to claim 10 or 11.   A method for producing a vector comprising incorporating the nucleic acid according to claim 10 or 11 into a vector capable of replicating in a host cell.   A transformant obtained by introducing the nucleic acid according to claim 10 or 11 or the vector according to claim 12 or 13 into a host cell.   The transformant according to claim 15, wherein the host cell is Escherichia coli.   A method for producing a transformant, wherein the nucleic acid according to claim 10 or 11 or the vector according to claim 12 or 13 is introduced into a host cell.