JP2003289874A - Conjugated polyketone reductase gene and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an enzyme, based on information concerning a gene which codes a conjugated polyketone reductase useful as an enzyme for catalyzing a stereoselective asymmetric reductive reaction of a carbonyl compound, and to provide a method for producing an optically active alcohol by using the enzyme. <P>SOLUTION: A gene which codes the conjugated polyketone reductase CPRC1 derived from Candida parapsilosis and another gene which codes the conjugated polyketone reductase CPRC2 are provided in the specification. An optically active 1-halo-3-amino-4-phenyl-2-butanol derivative is produced in the method for producing the optically active alcohol, by using a transformant containing the conjugated polyketone reductases. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a conjugated polyketone reductase, a recombinant vector DNA containing this gene, a transformant using the recombinant vector DNA, and a conjugated polyketone reduction using this transformant. The present invention relates to a method for producing an enzyme, and further to a method for producing an optically active alcohol using the present enzyme.

[0002]

BACKGROUND OF THE INVENTION Various optically active alcohols are compounds useful as raw materials for medicines, agricultural chemicals and the like. A method for producing an optically active alcohol by a stereoselective reduction reaction of a carbonyl compound using an enzyme or a microorganism has attracted attention as an efficient and industrial method. A conjugated polyketone reductase is known as an enzyme that catalyzes such a stereoselective reduction reaction (Biochim. Biophy.
s. Acta, 990, p. 175, 1989). Since this enzyme has wide substrate specificity and high stereoselectivity, it is expected to be an excellent catalyst for the production of optically active alcohol.

Examples of microorganisms having the ability to produce conjugated polyketone reductase include, for example, Candida parapsilosis IF.
The strain O0708 is known, but the microorganism is known to produce at least two types of conjugated polyketone reductase (Biosci. Biotechnol. B.
iochem. , 65, 2785, 2001).
When utilizing the bacterial cells of a microorganism that produces several kinds of reductases as a catalyst for the carbonyl asymmetric reduction reaction, there are concerns about problems such as deterioration of product quality due to differences in substrate specificity and stereoselectivity for carbonyl compounds. However, isolating only the required reductase activity from the microorganism requires a very complicated operation and is difficult to carry out industrially. Therefore, it is desirable to mass-produce only the required enzyme activity using genetic engineering technology.
However, information on genetic engineering for producing a conjugated polyketone reductase by genetic engineering is not known.

On the other hand, optically active 1-halo-3-amino-4
The -phenyl-2-butanol derivative is a compound useful as a synthetic raw material for drugs and the like. This compound is a compound that has two asymmetric carbons in the molecule. The optical activity of one asymmetric carbon is created by chemical synthesis, and the optical activity of the other asymmetric carbon is reduced by a carbonyl group reduction reaction. It is known that can be created by. A method for biologically performing this reduction reaction is known (J. Am. Oil. Chem. S.
oc. 76, 1275, 1999 and JP-A-9-285). It is disclosed that the former gives a threo body having a (1S, 2R) configuration, and the latter gives a threo body and an erythro body. In the latter case, Candida parapsilosis (Candida)
It is also disclosed that the cells of parapsilosis) IFO0585 give erythroforms. However, in any case, the amount of the target substance accumulated is small, and it is difficult to obtain an industrial production method.

[0005]

DISCLOSURE OF THE INVENTION The present invention is directed to genetic engineering of a gene encoding a conjugated polyketone reductase for the purpose of genetic engineering production of the conjugated polyketone reductase and efficient production of optically active alcohols by this enzyme. It is to provide a material, a method for producing a conjugated polyketone reductase using this material, and further a method for producing an optically active alcohol.

[0006]

The present inventors have succeeded in isolating a gene encoding two conjugated polyketone reductases produced by Candida parapsilosis and determining the structure of the gene. Recombinant DNA in which a gene was inserted into vector DNA was obtained, and this was introduced into Escherichia coli, and the transformant was cultured, thereby successfully producing polyketone reductase efficiently. Further, the recombinant enzyme also catalyzes the reduction reaction of the optically active 1-halo-3-amino-4-phenyl-2-butanone derivative, and the optically active 1-halo-3-amino-4-phenyl- It has also been found that it allows the production of 2-butanol derivatives.

That is, the present invention provides the following (a) or (b)
DNA of: (a) DNA encoding a protein consisting of the amino acid sequence shown by SEQ ID NO: 1 (b) One or several amino acids in the amino acid sequence shown by SEQ ID NO: 1 have been deleted, substituted, inserted or added It is a DNA consisting of an amino acid sequence and encoding a protein having a conjugated polyketone reductase activity.

The present invention also provides the following (a) or (b)
DNA of: (a) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 (b) One or several amino acids in the amino acid sequence represented by SEQ ID NO: 3 have been deleted, substituted, inserted or added It is a DNA consisting of an amino acid sequence and encoding a protein having a conjugated polyketone reductase activity.

The present invention also provides any of the above DNAs.
It is also a recombinant vector containing

The present invention is also a transformant containing the above recombinant vector, which is characterized by culturing the transformant in a medium and collecting the conjugated polyketone reductase from the culture. It is also a method for producing a conjugated polyketone reductase.

Further, the present invention provides a compound represented by the general formula (1):

[0012]

[Chemical 3]

(In the formula, X represents a halogen atom.
R ¹ and R ² independently represent a hydrogen atom, an optionally protected hydroxyl group, an alkoxyl group, an alkyl group, a nitro group, an optionally protected amino group, or a cyano group. P ¹ and P ^{2 each} independently represent a hydrogen atom or an amino group-protecting group, or P ¹ and P ² together represent a phthaloyl group. However, the case where P ¹ and P ² are simultaneously hydrogen atoms is excluded. ) (1S, 2S)-
A method for producing a 1-halo-3-amino-4-phenyl-2-butanol derivative, which comprises the following general formula (2):

[0014]

[Chemical 4]

1-halo-3-amino-4-phenyl-2-butanone derivative represented by the formula (wherein X, R ¹ , R ² , P ¹ and P ² are the same as above) and conjugated polyketone reduction Characterized by reacting with an enzyme or a culture of a microorganism capable of producing the enzyme or a treated product thereof (1S, 2
It is also a method for producing a (S) -1-halo-3-amino-4-phenyl-2-butanol derivative.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to two types of conjugated polyketone reductases (C) produced by Candida parapsilosis.
The present invention relates to isolation of genes encoding PRC1 and CPRC2) and elucidation of DNA base sequences of the genes.

The conjugated polyketone reductase activity in the present invention means a reductase activity acting on a conjugated polyketone such as isatin and ketopantoyl lactone. The presence or absence of the activity is, for example, 200 mM phosphate buffer (pH
7.0) substrate ketopantoyl lactone 0.4 mM, N
React in 2.5 ml reaction solution containing ADPH 0.32 mM and enzyme solution 0.05 ml at 30 ° C. for 3 minutes,
It can be confirmed by the presence or absence of a decrease in absorbance at a wavelength of 340 nm.

Next, an example of a method for obtaining the gene encoding the conjugated polyketone reductase according to the present invention will be described, but the present invention is not limited thereto.

First, the purified enzyme is digested with an appropriate endopeptidase, the fragment cleaved by reverse phase HPLC is purified, and then a part of the partial amino acid sequence is determined by a protein sequencer. Then, based on the obtained partial amino acid sequence, PCR (Polymerase)
Chain Reaction) Primer is synthesized.

Next, from the microorganism that is the origin of the DNA,
Conventional DNA isolation methods such as the Hereford method (Ce
11: 18, 1261, 1979), the chromosomal DNA of the microorganism is prepared. Using this chromosomal DNA as a template, PCR is carried out using the above-mentioned PCR primers, a part of the DNA encoding the enzyme is amplified (core sequence), and the nucleotide sequence is determined. The nucleotide sequence can be determined by the dideoxy chain termination method or the like. For example, ABI 373A DNA Sequence
cer (manufactured by Applied Biosystems)
And so on.

In order to clarify the nucleotide sequence of the peripheral region of the core sequence, the chromosomal DNA of the microorganism is digested with a restriction enzyme whose recognition sequence is not present in the core sequence, and the resulting DNA fragment is autologous with T4 ligase. Inverse PCR (Nucleic Acids Re
s. , 16, 8186, 1988) mold DN
Prepare A.

Next, based on the core sequence, a primer as a starting point of DNA synthesis toward the outside of the core sequence is synthesized, and the peripheral region of the core sequence is amplified by inverse PCR. By clarifying the base sequence of the DNA thus obtained, the DNA sequence of the entire coding region of the target enzyme can be determined.

The DNA of the present invention comprises a conjugated polyketone reductase (C having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing).
DNA encoding PRC1) or a conjugated polyketone reductase (CP) comprising the amino acid sequence shown in SEQ ID NO: 3
It is a DNA encoding RC2). These DNA are
It may be a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, or 1 in these salt sequences.
Alternatively, it may be a DNA consisting of a nucleotide sequence in which several bases are deleted, substituted, inserted or added, and coding for a protein having a conjugated polyketone reductase activity. Alternatively, it encodes a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing and having a conjugated polyketone reductase activity. It may be DNA.

As the vector DNA used for introducing the DNA of the enzyme of the present invention into the host microorganism and expressing it in the introduced host microorganism, the enzyme gene can be expressed in an appropriate host microorganism. Any one can be used. Examples of such vector DNA include a plasmid vector, a phage vector,
Examples include cosmid vectors. Also, a shuttle vector capable of gene exchange with another host strain may be used.

Such a vector includes operably linked promoters (T7 promoter, lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tuf).
B promoter, recA promoter, pL promoter, etc.) and can be suitably used as an expression vector containing an expression unit operably linked to the DNA of the present invention. For example, pET21a and the like can be preferably used.

As used herein, the term "regulatory element" refers to a nucleotide sequence having a functional promoter and any related transcription elements (eg enhancer, CCAAT box, TATA box, SPI site, etc.).

As used herein, the term “operably linked”
Means that the DNA is linked to various regulatory elements such as a promoter and an enhancer that regulate the expression so that the gene can be expressed so that the gene can be expressed in the host cell. It is well known to those skilled in the art that the type and kind of the regulatory factor can vary depending on the host.

Host cells into which the vector having the DNA of the present invention is introduced include bacteria, yeasts, filamentous fungi, plant cells,
Animal cells and the like can be mentioned, but Escherichia coli is particularly preferable.
The DNA of the present invention can be introduced into a host cell by a conventional method. When Escherichia coli is used as the host cell, the DNA of the present invention can be introduced by, for example, the calcium chloride method.
Examples of E. coli used as a host include E. coli JM109
Strain, E. coli HB101 strain, E. coli BL21 strain, E. coli B
L21 (DE3) strain and the like are mentioned, and Escherichia coli BL21 (DE3) strain is preferable. Next, a transformant producing a conjugated polyketone reductase, and the present enzyme and / or
Alternatively, a method for producing an optically active alcohol using this transformant will be described. The substrate of the conjugated polyketone reductase is represented by the following general formula (2):

[0029]

[Chemical 5]

(In the formula, X represents a halogen atom.
R ¹ and R ² independently represent a hydrogen atom, an optionally protected hydroxyl group, an alkoxyl group, an alkyl group, a nitro group, an optionally protected amino group, or a cyano group. P ¹ and P ^{2 each} independently represent a hydrogen atom or an amino group-protecting group, or P ¹ and P ² together represent a phthaloyl group. However, the case where P ¹ and P ² are simultaneously hydrogen atoms is excluded. ) Optical activity 1-
Halo-3-amino-4-phenyl-2-butanone derivatives can be used. Of these, substituents X, R ¹ , R ² ,
Examples of the combination of P ¹ and P ² include the following.

Examples of X include chlorine atom, bromine atom, fluorine atom and the like. Of these, chlorine atom is preferred. Examples of R ¹ and R ² include a hydrogen atom, a methyl group, a hydroxyl group, a methoxyl group, and an amino group.

Examples of P ¹ and P ² include a hydrogen atom or an amino group-protecting group. The protecting group for the amino group is not particularly limited, and examples thereof include a protecting group usually used for the protecting group for the amino group.
aggressive Groups in Organi
c Synthesis, Second Edition, John Wile
y & Sons, 1990, pages 309-384, methoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, ethoxycarbonyl, acetyl, trifluoroacetyl, benzyl, dibenzyl. , Phthaloyl group, tosyl group, benzoyl group and the like. Above all,
A methoxycarbonyl group, a t-butoxycarbonyl group, a benzyloxycarbonyl group, a phthaloyl group and the like are preferably used, and a methoxycarbonyl group and a t-butoxycarbonyl group are more preferable.

The above optically active 1-halo-3-amino-4-
The phenyl-2-butanone derivative is disclosed in JP-A-62-126.
It can be prepared by the method disclosed in JP-A No. 158 or JP-A-2-42048.

1-halo-3-amino-4-phenyl-2
-For the reduction of the butanone derivative, the transformant having the DNA of the present invention can be preferably used. Examples of such transformants include E. coli BL21 (DE3) in which the gene for the conjugated polyketone reductase CPRC1 (SEQ ID NO: 2 in the sequence listing) is introduced. coli BL21 (pETC
1) or E. coli BL21 (DE3) into which the gene for the conjugated polyketone reductase CPRC2 (SEQ ID NO: 4 in the sequence listing) was introduced. coli BL21 (pETC2) and the like. Among them, a conjugated polyketone reductase (CPRC2) consisting of the amino acid sequence represented by SEQ ID NO: 3 in Sequence Listing
A transformant having the ability to produce E. coli
BL21 (pETC2) is more preferred.

The transformant E. coli BL21
(PETC1) was assigned under the accession number FERMP-18763, and E. E. coli BL21 (pETC2) has been deposited at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, under the accession number FERM P-18764.

As the method of the reduction reaction, the substrate 1-halo-3-amino-4-phenyl-2-butanone derivative, the coenzyme NADPH, and a culture of the transformant or a treated product thereof, etc., in a suitable solvent. Is added, and the mixture is reacted with stirring under pH adjustment. The reaction can be carried out batchwise or continuously. Here, the treated product of the transformant and the like include, for example, a crude enzyme solution, cultured bacterial cells, freeze-dried bacterial cells, acetone-dried bacterial cells,
Alternatively, it means a ground product thereof, a mixture thereof, or the like. Furthermore, these cultures or treated products thereof can be used by being immobilized by the known means as the enzyme itself or the bacterial cells.

The temperature during the reaction is usually 10 to 60 ° C., preferably 20 to 40 ° C., and the pH during the reaction is 2.5 to
9, preferably in the range of 5-9. The amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates. The substrate concentration in the reaction solution is 0.01 to 50.
% (W / V) is preferable, and more preferably 0.1 to 3
It is 0% (W / V). The reaction time is appropriately determined depending on the substrate concentration, the amount of enzyme source and other reaction conditions. Normal,
It is preferable to set each condition so that the reaction is completed in 2 to 168 hours.

In order to promote the reduction reaction, the reaction can be promoted by adding the coenzyme NADPH required for the reduction reaction. In this case, specifically, these are added directly to the reaction solution.

Further, in order to accelerate the reduction reaction, N
It is preferable to carry out the reaction in the presence of an enzyme that reduces ADP to each reduced form and a substrate for reduction, because excellent results can be obtained. For example, glucose dehydrogenase is allowed to coexist as an enzyme that reduces to a reduced form, glucose is allowed to coexist as a substrate for reduction, or formate dehydrogenase is allowed to coexist as an enzyme to reduce to a reduced form and formic acid is allowed to coexist as a substrate for reduction.

Furthermore, it is also effective to add a surfactant such as Triton (manufactured by Nacalai Tesque), Span (manufactured by Kanto Kagaku), Tween (manufactured by Nacalai Tesque) to the reaction solution. Further, for the purpose of avoiding the inhibition of the reaction by the substrate and / or the alcohol product which is the product of the reduction reaction, ethyl acetate, butyl acetate, isopropyl ether,
A water-insoluble organic solvent such as toluene or hexane may be added to the reaction solution. Further, for the purpose of enhancing the solubility of the substrate, a water-soluble organic solvent such as methanol, ethanol, acetone, tetrahydrofuran or dimethylsulfoxide may be added.

Optically active 1-halo-obtained by the above method
The collection of the 3-amino-4-phenyl-2-butanol derivative is not particularly limited, but may be carried out directly from the reaction solution or after separating the bacterial cells with a solvent such as ethyl acetate, toluene, t-butyl methyl ether or hexane. After extraction, dehydration, and purification by crystallization, silica gel column chromatography, or the like, highly pure optically active 1-halo-3-amino-
A 4-phenyl-2-butanol derivative can be easily obtained.

[0042]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

In the following Examples, the polyketone reductase activity was measured by using a 200 mM phosphate buffer solution (pH 7.
0) substrate ketopantoyl lactone 0.4 mM, NAD
1. containing PH 0.32 mM and enzyme solution 0.05 ml
React in 5 ml of reaction solution at 30 ℃ for 3 minutes,
This was done by measuring the decrease in absorbance at 0 nm. This was used as a standard reaction condition for reducing activity measurement. Under these reaction conditions, 1 μmol of NADPH was added to NADP for 1 minute.
The enzyme activity that oxidizes to 1 was defined as 1 unit.

The detailed operation method and the like regarding the recombinant DNA technology used in the following examples are described in the following textbooks. 1) Molecular Cloning, Second Edition, C
old Spring Harbor Laborato
RY Publishing, 1989. 2) Current Protocols in Mo
regular Biology, John Wile
Published by y & Sons, 1987.

Example 1 Purification of Enzymes Candida parapsilosis (Candida pa)
rapsilosis) IFO708 cells were crushed,
A cell-free extract obtained by centrifugation was obtained. This was fractionated by ammonium sulfate precipitation, and the precipitate generated in the 40-60% saturated fraction was dissolved in a buffer solution and dialyzed. This was applied to a MonoQHR10 / 10 column (Amersham Pharmacia Biotech) and potassium chloride (0-
Elution at 1M). Active fractions were collected and sodium chloride was added to 4 M to add Phenyl-sepha.
Rose HR10 / 10 column (manufactured by Amersham Pharmacia Biotech Co., Ltd.) was used for sodium chloride (4-
Elute at 0M). The active fraction eluted earlier was used as CPRC.
1, the active fraction eluted later was designated as CPRC2. Each was gel-filtered by Superdex 75HR10 / 30 column (Amersham Pharmacia Biotech). After the active fraction was dialyzed, it was applied to a MonoQHR5 / 5 column (manufactured by Amersham Pharmacia Biotech) and eluted with potassium chloride (0-0.6M), and the active fraction was used as a purified enzyme.

Example 2 Gene encoding CPRC1
Cloning (acquisition of chromosomal DNA) Candida parapsilosis IFO
Hereford (Cell, 18
Chromosomal DNA was extracted according to the method described in Vol. (Cloning of gene encoding CPRC1 by PCR) Candida parapsilosis IFO070
After denaturing CPRC1 purified from 8 in the presence of 8M urea, it was digested with lysyl endopeptidase derived from Achromobacter (manufactured by Wako Pure Chemical Industries, Ltd.), and the sequence of the obtained peptide fragment was determined by the Edman method. Considering the DNA sequence predicted from this amino acid sequence, two PCR primers (primer 1: 5'CAYATHGAYGGI
GCIGARATHTAYGG 3 ', primer 2:
5'TAYTCIACDATICCIGGGNGTYTG
RTCYTG 3 ') was synthesized. 250 pmol each of two primers (primer 1 and primer 2), 2300 ng of chromosomal DNA, 10 nmol of dNTP each, Ex
50 μl of ExTaq buffer containing 1.5 U of Taq (Takara Shuzo) was prepared, and heat denaturation (94 ° C., 1 min), annealing (55 ° C., 1 min), extension reaction (72 ° C., 2 min).
35 cycles, and after cooling to 4 ° C., the amplified DNA was confirmed by agarose gel electrophoresis. (Subcloning of DNA fragment amplified by PCR method) The amplified DNA was cloned into pT7Blue Vector (No.
(manufactured by Vagen) and the base sequence was determined. As a result, the amplified DNA was composed of 443 bases including the primer sequence. The sequence is a DNA sequence part underlined in the DNA sequence shown in FIG. Hereinafter, this sequence is referred to as "core sequence". (Cloning of sequences around core sequence by inverse PCR method)
Complementary sequence 5'TCATA near the 5'side of the core sequence
AGTATGGTTACCCGAATTG 3 '(primer 3) and the sequence 5'TGGTG near the 3'side
TTTCCAATTTCACTATTGAG 3 '(primer 4) was prepared. As a template for the inverse PCR, first, chromosome D of Candida parapsilosis IFO708
NA was digested with the restriction enzyme HincII and the digest was digested with T
Self-circularization was performed using 4 DNA ligase. 500 ng of this self-cyclized product, 50 pmol each of two primers (primer 3 and primer 4), 10 nmol each of dNTP,
ExTaq including 2.5U of ExTaq (Takara Shuzo)
Prepare 50 μl of buffer solution and heat denaturate (94 ° C, 1 min),
Annealing (48 ° C, 1 min), extension reaction (74 ° C, 4
Min.) For 30 cycles, and after cooling to 4 ° C., the amplified DNA was confirmed by agarose gel electrophoresis.

The amplified DNA was labeled with pT7Blue Vector.
r (manufactured by Novagen) was subcloned and its nucleotide sequence was determined. From this result and the result of the core array,
The entire nucleotide sequence of the DNA encoding CPRC1 was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are summarized in Fig. 1. Candida parapsilosis IFO0708-derived conjugated polyketone reductase CPRC
The amino acid sequence of 1 is shown in SEQ ID NO: 1 in the sequence listing. The nucleotide sequence of the DNA encoding this enzyme is shown in SEQ ID NO: 2 in the sequence listing.

Example 3 Recombination containing CPRC1 gene
Preparation of vector A recombinant vector used for transformation was prepared in order to express CPRC1 in E. coli. First, CPRC
NdeI site is added to the start codon portion of the structural gene of 1 and a new stop codon and Xho are added immediately after the stop codon.
Double-stranded DNA with I site added was obtained by the following method. Based on the nucleotide sequence determined in Example 2, CPRC
Primer 5 (5'ATACATATGTCACT) in which an NdeI site was added to the start codon of the structural gene of 1
TGCTGGAAAAGAATTT 3 ') and CPR
XhoI is located 6 bases downstream of the stop codon of the C1 structural gene.
Primer 6 with added site (5'TATCTCGAG
GTATCCTCAATCATACTTGGA 3 ')
And were synthesized.

100 pmol each of the above two primers (primer 5 and primer 6), 2300 ng of chromosomal DNA of Candida parapsilosis IFO708,
dNTP for each 10 nmol, ExTaq (Takara Shuzo)
50 μl of ExTaq buffer containing 2.5 U was prepared, and heat denaturation (94 ° C., 1 minute) and annealing (60 ° C.,
1 minute) and extension reaction (74 ° C., 2 minutes) for 30 cycles, cooled to 4 ° C., and the amplified DNA was confirmed by agarose gel electrophoresis. This amplified fragment was designated as NdeI and Xh.
digested with oI and plasmid pET21a (Novage
NdeI, Xho downstream of the T7 promoter
By inserting into the I site, the recombinant vector pETC
Got 1. The production method and structure of pETC1 are shown in FIG.

Example 4 Production of Recombinant Escherichia coli Escherichia coli BL21 (DE3) (manufactured by Novagen) was transformed with the recombinant vector pETC1 obtained in Example 3 to obtain recombinant Escherichia coli BL21 (pETC1). It was The transformant E. coli BL2 thus obtained
1 (pETC1) is accession number FERM P-1876
3 has been deposited at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology.

Example 5 CPRC in recombinant E. coli
Expression of 1 Recombinant E. coli BL21 (pETC1) obtained in Example 4
2YT medium (bact tryptone 1.6% (w /
v), Bact yeast extract 1.0% (w / v), N
After culturing for 14 hours in aCl 0.5% (w / v), pH 7.0 and collecting the cells, 10 mM Tris-HCl buffer (pH 7.
4) and suspended in ultrasonic waves to obtain a cell-free extract.
The CPRC1 activity of this cell-free extract was measured by the method described above. No activity was observed in Escherichia coli BL21 (pET21a), which is a transformant containing only the vector plasmid, whereas in E. coli BL21 (pETC1), the specific activity was 21.4 units / mg.

Example 6 Gene encoding CPRC2
Cloning (acquisition of chromosomal DNA) in the same manner as in Example 2 from the cells of Candida parapsilosis IFO708 to chromosome DN.
A was extracted. (Cloning of CPRC2 Gene by PCR Method) After denaturing CPRC2 purified from Candida parapsilosis IFO0708 in the presence of 8M urea, it was digested with lysyl endopeptidase derived from Achromobacter (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain The sequence of the obtained peptide fragment was determined by the Edman method. Considering the DNA sequence predicted from this amino acid sequence, two PCR primers (primer 7: 5'GAYGTICCNMGIGARGAY
ATHTGGGT 3 ', primer 8: 5' TAIC
CRTGNGTYTGYTCCIGTIGTRAARAA
3 ') was synthesized. 250 pmol each of the above two primers (primer 7 and primer 8), chromosome DN
A2300 ng, dNTP 10 nmol each, ExTaq
ExTaq buffer 5 containing 1.5U (manufactured by Takara Shuzo)
0 μl was prepared and subjected to heat denaturation (94 ° C., 1 min), annealing (55 ° C., 1 min), extension reaction (72 ° C., 2 min).
After cycling and cooling to 4 ° C, the amplified DNA was confirmed by agarose gel electrophoresis. (Subcloning of DNA fragment amplified by PCR method) The amplified DNA was cloned into pT7Blue Vector (No.
(manufactured by Vagen) and the base sequence was determined. As a result, the amplified DNA was composed of 182 bases including the primer sequence. The sequence is a DNA sequence part underlined in the DNA sequence shown in FIG. Hereinafter, this sequence is referred to as "core sequence". (Cloning of sequences around core sequence by inverse PCR method)
Complementary sequence 5'CCAAC in the portion close to the 5'side of the core sequence
CTGGACATATATTTTGTGG 3 '(primer 9) and the sequence 5'TGATAA near the 3'side
AGCTTTGGCACACGCTTGG 3 '(primer 10) was prepared. First, as a template for inverse PCR, chromosome DN of Candida parapsilosis IFO708 was used.
A was digested with the restriction enzyme HincII, and the digest was digested with T4.
Self-closing was carried out using DNA ligase. 500 ng of this self-cyclized product, 50 pmol each of the above-mentioned two primers (primer 9 and primer 10), and 10 nm each for dNTP.
ol, ExTaq (manufactured by Takara Shuzo) including 2.5U Ex
Prepare 50 μl of Taq buffer and heat denaturate (96 ° C., 1
Min), annealing (57 ° C, 1 min), extension reaction (74
After 30 cycles of 4 ° C.) and cooling to 4 ° C., amplified DNA was confirmed by agarose gel electrophoresis. The amplified DNA was cloned into pT7Blue Vector (Novage
(manufactured by company n) and the base sequence was determined. From this result and the result of the core sequence, the entire base sequence of the DNA encoding CPRC2 was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are summarized in Fig. 3. The amino acid sequence of the conjugated polyketone reductase CPRC2 derived from Candida parapsilosis IFO0708 is shown in SEQ ID NO: 3 in the sequence listing. The nucleotide sequence of the DNA encoding this enzyme is shown in SEQ ID NO: 4 in the sequence listing.

Example 7 Recombination containing CPRC2 gene
Preparation of vector A recombinant vector used for transformation was prepared in order to express CPRC2 in E. coli. First, CPRC
NdeI site was added to the start codon of the structural gene of 2 and a new stop codon and Xho were added immediately after the stop codon.
Double-stranded DNA with I site added was obtained by the following method. Based on the nucleotide sequence determined in Example 6, CPRC
Primer 11 (5'TTACATATAGTCTC with an NdeI site added to the start codon of the structural gene of 2)
AAGTAACTTACTACCA 3 ') and CPR
XhoI is located 5 bases downstream of the stop codon of the C2 structural gene.
Primer 12 with added site (5'TTTCTCGA
GTGTCTCTACAAATCTTTTAAAT
3 ') and were synthesized. Two kinds of primers (primer 11
And primer 12) 100 pmol each, chromosomal DNA 2300 of Candida parapsilosis IFO708
50 μl of ExTaq buffer containing ng and 10 nmol of dNTP each and 2.5 U of ExTaq (manufactured by Takara Shuzo) was prepared and subjected to heat denaturation (94 ° C., 1 minute) and annealing (60 minutes).
C., 1 minute) and extension reaction (74.degree. C., 2 minutes) for 30 cycles. After cooling to 4.degree. C., amplified DNA was confirmed by agarose gel electrophoresis. This amplified fragment was designated as NdeI and X
Digested with hoI and plasmid pET21a (Novag
NdeI, Xh downstream of the T7 promoter
By inserting into the oI site, the recombinant vector pET
C2 was obtained. The production method and structure of pETC2 are shown in FIG.

Example 8 Preparation of Recombinant Escherichia coli Escherichia coli BL21 (DE3) (manufactured by Novagen) was transformed with the recombinant vector pETC2 obtained in Example 7, and recombinant Escherichia coli BL21 (pETC2) was obtained from each. It was The transformant E. coli BL2 thus obtained
1 (pETC2) is accession number FERM P-1876
4 has been deposited at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology.

Example 9 CPRC in recombinant E. coli
Expression of 2 The recombinant E. coli BL21 (pETC2) obtained in Example 8 was treated with 2YT medium (Bacto tryptone 1.6% (w / v),
Bact yeast extract 1.0% (w / v), NaCl
Incubate at 0.5% (w / v), pH 7.0) for 14 hours,
After collecting the cells, the cells were suspended in 10 mM Tris-HCl buffer (pH 7.4) and ultrasonically disrupted to obtain a cell-free extract. The CPRC2 activity of this cell-free extract was measured by the method described above. Escherichia coli BL2 which is a transformant containing only vector plasmid
1 (pET21a) showed no activity, whereas E. coli BL21 (pETC2) had a specific activity of 4.
26 units / mg.

Example 10 Optically active 1-chloro-3-a
Synthesis of mino-4-phenyl-2-butanol 0.6 mg / ml NADP, 0.2 mg / ml glucose dehydrogenase (manufactured by Amano Enzyme), 5% (w /
v) glucose, 50 mM phosphate buffer (pH 7.
0) Reaction solution 0.5 containing 2 mM 1-chloro-3-amino-4-phenyl-2-butanone (ethanol solution)
E. coli BL21 (pETC2) described in Example 9 was added to ml.
2.5 mg of the freeze-dried product was added and reacted at 28 ° C. for 1 hour. 0.5 ml of acetonitrile was added to the reaction solution and the mixture was centrifuged, and the supernatant was analyzed by HPLC (column: YM
C-Pack ODS-A A-303, manufactured by YMC, column temperature: 40 ° C., mobile phase: water / acetonitrile = 55/45 (v / v), flow rate 1.5 ml / min,
Detection: 210 nm, elution time: (2S, 3S) -1-chloro-3-amino-4-phenyl-2-butanol, 1
0 minutes, (2S, 3R) -1-chloro-3-amino-4-
Phenyl-2-butanol, 12.5 minutes, 1-chloro-
3-Amino-4-phenyl-2-butanone, 18 minutes).
As a result, the diastereomer excess rate of 99% or more ((2
S, 3R) -1-Chloro-3-amino-4-phenyl-
2-Butanol was produced in a yield of 66%.

[0057]

Industrial Applicability According to the present invention, a conjugated polyketone reductase having broad substrate specificity and high stereoselectivity can be easily produced in a large amount. Further, an industrial production method of an optically active alcohol such as optically active 1-halo-3-amino-4-phenyl-2-butanol becomes possible.

[Brief description of drawings]

FIG. 1 shows a DNA sequence containing a gene encoding a conjugated polyketone reductase CPRC1 and its deduced amino acid sequence.

FIG. 2 shows a method for constructing an expression vector of a gene encoding a conjugated polyketone reductase CPRC1 and its structure.

FIG. 3 shows a DNA sequence containing a gene encoding a conjugated polyketone reductase CPRC2 and its deduced amino acid sequence.

FIG. 4 shows a method for constructing an expression vector of a gene encoding a conjugated polyketone reductase CPRC2 and its structure.

[Sequence list] SEQUENCE LISTING <110> Kanegafuchi Chemical Industry Co., Ltd. <120> Conjugated polyketone reductase gene and its use <130> TKS-4729 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 304 <212> PRT <213> Candida parapsilosis <400> 1 Met Ser Leu Ala Gly Lys Glu Phe Lys Leu Ser Asn Gly Asn Lys Ile   1 5 10 15 Pro Ala Val Ala Phe Gly Thr Gly Thr Lys Tyr Phe Lys Arg Gly His              20 25 30 Asn Asp Leu Asp Lys Gln Leu Ile Gly Thr Leu Glu Leu Ala Leu Arg          35 40 45 Ser Gly Phe Arg His Ile Asp Gly Ala Glu Ile Tyr Gly Thr Asn Lys      50 55 60 Glu Ile Gly Ile Ala Leu Lys Asn Val Gly Leu Asn Arg Lys Asp Val  65 70 75 80 Phe Ile Thr Asp Lys Tyr Asn Ser Gly Asn His Thr Tyr Asp Gly Lys                  85 90 95 His Ser Lys His Gln Asn Pro Tyr Asn Ala Leu Lys Ala Asp Leu Glu             100 105 110 Asp Leu Gly Leu Glu Tyr Val Asp Leu Tyr Leu Ile His Phe Pro Tyr         115 120 125 Ile Ser Glu Lys Ser His Gly Phe Asp Leu Val Glu Ala Trp Arg Tyr     130 135 140 Leu Glu Arg Ala Lys Asn Glu Gly Leu Ala Arg Asn Ile Gly Val Ser 145 150 155 160 Asn Phe Thr Ile Glu Asn Leu Lys Ser Ile Leu Asp Ala Asn Thr Asp                 165 170 175 Ser Ile Pro Val Val Asn Gln Ile Glu Phe Ser Ala Tyr Leu Gln Asp             180 185 190 Gln Thr Pro Gly Ile Val Glu Tyr Ser Gln Gln Gln Gly Ile Leu Ile         195 200 205 Glu Ala Tyr Gly Pro Leu Gly Pro Ile Thr Gln Gly Arg Pro Gly Pro     210 215 220 Leu Asp Lys Val Leu Ser Lys Leu Ser Glu Lys Tyr Lys Arg Asn Glu 225 230 235 240 Gly Gln Ile Leu Leu Arg Trp Val Leu Gln Arg Gly Ile Leu Pro Ile                 245 250 255 Thr Thr Thr Ser Lys Glu Glu Arg Ile Asn Asp Val Leu Glu Ile Phe             260 265 270 Asp Phe Glu Leu Asp Lys Glu Asp Glu Asp Gln Ile Thr Lys Val Gly         275 280 285 Lys Glu Lys Thr Leu Arg Gln Phe Ser Lys Glu Tyr Ser Lys Tyr Asp     290 295 300 <210> 2 <211> 915 <212> DNA <213> Candida parapsilosis <400> 2 atg tca ctt gct gga aaa gaa ttt aaa tta tcc aac gga aac aaa atc 48 Met Ser Leu Ala Gly Lys Glu Phe Lys Leu Ser Asn Gly Asn Lys Ile   1 5 10 15 cca gct gtt gca ttt gga act gga act aaa tac ttt aag cgc ggc cac 96 Pro Ala Val Ala Phe Gly Thr Gly Thr Lys Tyr Phe Lys Arg Gly His              20 25 30 aac gat ctc gac aag caa ttg att ggt act ttg gaa ctt gct ttg aga 144 Asn Asp Leu Asp Lys Gln Leu Ile Gly Thr Leu Glu Leu Ala Leu Arg          35 40 45 agt ggg ttc agg cac att gac ggt gct gag att tac ggt acc aac aag 192 Ser Gly Phe Arg His Ile Asp Gly Ala Glu Ile Tyr Gly Thr Asn Lys      50 55 60 gaa att gga atc gct tta aaa aac gtt ggt tta aac aga aaa gat gtc 240 Glu Ile Gly Ile Ala Leu Lys Asn Val Gly Leu Asn Arg Lys Asp Val  65 70 75 80 ttc atc act gac aaa tac aat tcg ggt aac cat act tat gac gga aaa 288 Phe Ile Thr Asp Lys Tyr Asn Ser Gly Asn His Thr Tyr Asp Gly Lys                  85 90 95 cac tcg aaa cac caa aac cca tac aat gca tta aag gct gat ttg gaa 336 His Ser Lys His Gln Asn Pro Tyr Asn Ala Leu Lys Ala Asp Leu Glu             100 105 110 gat ttg gga ttg gag tat gtc gat tta tac ttg atc cat ttc cca tac 384 Asp Leu Gly Leu Glu Tyr Val Asp Leu Tyr Leu Ile His Phe Pro Tyr         115 120 125 att tct gaa aaa tct cac ggg ttc gac ttg gtt gaa gct tgg agg tat 432 Ile Ser Glu Lys Ser His Gly Phe Asp Leu Val Glu Ala Trp Arg Tyr     130 135 140 ttg gaa aga gcc aag aac gaa ggt tta gct cgc aat att ggt gtt tcc 480 Leu Glu Arg Ala Lys Asn Glu Gly Leu Ala Arg Asn Ile Gly Val Ser 145 150 155 160 aat ttc act att gag aat ttg aaa tct atc ttg gat gcc aac acc gat 528 Asn Phe Thr Ile Glu Asn Leu Lys Ser Ile Leu Asp Ala Asn Thr Asp                 165 170 175 tcg atc cca gtg gtt aat caa att gaa ttt agt gct tac ttg caa gac 576 Ser Ile Pro Val Val Asn Gln Ile Glu Phe Ser Ala Tyr Leu Gln Asp             180 185 190 caa act cca gga att gtt gaa tat tca cag caa caa gga att ctt att 624 Gln Thr Pro Gly Ile Val Glu Tyr Ser Gln Gln Gln Gly Ile Leu Ile         195 200 205 gaa gct tat ggt cca ttg ggc cca att act caa gga aga cct ggg ccc 672 Glu Ala Tyr Gly Pro Leu Gly Pro Ile Thr Gln Gly Arg Pro Gly Pro     210 215 220 ttg gat aag gtt ttg tca aaa ttg tca gaa aag tac aag aga aat gaa 720 Leu Asp Lys Val Leu Ser Lys Leu Ser Glu Lys Tyr Lys Arg Asn Glu 225 230 235 240 ggt cag atc tta ttg aga tgg gtg ttg caa aga ggt att ttg ccc att 768 Gly Gln Ile Leu Leu Arg Trp Val Leu Gln Arg Gly Ile Leu Pro Ile                 245 250 255 act aca acc tct aaa gag gaa cgt att aat gat gtt ttg gaa att ttc 816 Thr Thr Thr Ser Lys Glu Glu Arg Ile Asn Asp Val Leu Glu Ile Phe             260 265 270 gat ttc gag ttg gat aaa gaa gat gag gat cag atc act aaa gtc gga 864 Asp Phe Glu Leu Asp Lys Glu Asp Glu Asp Gln Ile Thr Lys Val Gly         275 280 285 aag gaa aaa act ctt aga caa ttt tcg aaa gag tat tcc aag tat gat 912 Lys Glu Lys Thr Leu Arg Gln Phe Ser Lys Glu Tyr Ser Lys Tyr Asp     290 295 300 tga 915 <210> 3 <211> 307 <212> PRT <213> Candida parapsilosis <400> 3 Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly   1 5 10 15 Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala              20 25 30 Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu          35 40 45 Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn      50 55 60 Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg  65 70 75 80 Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile                  85 90 95 Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala             100 105 110 Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe         115 120 125 Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu     130 135 140 Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile 145 150 155 160 Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro                 165 170 175 Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe             180 185 190 Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly         195 200 205 Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Glu     210 215 220 Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys 225 230 235 240 Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile                 245 250 255 Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu             260 265 270 Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn         275 280 285 Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe     290 295 300 Lys Asp Leu 305 <210> 4 <211> 924 <212> DNA <213> Candida parapsilosis <400> 4 atg act caa agt aac tta cta cca aaa aca ttt cgt acc aaa tct ggg 48 Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly   1 5 10 15 aag gag ata tca att gca ctt gga aca ggg acc aaa tgg aaa caa gcc 96 Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala              20 25 30 caa aca att aac gat gtt agt act gag ttg gtg gac aat atc ctt ttg 144 Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu          35 40 45 ggg tta aag ttg ggt ttt aga cac att gat act gct gaa gct tac aac 192 Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn      50 55 60 acg caa aag gaa gtt ggt gaa gcc ctc aaa aga acc gat gtt cca aga 240 Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg  65 70 75 80 gag gat att tgg gtt acc aca aaa tat agt cca ggt tgg ggt tca atc 288 Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile                  85 90 95 aag gca tac agt aaa tcg cca agt gat tca att gat aaa gct ttg gca 336 Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala             100 105 110 cag ctt ggt gtt gac tac gtt gat tta ttt ttg att cac tcc cca ttc 384 Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe         115 120 125 ttc acc act gag caa act cat gga tat aca tta gag caa gct tgg gaa 432 Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu     130 135 140 gct ttg gtt gaa gca aag aag gcg gga aag gtt aga gaa att ggt atc 480 Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile 145 150 155 160 tca aat gct gct att cca cac ttg gaa aaa ctc ttt gct gcc tcc ccg 528 Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro                 165 170 175 agt cct gag tac tac cct gtt gtc aac caa att gaa ttc cat cca ttc 576 Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe             180 185 190 ttg caa aac caa tct aaa aac att gtt aga ttt tgt caa gag cat ggg 624 Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly         195 200 205 atc ttg gtc gaa gct ttt tcg cca ttg gcg cca ttg gca aga gtt gaa 672 Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Glu     210 215 220 act aat gct ctc gct gag aca tta aag aga ttg gcg gaa aag tac aaa 720 Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys 225 230 235 240 aag acc gaa gct caa gtt tta ttg agg tat act ttg caa aga ggt att 768 Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile                 245 250 255 ttg cca gtg aca aca tcg tca aag gaa agc aga tta aaa gag tct ttg 816 Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu             260 265 270 aat ttg ttt gac ttt gaa ttg act gac gag gag gtt aat gaa atc aac 864 Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn         275 280 285 aag att ggc gat gct aat ccc tat aga gct ttc ttc cat gag caa ttt 912 Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe     290 295 300 aaa gat ttg tag 924 Lys Asp Leu 305

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/02 C12N 15/00 ZNAA C12P 13/00 5/00 AF term (reference) 4B024 AA03 BA08 CA02 DA06 4B050 CC03 DD04 FF01 FF04 FF05 FF11 FF12 LL05 4B064 AE01 CA19 CB18 CC24 CD12 DA01 4B065 AA26X AA73Y AB01 CA28 CA44

Claims (14)

[Claims] 1. A DNA of the following (a) or (b): (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 (b) 1 or a number in the amino acid sequence of SEQ ID NO: 1 A DNA consisting of an amino acid sequence in which individual amino acids have been deleted, substituted, inserted or added, and encoding a protein having a conjugated polyketone reductase activity. 2. The following DNA (a) or (b): (a) DNA consisting of the base sequence shown in SEQ ID NO: 2 (b) One or several bases in the base sequence shown in SEQ ID NO: 2 A DNA consisting of a deleted, substituted, inserted or added nucleotide sequence and encoding a protein having a conjugated polyketone reductase activity. 3. A recombinant vector containing the DNA according to claim 1 or 2. 4. A transformant containing the recombinant vector according to claim 3. 5. A method for producing a conjugated polyketone reductase, which comprises culturing the transformant according to claim 4 in a medium and collecting the conjugated polyketone reductase from the culture. 6. The following DNA (a) or (b): (a) DNA encoding a protein consisting of the amino acid sequence shown by SEQ ID NO: 3 (b) 1 or a number in the amino acid sequence shown by SEQ ID NO: 3 A DNA consisting of an amino acid sequence in which individual amino acids have been deleted, substituted, inserted or added, and encoding a protein having a conjugated polyketone reductase activity. 7. The following DNA (a) or (b): (a) DNA consisting of the base sequence shown in SEQ ID NO: 4 (b) One or several bases in the base sequence shown in SEQ ID NO: 4 A DNA consisting of a deleted, substituted, inserted or added nucleotide sequence and encoding a protein having a conjugated polyketone reductase activity. 8. A recombinant vector containing the DNA according to claim 6 or 7. 9. A transformant containing the recombinant vector according to claim 8. 10. A method for producing a conjugated polyketone reductase, which comprises culturing the transformant according to claim 9 in a medium and collecting the conjugated polyketone reductase from the culture. 11. General formula (1): (In the formula, X represents a halogen atom. R ¹ and R ² are independently a hydrogen atom, an optionally protected hydroxyl group,
It represents an alkoxyl group, an alkyl group, a nitro group, an amino group which may be protected, or a cyano group. P ¹ , P ²
Each independently represent a hydrogen atom or a protecting group for an amino group, or P ¹ and P ² together represent a phthaloyl group. However, the case where P ¹ and P ² are simultaneously hydrogen atoms is excluded. ) Is represented by (1S, 2S) -1-halo-3-
A method for producing an amino-4-phenyl-2-butanol derivative, comprising the general formula (2): (In the formula, X, R ¹ , R ² , P ¹ and P ² are the same as above.) And a 1-halo-3-amino-4-phenyl-2-butanone derivative and a conjugated polyketone reductase or A method for producing a (1S, 2S) -1-halo-3-amino-4-phenyl-2-butanol derivative, which comprises reacting a culture of a microorganism having enzyme-producing ability or a treated product thereof. 12. The conjugated polyketone reductase is
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 and conjugated The method according to claim 11, which is a protein having polyketone reductase activity. 13. The method according to claim 11, wherein the microorganism is the transformant according to claim 9. 14. X is a chlorine atom, R ¹ and R ² are hydrogen atoms,
14. The method according to claim 11, wherein P ¹ is a t-butoxycarbonyl group and P ² is a hydrogen atom.