Carbonyl reductase, nucleic acid encoding the enzyme, and method for producing optically active compounds using the same

Abstract

The present invention provides a carbonyl reductase having an activity of reducing a carbonyl-containing compound to convert it into an optically active compound, and a method for producing an optically active compound using the enzyme. Specifically, the present invention provides a carbonyl reductase having one or more of mutations in which aspartic acid at position 54, methionine at position 157, alanine at position 170, isoleucine at position 211, methionine at position 214, and methionine at position 249 are each substituted with other specific amino acids in the amino acid sequence shown in SEQ ID NO: 1 or a homolog of the amino acid sequence, and a method for producing an optically active compound using the carbonyl reductase.

Description

Technical Field

[0001] The present invention relates to a carbonyl reductase having the activity of reducing a carbonyl-containing compound to convert it into an industrially useful compound as an intermediate raw material for medicines, pesticides, etc., namely, an optically active compound, a nucleic acid encoding the carbonyl reductase, a recombinant vector containing the nucleic acid, a transformant containing the recombinant vector, and a method for producing an optically active compound using the carbonyl reductase, etc. Background Art

[0002] Statins such as rosuvastatin, pitavastatin, atorvastatin, fluvastatin, pravastatin, or their salts are HMG-CoA reductase inhibitors and are useful in the treatment of hypercholesterolemia, combined hyperlipidemia, etc. In recent years, as generic drugs have been launched on the market, a method for industrially manufacturing drugs using these statins or their salts at a lower price is desired.

[0003] These statin compounds are optically active alcohols or salts thereof having the following structures.

[0004]

[0005] There is known a method for producing a carbonyl-containing compound by stereoselectively reducing a carbonyl group-containing compound having a structure in which carbonyl groups are continuously present as follows using an organic synthesis method or a biochemical method.

[0006]

[0007] For example, Patent Document 1 describes a method for producing optically active compounds such as optically active alcohols with high optical purity and high concentration by allowing a specific carbonyl reductase (hereinafter sometimes referred to as "OCR1") to act on a carbonyl-containing compound. Patent Document 2 describes a method for producing rosuvastatin calcium using OCR1, and Patent Document 3 describes a method for producing pitavastatin calcium using OCR1.

[0008] Here, OCR1 has poor thermal stability and the stability of the enzyme decreases during reaction (when heated), so a large amount of OCR1 must be used. Therefore, studies on mutants have been conducted for the purpose of improving the thermal stability of OCR1 (Non-Patent Document 1).

[0009] However, in order to industrially produce optically active compounds useful as intermediate raw materials for pharmaceuticals, pesticides, etc., particularly statins, with high purity and high optical purity at low cost, it is desired to further improve the performance of OCR1.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent No. 4270918

[0013] Patent Document 2: International Publication No. 2015 / 119261

[0014] Patent Document 3: International Publication No. 2017 / 022846

[0015] Non-patent literature

[0016] Non-patent literature 1: Journal of Bioscience and Bioengineering, VOL.123 No.6, 673-678, 2017 Summary of the invention

[0017] Problems to be solved by the invention

[0018] The problem to be solved by the present invention is to develop a carbonyl reductase having higher carbonyl reducing activity, thermal stability, stereoselectivity, optical selectivity, etc. than OCR1. In addition, the problem to be solved by the present invention is to obtain an optically active compound industrially with high purity, high optical purity and low price by using the carbonyl reductase.

[0019] Methods used to solve problems

[0020] The present inventors have conducted intensive research to solve the above problems and have found that by replacing the specific amino acid of OCR1 with other specific amino acids, a carbonyl reductase having high carbonyl reduction activity, thermal stability, stereoselectivity, optical selectivity, etc. can be obtained, thereby completing the present invention. It has also been found that by using the carbonyl reductase, an optically active compound can be obtained industrially with high purity, high optical purity and low price, thereby completing the present invention.

[0021] That is, the gist of the present invention is as follows.

[0022] [1] A carbonyl reductase comprising a polypeptide consisting of an amino acid sequence having at least one mutation selected from the group consisting of the following (a) to (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence,

[0023] (a) a mutation in which aspartic acid at position 54 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine,

[0024] (b) a mutation in which methionine at position 157 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine,

[0025] (c) a mutation in which alanine at position 170 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine,

[0026] (d) a mutation in which isoleucine at position 211 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine,

[0027] (e) a mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine,

[0028] (f) A mutation in which methionine at position 249 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine.

[0029] [2] The carbonyl reductase according to [1], characterized in that it has at least two mutations selected from the group consisting of (a) to (f) above.

[0030] [3] The carbonyl reductase according to [1] or [2], which comprises a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence, and further comprising (g) a mutation in which valine at position 166 in the amino acid sequence represented by SEQ ID NO: 1 is replaced by leucine.

[0031] [4] A method for producing an optically active compound represented by general formula (IV), characterized in that the carbonyl-containing compound is contacted with a carbonyl reductase according to any one of [1] to [3], a microorganism or cell capable of producing the above-mentioned enzyme, a processed product of the above-mentioned microorganism or cell, and / or a culture solution containing the above-mentioned enzyme obtained by culturing the above-mentioned microorganism or cell, thereby asymmetric reduction of the carbonyl-containing compound is carried out.

[0032]

[0033] (In the above formulae (I), (II) and (III), R represents a hydrogen atom, an alkyl group or an aryl group, represents a substituent having an aromatic ring and / or a heterocyclic ring)

[0034]

[0035] (In formula (IV), R and Same meaning as above).

[0036] [5] The production method according to [4], characterized in that the carbonyl-containing compounds represented by the above formula (II) and the above formula (III) are optically active substances represented by the following formula (II') and the following formula (III'), respectively.

[0037]

[0038] (In the formula, R and Same meaning as above)

[0039]

[0040] (In the formula, R and Same meaning as above).

[0041] [6] The method according to [4] or [5], wherein the substituent for

[0042] (Where, represents a substituent having an aromatic ring and / or a heterocyclic ring),

[0043]

[0044]

[0045] [7] The method according to [6], wherein the substituent for

[0046]

[0047] [8] The production method according to any one of [4] to [7], wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding the carbonyl reductase according to any one of [1] to [3], wherein the nucleic acid contains the base sequence shown in (p), (q) or (r) below,

[0048] (p) a base sequence having a base sequence in which one to a plurality of bases are substituted, deleted and / or added to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity,

[0049] (q) a base sequence having a sequence identity of 90% or more to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity,

[0050] (r) A base sequence having a base sequence that hybridizes with a complementary chain of the base sequence shown in SEQ ID NO: 2 under stringent conditions and encoding a polypeptide having carbonyl reductase activity.

[0051] It should be noted that in this specification, the substituents are sometimes is called "substituent A", and the substituent It is called "substituent B".

[0052] Effects of the Invention

[0053] According to the present invention, a carbonyl reductase having the activity of reducing a carbonyl-containing compound to convert it into an industrially useful compound as an intermediate raw material for medicines, pesticides, etc., i.e., an optically active compound, a nucleic acid encoding the carbonyl reductase, a recombinant vector containing the nucleic acid, and a transformant containing the recombinant vector can be provided. Furthermore, according to the present invention, a method for industrially obtaining an optically active compound that is industrially useful as an intermediate raw material for medicines, pesticides, etc., with high purity and high optical purity and at low price can be provided. DETAILED DESCRIPTION

[0054] The present invention is described in detail below.

[0055] 1. Carbonyl reductase of the present invention

[0056] The carbonyl reductase of the present invention comprises a polypeptide consisting of an amino acid sequence having at least one mutation selected from the group consisting of the following (a) to (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence, and has carbonyl reductase activity.

[0057] (a) Mutation in which aspartic acid at position 54 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine

[0058] (b) Mutation in which methionine at position 157 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine

[0059] (c) Mutation in which alanine at position 170 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine

[0060] (d) Mutation in which the isoleucine at position 211 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine

[0061] (e) Mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine

[0062] (f) Mutation in which methionine at position 249 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine

[0063] In the present invention, carbonyl reductase activity refers to the activity of converting asymmetric reduction of carbonyl groups in carbonyl-containing compounds into optically active compounds. Whether there is carbonyl reductase activity can be determined by measuring the activity of converting asymmetric reduction of carbonyl groups in carbonyl-containing compounds into optically active compounds using conventional test methods. For example, the carbonyl reductase as the object of measurement, the microorganism or cell having the ability to produce the enzyme, the processed material of the microorganism or cell, and / or the culture solution containing the above enzyme obtained by culturing the microorganism or cell is acted on the carbonyl-containing compound shown in the general formula (I), (II) or (III), and the amount of the compound shown in the general formula (IV) converted from these carbonyl-containing compounds is directly measured, thereby confirming the carbonyl reductase activity. In addition, in the case of a measurement system containing NADPH as a coenzyme, the carbonyl reductase activity can be confirmed by measuring the initial reduction rate of NADPH.

[0064] In the present invention, the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence (OCR1) derived from methanol-inducible yeast nonfermentative variant (Ogataea minuta var. nonfermentans) NBRC (formerly IFO) 1473 described in Japanese Patent No. 4270918.

[0065] In the present invention, a homolog of the amino acid sequence shown in SEQ ID NO: 1 refers to a polypeptide having an amino acid sequence in which one to a plurality of amino acids are deleted, inserted, substituted and / or added to the amino acid sequence shown in SEQ ID NO: 1 and having carbonyl reductase activity.

[0066] “One to a plurality of amino acids” means usually 1 to 100, preferably 1 to 50, more preferably 1 to 20, further preferably 1 to 10, particularly preferably 1 to 5 amino acids.

[0067] In addition, in the present invention, a homolog of the amino acid sequence shown in SEQ ID NO: 1 refers to a polypeptide having an amino acid sequence having a sequence identity of 90% or more to the entire length of the amino acid sequence shown in SEQ ID NO: 1 and having carbonyl reductase activity. Preferably, the polypeptide has an amino acid sequence having a sequence identity of 95% or more, more preferably 98% or more, and even more preferably 99% or more to the entire length of the amino acid sequence shown in SEQ ID NO: 1 and having carbonyl reductase activity.

[0068] The homology (also referred to as identity or similarity) of the amino acid sequences in the present specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), for example, under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filter = off). Other algorithms for determining the homology of amino acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [this algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48:444-453 (1970) [this algorithm has been integrated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4:11-17 (1988) [this algorithm has been integrated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988) [this algorithm has been integrated into the FASTA program in the GCG software package], etc., can also be preferably used.

[0069] The amino acid sequence represented by the above SEQ ID NO: 1 and homologues of the amino acid sequence can be obtained by the method described in Japanese Patent No. 4270918.

[0070] The carbonyl reductase of the present invention has excellent properties because it has a mutation (a), (b), (c), (d), (e) or (f) in the amino acid sequence shown in SEQ ID NO: 1 or a homolog of the amino acid sequence. Each mutation is described below in the order of (d), (c), (f), (a), (b), and (e).

[0071] (d) A mutation in which isoleucine at position 211 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine. This mutation improves stereoselectivity and optical selectivity.

[0072] Specifically, the performance of selectively reducing the 3-position carbonyl of the carbonyl-containing compound represented by the following general formula (I) or (II) is improved. Therefore, the optically active compound represented by the general formula (IV) can be obtained from the carbonyl-containing compound represented by the general formula (I) or (II) with high yield and high chemical purity. In addition, the (d) mutation also improves the optical selectivity, and therefore, the optically active compound represented by the general formula (IV) can be obtained from the carbonyl-containing compound represented by the general formula (I), (II) or (III) with high yield and high optical purity.

[0073]

[0074] In particular, the substituent A is When , the performance of selective reduction of the 3-position carbonyl group is high.

[0075] Therefore, for example, the optically active compounds shown below can be obtained with high yield, high chemical purity and high optical purity by the following reaction.

[0076]

[0077] (c) A mutation in which alanine at position 170 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine. This mutation improves thermal stability.

[0078] (f) is a mutation in which methionine at position 249 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine. This mutation improves thermal stability.

[0079] Through these mutations, even in the reduction reaction under heating conditions, the activity of the carbonyl reductase does not decrease but maintains high enzyme activity, so that the optically active compound shown in the general formula (IV) can be obtained with high yield and high chemical purity from the carbonyl-containing compound shown in the general formula (I), (II) or (III). In addition, when there are two mutations (c) and (f), the thermal stability is further improved, so it is preferred.

[0080] (a) A mutation in which aspartic acid at position 54 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine. This mutation improves stereoselectivity.

[0081] Specifically, the performance of selectively reducing the 3-position carbonyl group of the carbonyl-containing compound represented by the general formula (I) or (II) is improved, so that the optically active compound represented by the general formula (IV) can be obtained with high yield and high chemical purity.

[0082] In particular, the substituent A is When the reaction rate and efficiency of carbonyl-containing compounds are improved.

[0083] Therefore, for example, by the following reaction, the production rate and production efficiency of the optically active compound shown below are improved, and the optically active compound can be obtained with high yield, high chemical purity and high optical purity.

[0084]

[0085] (b) A mutation in which methionine at position 157 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine. This mutation improves stereoselectivity and optical selectivity.

[0086] (e) A mutation is a mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine. This mutation improves stereoselectivity and optical selectivity.

[0087] Here, when a carbonyl reductase having an amino acid sequence shown in sequence number 1 is allowed to act on a carbonyl-containing compound represented by the following general formula (I), the carbonyl group at position 3 is preferentially reduced compared to the carbonyl group at position 5, and thus, a compound represented by the following general formula (III) is generated in greater amounts than a compound represented by the following general formula (II). In addition, the reaction rate of the compound represented by the general formula (III) to the compound represented by the following general formula (IV) is slower than the reaction rate of the compound represented by the general formula (II) to the optically active compound represented by the general formula (IV), and thus the compound represented by the general formula (III) may remain.

[0088]

[0089]

[0090] When the carbonyl reductase having the mutation (b) and the mutation (e) is allowed to act on the carbonyl-containing compound represented by the general formula (I), the reduction of the carbonyl group at position 3 is suppressed, and the carbonyl group at positions 3 and 5 are reduced in a well-balanced manner. In addition, the reaction rate and reaction efficiency of converting the compounds represented by the general formulas (II) and (III) to the optically active compound represented by the general formula (IV) are improved, so that the optically active compound represented by the general formula (IV) can be efficiently obtained with high yield and high chemical purity.

[0091] In particular, the substituent A is When the carbonyl-containing compound is converted into an optically active compound, the reaction rate and reaction efficiency are further improved.

[0092] Therefore, for example, by the following reaction, the production rate and production efficiency of the optically active compound shown below are improved, and the optically active compound can be obtained with high yield, high chemical purity and high optical purity.

[0093]

[0094] In addition, the presence of both mutations (b) and (e) is more preferred because the reaction efficiency with respect to the carbonyl-containing compound is also improved.

[0095] The carbonyl reductase of the present invention is a carbonyl reductase having a polypeptide consisting of an amino acid sequence having at least one mutation selected from the group consisting of (a) to (f) in the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having carbonyl reductase activity as a homolog of the amino acid sequence. Among the mutations (a), (b), (c), (d), (e) or (f), one or more mutations can be appropriately selected according to the type of the optically active compound represented by the above formula (IV) as the target, preferably at least two mutations selected from the group consisting of (a) to (f), and more preferably at least three mutations.

[0096] In addition, the carbonyl reductase of the present invention may have a (g) mutation. The (g) mutation is a mutation described in Non-Patent Document 1, in which valine at position 166 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. This mutation improves the thermal stability of the carbonyl reductase.

[0097] The carbonyl reductase of the present invention can be produced from the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having carbonyl reductase activity which is a homolog of the amino acid sequence by methods well known to those skilled in the art, such as site-directed mutagenesis, PCR, and other well-known techniques.

[0098] In addition, the carbonyl reductase of the present invention can also be produced by culturing a transformant containing a nucleic acid encoding the enzyme, and isolating and purifying the carbonyl reductase from the obtained culture. The nucleic acid encoding the carbonyl reductase of the present invention can be either DNA or RNA, or can be a DNA / RNA chimera. DNA is preferably cited. In addition, the nucleic acid can be either double-stranded or single-stranded. When it is double-stranded, it can be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. When it is single-stranded, it can be either a sense strand (i.e., a coding strand) or an antisense strand (i.e., a non-coding strand).

[0099] Examples of DNA encoding the carbonyl reductase of the present invention include synthetic DNA. For example, the full-length carbonyl reductase cDNA obtained by direct amplification by reverse transcriptase-PCR using total RNA or mRNA fraction prepared from cells or tissues derived from methanol-inducible yeast non-fermentative variant NBRC1473 as a template can be prepared using a known kit, such as Mutan TM -super Express Km(TAKARA BIO INC.)、MutanTM -K (TAKARABIO INC.) etc. according to the ODA-LA PCR method, Gapped duplex method, Kunkel method etc. known methods themselves or methods based on these methods are transformed and obtained. Alternatively, a cDNA library prepared by inserting the above-mentioned total RNA or mRNA fragments into an appropriate vector is cloned by colony hybridization method, plaque hybridization method or PCR method etc., and the obtained cDNA is transformed according to the above-mentioned method. The vector used for the library can be any one of phage, plasmid, cosmid, phagemid etc.

[0100] The nucleic acid encoding the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is not limited as long as it encodes the polypeptide having carbonyl reductase activity of the present invention, and in particular, nucleic acids having the base sequence shown in the following (p), (q) or (r) can be mentioned.

[0101] (p) A nucleic acid having a base sequence in which one to a plurality of bases are substituted, deleted and / or added to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention

[0102] (q) A nucleic acid having a base sequence having a sequence identity of 90% or more to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention

[0103] (r) A nucleic acid having a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention

[0104] As homologs of the nucleic acid shown in (p) above, there can be mentioned nucleic acids encoding the polypeptide having carbonyl reductase activity of the present invention, which contain a base sequence in which one to multiple bases are deleted, substituted, inserted and / or added to the base sequence shown in SEQ ID NO: 2. In the case of substitution, insertion or addition, one to multiple bases are preferably substituted, inserted or added. Here, "one to multiple bases" is, for example, 1 to 60 bases, preferably 1 to 30 bases, more preferably 1 to 15 bases, further preferably 1 to 10 bases, and particularly preferably 1 to 5 bases.

[0105] As a homolog of the nucleic acid shown in (q) above, there can be mentioned a nucleic acid having a base sequence having a sequence identity of 90% or more with the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention. Preferably, the nucleic acid has a base sequence having a homology (also referred to as identity) of 95% or more, more preferably 98% or more, and further preferably 99% or more with the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention.

[0106] The homology (also referred to as identity) of the base sequence in this specification can be calculated using the homology calculation algorithm NCBIBLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), for example, under the following conditions (expected value = 10; gaps allowed; filter = open; match score = 1; mismatch score = -3). As other algorithms for determining the homology of base sequences, the same preferred examples as the above-mentioned homology calculation algorithm for amino acid sequences can be used.

[0107] As a homolog of the nucleic acid shown in (r) above, as long as it encodes a polypeptide having carbonyl reductase activity of the present invention, it may be a nucleic acid that hybridizes under stringent conditions with the complementary strand of the base sequence of SEQ ID NO: 2. Here, the "stringent conditions" can be appropriately set with reference to the conditions reported (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.16.3.6, 1999), and specifically, for example, conditions such as washing once, more preferably 1 to 3 times, at a salt concentration and temperature equivalent to the washing conditions for ordinary Southern blot hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, more preferably 65°C, 0.1×SSC, 0.1% SDS, 68°C, 0.1×SSC, 0.1% SDS (high stringent conditions), etc.

[0108] For those skilled in the art, by using site-directed mutagenesis (Nucleic Acids Res.10, pp.6487 (1982), Methods in Enzymol.100, pp.448 (1983), Molecular Cloning, PCR APractical Approach IRL Press pp.200 (1991)) and the like to appropriately replace, delete, insert and / or add the nucleic acid represented by sequence number 2 to introduce the desired mutation, the nucleic acid homolog as described above can be obtained.

[0109] The nucleic acid of the present invention can encode a polypeptide having carbonyl reductase activity of the present invention. When the nucleic acid of the present invention has the base sequence shown in SEQ ID NO: 2 or a base sequence having a high identity with the base sequence shown in SEQ ID NO: 2, the degree of carbonyl reductase activity of the carbonyl reductase containing the polypeptide encoded by the nucleic acid may be quantitatively equivalent to the carbonyl reductase containing the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or the carbonyl reductase containing the polypeptide having a homolog of the amino acid sequence, or may be different within an acceptable range (e.g., about 0.1 to about 5 times, preferably about 0.3 to about 3 times).

[0110] In addition, based on the amino acid sequence or a part thereof in the amino acid sequence of sequence number 1, or the base sequence or a part thereof shown in sequence number 2, a homology search can be performed on a database such as the DNA Databank of JAPAN (DDBJ) to obtain the amino acid sequence information of a polypeptide having carbonyl reductase activity or the base sequence information of the DNA encoding the polypeptide.

[0111] In the production method of the present invention described later, the above-mentioned carbonyl reductase can be used to directly react with a carbonyl-containing compound as a substrate, but it is preferred to use a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell.

[0112] As the microorganism or cell having the ability to produce the carbonyl reductase of the present invention, either a microorganism or cell that originally has the ability to produce the carbonyl reductase can be used, or a microorganism or cell that has been endowed with the above-mentioned production ability through breeding can be used. As the microorganism or cell, it can be dead or alive, for example, dormant cells can be preferably used. As the type of microorganism or cell having the ability to produce the carbonyl reductase of the present invention, "host microorganism" or "host cell" described later can be cited.

[0113] As a means of imparting the above-mentioned production ability through breeding, known methods such as gene recombination treatment (transformation) and mutation treatment can be adopted. As the method of transformation, a method of introducing a target DNA, a method of enhancing the expression of the target DNA by modifying an expression control sequence such as a promoter on a chromosome, etc. can be cited.

[0114] Among these, microorganisms or cells transformed with the above-mentioned DNA encoding the polypeptide of the present invention are preferably used.

[0115] As described above, the nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention can be cloned by using chromosomal DNA derived from the methanol-inducible yeast non-fermenting variant NBRC1473 strain as a template and performing PCR using appropriate primers.

[0116] In addition, as described above, the nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention can be cloned by preparing the full-length carbonyl reductase cDNA using total RNA or mRNA derived from the methanol-inducible yeast non-fermentative variant NBRC1473 strain as a template, directly amplifying it by RT-PCR, and then performing PCR using appropriate primers.

[0117] For example, by inserting the DNA encoding the polypeptide of the present invention obtained as above into a known expression vector in an expressible configuration, a polypeptide gene expression vector of the present invention can be provided. In addition, by transforming a host cell with the expression vector, a transformant into which the DNA encoding the polypeptide of the present invention is introduced can be obtained. The transformant can also be obtained by integrating the DNA encoding the polypeptide of the present invention into the host's chromosomal DNA in an expressible manner using methods such as homologous recombination.

[0118] In this specification, "expression vector" refers to a genetic factor used to replicate and express a protein with a desired function in the host organism by integrating and introducing a polynucleotide encoding a protein with a desired function into the host organism. Examples include plasmids, viruses, bacteriophages, cosmids, etc., but are not limited to these. The expression vector is preferably a plasmid.

[0119] In the present specification, the term "transformant" refers to a microorganism or cell into which a target gene is introduced using the above-mentioned expression vector or the like, thereby being capable of expressing a desired trait associated with a protein having a desired function.

[0120] As a method for making a transformant, specifically, the following method can be exemplified: a method of introducing a DNA encoding a polypeptide of the present invention into a plasmid vector, a phage vector, or a viral vector stably present in a host cell, and introducing the expression vector thus constructed into the host cell; a method of directly introducing the DNA into the host genome, and transcribing and translating its genetic information. In this case, it is preferred that a promoter suitable for the host be connected to the 5'-side upstream of the DNA, and further, it is more preferred that a terminator be connected to the 3'-side downstream. As such a promoter and terminator, as long as they are known to function in cells used as hosts, there is no particular limitation, for example, the vectors, promoters, and terminators described in detail in "Microbiology Basics Lecture 8 Genetic Engineering·Kyoritsu Publishing" can be used.

[0121] The host microorganism to be transformed for expressing the carbonyl reductase of the present invention is not particularly limited as long as the host itself does not adversely affect the carbonyl-containing compound as a substrate or the optically active compound as a target, and examples thereof include the following microorganisms.

[0122] Bacteria for which a host-vector system has been established belong to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc.

[0123] Actinomycetes belonging to the genera Rhodococcus and Streptomyces for which host-vector systems have been established.

[0124] Yeasts with established host-vector systems belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, Candida, etc.

[0125] Fungi belonging to the genera Neurospora, Aspergillus, Cephalosporium, Trichoderma, etc., for which a host-vector system has been established.

[0126] The steps for preparing transformants, the construction of recombinant vectors suitable for the host and the host culture methods can be carried out according to the commonly used techniques in the fields of molecular biology, bioengineering and genetic engineering (for example, Green et al., Molecular Cloning: A Laboratory Manual (4) th ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)).

[0127] Preferred host microorganisms, preferred transformation methods for each microorganism, vectors, promoters, terminators, and the like are specifically listed below, but the present invention is not limited to these examples.

[0128] In the case of Escherichia, particularly Escherichia coli, plasmid vectors include pBR and pUC plasmids, and promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (fusion of lac and trp), lambda phage PL and PR, etc. In addition, terminators include terminators derived from trpA, phage, and rrnB ribosomal RNA.

[0129] In the case of Bacillus, examples of vectors include pUB110 plasmids and pC194 plasmids, and integration into chromosomes is also possible. Promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and α-amylase can be used.

[0130] In the case of the genus Pseudomonas, examples of vectors include established common host-vector systems such as Pseudomonas putida and Pseudomonas cepacia, plasmids involved in the decomposition of toluene compounds, and TOL plasmid-based broad-host vectors (containing genes required for autonomous replication such as those derived from RSF1010) pKT240 (Gene, 26, 273-82 (1983)).

[0131] In the case of Brevibacterium, in particular, Brevibacterium lactofermentum, examples of vectors include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). Various promoters and terminators used in Escherichia coli can be used as promoters and terminators.

[0132] In the case of Corynebacterium, particularly Corynebacterium glutamicum, examples of the vector include plasmid vectors such as pCS11 (Japanese Unexamined Patent Publication No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)).

[0133] In the case of the genus Saccharomyces, particularly Saccharomyces cerevisiae, examples of vectors include YRp-based, YEp-based, YCp-based, and YIp-based plasmids, etc. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase can be used.

[0134] In the case of the genus Schizosaccharomyces, examples of vectors include plasmid vectors derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986) and the like. In particular, pAUR224 is commercially available from Takara Bio Co., Ltd. and can be easily used.

[0135] In the case of the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied Aspergillus, and they can be integrated with plasmids or chromosomes for use, and promoters derived from extracellular proteases and amylases can be used (Trends in Biotechnology 7, 283-287 (1989)).

[0136] In addition to the above, host-vector systems corresponding to various microorganisms have been established, and these can also be used appropriately.

[0137] In addition to microorganisms, various host / vector systems have also been established for plants and animals. In particular, systems for expressing large amounts of heterologous proteins in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)) and plants such as rapeseed, corn, and potatoes have been established, as well as cell-free protein synthesis systems using Escherichia coli cell-free extracts, wheat germ, etc., which can also be appropriately utilized.

[0138] Examples of the treated products of the microorganism or cells having the ability to produce the carbonyl reductase of the present invention include treated products obtained by treating the microorganism or cells with an organic solvent such as acetone, dimethyl sulfoxide (DMSO) or toluene, or a surfactant, treated products obtained by freeze-drying, cell preparations such as those obtained by physical disruption or enzymatic disruption, treated products obtained by extracting an enzyme fraction from the microorganism or cells in the form of a crude or purified product, and products obtained by immobilizing these substances on a carrier represented by polyacrylamide gel, carrageenan, and the like.

[0139] Examples of the culture solution containing the enzyme obtained by culturing a microorganism or cell having the ability to produce the carbonyl reductase of the present invention include a suspension of the cells and a liquid culture medium, a supernatant obtained by removing the cells by centrifugation when the cells are secretory expression cells, and a concentrate thereof.

[0140] 2. Compositions of the present invention

[0141] The composition (enzyme agent) of the present invention contains the carbonyl reductase of the present invention, a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the above enzyme obtained by culturing the microorganism or cell, and is a substance that catalyzes a reaction of generating an optically active compound represented by the general formula (IV) using a carbonyl-containing compound represented by the general formula (I), (II) or (III) as a substrate. The composition of the present invention is useful because it can be used as a catalyst to industrially produce the optically active compound that is industrially useful as an intermediate raw material for drugs, pesticides, etc. with high purity and high optical purity at a low price.

[0142]

[0143]

[0144] The composition of the present invention may contain, in addition to the active ingredient (enzyme, etc.), an excipient, a buffer, a suspending agent, a stabilizer, a preservative, a preservative, a physiological saline, etc. As an excipient, lactose, sorbitol, D-mannitol, sucrose, etc. may be used. As a buffer, phosphates, citrates, acetates, etc. may be used. As a stabilizer, propylene glycol, ascorbic acid, etc. may be used. As a preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. may be used. As a preservative, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. may be used.

[0145] 3. Method for producing the optically active compound of the present invention

[0146] According to the present invention, a method for producing an optically active compound represented by the following general formula (IV) can be provided, which comprises the step of allowing the carbonyl reductase of the present invention to act on a carbonyl-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II) and (III) to produce an optically active compound represented by the following general formula (IV).

[0147]

[0148]

[0149] In addition, the carbonyl group-containing compounds represented by the above formula (II) and the above formula (III) are preferably optically active substances represented by the following formula (II') and the following formula (III'), respectively.

[0150]

[0151] In the present specification, R represents a hydrogen atom, an alkyl group or an aryl group. The alkyl group is preferably a linear or branched alkyl group having 1 to 8 carbon atoms, more preferably a linear alkyl group having 1 to 4 carbon atoms, and particularly preferably an ethyl group or an n-propyl group.

[0152] In addition, the substituent A represents a substituent having an aromatic ring and / or a heterocyclic ring. Specifically, as the substituent A, a substituent having an aromatic ring containing a fluorine atom as a substituent, a substituent having a heterocyclic ring containing a nitrogen atom, and / or a substituent having a naphthalene ring is preferred, among which

[0153] Especially preferred

[0154] Here, the substituent B represents a substituent having an aromatic ring and / or a heterocyclic ring. Specifically, as the substituent B, a substituent having a heterocyclic ring containing a nitrogen atom is preferred, and

[0155]

[0156] In the present invention, the substituent A is particularly preferably

[0157] When the carbonyl reductase of the present invention is brought into contact with a carbonyl-containing compound represented by the above-mentioned general formula (I), (II) or (III), the optically active compound represented by the above-mentioned general formula (IV) can be produced by bringing the purified or roughly purified carbonyl reductase of the present invention, a microorganism or cell having the ability to produce the carbonyl reductase of the present invention (e.g., a transformant having a DNA encoding the polypeptide of the present invention), a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell into contact with the carbonyl-containing compound represented by the above-mentioned general formula (I), (II) or (III).

[0158] The carbonyl reductase of the present invention can be used directly in the reaction, but it is preferred to use a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell, among which it is preferred to use a transformant having a DNA encoding the polypeptide of the present invention.

[0159] The amount of the microorganism or cell added to the reaction solution, the processed material of the microorganism or cell, and / or the culture solution containing the enzyme obtained by culturing the microorganism or cell can be appropriately selected according to the carbonyl-containing compound as the substrate. For example, when adding a microorganism or cell, the concentration of the microorganism or cell in the reaction solution is usually about 0.1w / v% to 50w / v%, preferably 1w / v% to 20w / v%, and when using a processed material or culture solution, the specific activity of the enzyme is obtained, and when adding, the amount reaching the above-mentioned cell concentration is added. Here, w / v% means weight / volume%.

[0160] In addition, from the perspective of good handling and easy addition to the reaction solution, the carbonyl reductase added to the reaction solution, the microorganism or cell having the ability to produce the enzyme, the processed product of the microorganism or cell, and / or the culture solution containing the enzyme obtained by culturing the microorganism or cell can also be used in a frozen state. When used in a frozen state, its shape is not particularly limited, and it can be, for example, prism-shaped, cylindrical, block-shaped, spherical, etc.

[0161] The reaction method is not particularly limited, and a carbonyl-containing compound as a reaction substrate can be added to a liquid containing the carbonyl reductase of the present invention, and the reaction can be carried out at an appropriate temperature and pressure (e.g., atmospheric pressure). Thus, an optically active compound represented by the above general formula (IV) can be produced.

[0162] The amount of the carbonyl-containing compound as a reaction substrate can be appropriately selected according to the type of the compound. For example, the carbonyl-containing compound as a reaction substrate can be used at a substrate concentration in the range of 0.01 w / v% to 90 w / v%, preferably 0.1 w / v% to 30 w / v%.

[0163] The reaction substrate may be added all at once at the start of the reaction, but is preferably added continuously or intermittently from the viewpoint of reducing the influence of substrate inhibition of the enzyme and increasing the cumulative concentration of the product.

[0164] The reaction medium can be appropriately selected according to the type of the carbonyl-containing compound as the reaction substrate. For example, an aqueous medium or a mixture of an aqueous medium and an organic solvent can be used. As the aqueous medium, for example, water or a buffer solution can be cited. As the organic solvent, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butyl alcohol, acetone, dimethyl sulfoxide, etc. can be used as organic solvents with high solubility of the carbonyl-containing compound as the reaction substrate. In addition, as the organic solvent, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, etc., which have the effect of removing reaction by-products, etc., can also be used.

[0165] The reaction temperature, the pH during the reaction, and the reaction time can be appropriately selected according to the type of the carbonyl-containing compound as the reaction substrate. For example, the reaction temperature is 4°C to 80°C, preferably 10°C to 70°C, the pH during the reaction is pH 311, preferably pH 4 to 8, and the reaction time is about 0.5 hour to 72 hours.

[0166] With regard to the optically active compound represented by the above-mentioned general formula (IV) produced by the production method of the present invention, after the reaction is completed, the bacterial cells, proteins, etc. in the reaction solution can be separated by a separation or purification method known to those skilled in the art, such as centrifugation and membrane treatment, and then purified by an appropriate combination of extraction with an organic solvent, distillation, column chromatography using an ion exchange resin or silica gel, crystallization at the isoelectric point, crystallization using a monohydrochloride, a dihydrochloride, a calcium salt, etc.

[0167] In addition, the present invention relates to a method for producing an optically active compound, wherein the optically active compound is produced by allowing the transformant cell of the present invention obtained by the above method, a culture of the transformant cell, or a processed product of the transformant cell to act on a carbonyl-containing compound represented by the following general formula (I), (II), or (III) as a reaction substrate to asymmetric reduction of the carbonyl group of the compound. The transformant cell, the culture of the transformant cell, or the processed product of the transformant cell may be used alone or in combination.

[0168] In particular, the production method of the present invention can be preferably used for the case where the substituent A is , namely the manufacture of pitavastatin or rosuvastatin.

[0169] (1) Pitavastatin

[0170] Examples of methods for producing pitavastatin include methods described in Japanese Patent No. 4270918 and International Publication No. 2017 / 022846.

[0171] The carbonyl-containing compound represented by the following general formula (I′), (II″) or (III″) as the reaction substrate can be arbitrarily produced by combining the methods described in Japanese Patent Publication No. 1-279866, Japanese Patent Publication No. 8-127585, Japanese Patent Publication No. 5-178841, International Publication No. 2017 / 022846, etc. with known methods. These compounds can be used as raw materials in combination of one or more.

[0172]

[0173] When the compound represented by the general formula (I') is used as a reaction substrate, there are cases where the compound represented by the general formula (II") is used as its production intermediate and cases where the compound represented by the general formula (III") is used as its production intermediate. Therefore, the compound represented by the general formula (II") and the compound represented by the general formula (III") can be pre-produced from the compound represented by the general formula (I'), and they can be isolated and further derived into the optically active compound represented by the general formula (IV'), or the compound represented by the general formula (IV') can be directly produced without isolating the compound represented by the general formula (II") and the compound represented by the general formula (III")

[0174]

[0175] The carbonyl-containing compound represented by the general formula (I′), (II″) or (III″) as the reaction substrate is usually used in a substrate concentration range of 0.01 w / v% to 20 w / v%, preferably 0.1 w / v% to 10 w / v%. The reaction substrate may be pre-existing in the reaction system or added all at once at the start of the reaction. In addition, from the viewpoint of reducing the influence of substrate inhibition of the enzyme when it exists and increasing the cumulative concentration of the product, it may be added continuously or intermittently from the start of the reaction.

[0176] The reaction is preferably carried out in the presence of a coenzyme NAD(P)+ or NAD(P)H. In this case, the coenzyme is preferably added to a concentration of generally 0.001 mmol / L to 100 mmol / L, preferably 0.01 mmol / L to 10 mmol / L.

[0177] When the above coenzyme is added, it is preferred to generate NAD(P)H in the reaction system in order to improve production efficiency. + Regeneration to NAD(P)H. As the regeneration method, the following can be cited:

[0178] 1) Utilizing the NAD(P) produced by the microorganism or cell of the present invention + The ability to generate NAD(P)H, i.e. NAD(P) + Methods of restoring capacity;

[0179] 2) Add NAD(P) to the reaction system + A method of using one or more of a microorganism capable of generating NAD(P)H, a processed product thereof, or an enzyme capable of regenerating NAD(P)H such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.) (hereinafter referred to as "regeneration enzyme");

[0180] 3) When producing the microorganism or cell of the present invention, a method in which one or more genes of the above-mentioned regeneration enzymes are introduced into a host organism or host cell at the same time; etc.

[0181] In the above method 1), it is preferred to add glucose, ethanol, 2-propanol, formic acid or the like to the reaction system from the viewpoint of reaction efficiency.

[0182] In the above-mentioned method 2), a microorganism having the ability to produce the above-mentioned regenerated enzyme, a microorganism-treated product such as an acetone-treated product, a glutaraldehyde-treated product, a freeze-dried product, a physically disrupted product or an enzyme-disrupted product of the microorganism, a product obtained by removing the enzyme fraction in the form of a crude product or a purified product, and a product obtained by immobilizing these on a carrier such as a polyacrylamide gel or carrageenan, etc., and commercially available enzymes can also be used.

[0183] The regenerating enzyme may be added in an amount generally 0.01 to 100 times, preferably about 0.5 to 20 times, the carbonyl reducing activity of the enzyme having the ability to stereoselectively reduce a carbonyl group of the present invention.

[0184] In addition, it is necessary to add a compound that serves as a substrate for the above-mentioned regeneration enzyme, such as glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. The amount added is usually 0.1 mol to 20 mol, preferably 1 mol to 10 mol, relative to 1 mol of the carbonyl-containing compound that serves as the reaction substrate.

[0185] In the above method 3), a method of integrating the DNA of the above regeneration enzyme into chromosomes together with the DNA encoding the enzyme used in step (i), a method of introducing two DNAs into one expression vector and transforming a host organism or cell, or a method of transforming a host organism or host cell after introducing two DNAs into different expression vectors, etc. In the case of a method of transforming a host organism or host cell after introducing two DNAs into different expression vectors, the incompatibility of the two expression vectors needs to be considered when selecting expression vectors.

[0186] When introducing multiple genes into one expression vector, regions involved in expression regulation such as promoters and terminators may be linked to each gene, or the genes may be expressed in the form of an operon containing multiple cistrons such as the lactose operon.

[0187] The reaction is carried out in an aqueous medium or in a mixture of an aqueous medium and an organic solvent. The aqueous medium or the mixture of an aqueous medium and an organic solvent contains a carbonyl compound as a reaction substrate and the above-mentioned enzyme, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by cultivating the microorganism or cell. In addition, various coenzymes may be contained as required. When containing a coenzyme, it is more preferred to form a regeneration system, i.e., to enable the coenzyme to be regenerated.

[0188] In addition, the carbonyl group-containing compound which is a reaction substrate can also be produced by the method mentioned later.

[0189] Examples of the aqueous medium include water or pH buffer solutions such as potassium phosphate buffer, sodium citrate buffer, and Tris-HCl buffer.

[0190] As the organic solvent, ethyl acetate, isopropyl acetate, butyl acetate, toluene, chloroform, n-hexane, n-heptane, dimethyl sulfoxide, methanol, ethanol, n-propanol, 2-propanol, etc. can be used as an organic solvent with high solubility of the carbonyl-containing compound as the reaction substrate. Among them, as the organic solvent, dimethyl sulfoxide, methanol, and ethanol are preferred from the perspective of high solubility of the carbonyl-containing compound as the reaction substrate. In addition, dimethyl sulfoxide is more preferred from the perspective of high conversion rate.

[0191] The reaction is carried out at a reaction temperature of usually 4 to 70°C, preferably 30 to 60°C, and usually pH 3 to 11, preferably pH 4 to 8. The reaction time is usually 0.5 to 48 hours, preferably 0.5 to 24 hours.

[0192] The obtained compound represented by the general formula (IV′) can be purified by adjusting the pH to an appropriate level after separating the bacteria, polypeptides, etc. by centrifugation, filtration, etc., and by a suitable combination of extraction with an organic solvent such as hexane, ethyl acetate, toluene, purification by column chromatography, crystallization, etc.

[0193] For example, in the reaction of the following formula, it is preferred to use a carbonyl reductase having one or more, preferably two or more, of the mutations (d), (c) or (f) in the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having carbonyl reductase activity as a homologue of the amino acid sequence. In addition, it may further have a mutation (g). In particular, in the reaction of the following formula, a carbonyl reductase having at least a mutation (d) is preferred.

[0194]

[0195] In addition, for example, in the reaction of the following formula, a carbonyl reductase having one or more, preferably two or more, and particularly preferably three or more of the mutations (a), (b), (c), (e) or (f) in the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having carbonyl reductase activity as a homolog of the amino acid sequence can be preferably used. In addition, it can further have a mutation (g). In particular, in the reaction of the following formula, a carbonyl reductase having mutations (b), (e) and (f) is preferred.

[0196]

[0197] (2) Rosuvastatin

[0198] Examples of methods for producing rosuvastatin include methods described in International Publication No. 2015 / 119261 and the like.

[0199] As the reaction substrate, for example, a carbonyl group-containing compound represented by the following general formula (I") can be used.

[0200]

[0201] The carbonyl-containing compound represented by the general formula (I") as the reaction substrate is usually used in a substrate concentration range of 0.01 w / v% to 20 w / v%, preferably 0.1 w / v% to 10 w / v%. The reaction substrate can be added all at once at the start of the reaction. In addition, from the viewpoint of reducing the influence of substrate inhibition of the enzyme when there is substrate inhibition and increasing the cumulative concentration of the product, it can also be added continuously or intermittently.

[0202] The reaction is preferably carried out in the presence of the coenzyme NAD(P) + In this case, the coenzyme is added to a concentration of usually 0.001 mmol / L to 100 mmol / L, preferably 0.01 mmol / L to 10 mmol / L.

[0203] When the above coenzyme is added, it is preferred to generate NAD(P)H in the reaction system in order to improve production efficiency. + Regeneration to NAD(P)H. As the regeneration method, the following can be cited:

[0204] 1) Utilizing the NAD(P) produced by the microorganism or cell of the present invention + The ability to generate NAD(P)H, i.e. NAD(P) + Methods of restoring capacity;

[0205] 2) Add NAD(P) to the reaction system +A method of using one or more of a microorganism capable of generating NAD(P)H, a processed product thereof, or an enzyme capable of regenerating NAD(P)H such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.) (hereinafter referred to as "regeneration enzyme");

[0206] 3) When producing the microorganism or cell of the present invention, a method in which one or more genes of the above-mentioned regeneration enzymes are introduced into a host organism or host cell at the same time; etc.

[0207] In the above method 1), it is preferred to add glucose, ethanol, 2-propanol or formic acid to the reaction system.

[0208] In the above-mentioned method 2), a microorganism having the ability to produce the above-mentioned regenerated enzyme, an acetone-treated product of the microorganism, a freeze-dried product, a physically disrupted product or an enzyme disrupted product, etc., a product obtained by removing the enzyme fraction in the form of a crude product or a purified product, and a product obtained by immobilizing these on a carrier such as polyacrylamide gel or carrageenan, etc., can be used. In addition, commercially available enzymes can also be used.

[0209] In this case, the regenerating enzyme is added in an amount generally 0.01 to 100 times, preferably about 0.5 to 20 times, the carbonyl reducing activity of the enzyme having the ability to stereoselectively reduce a carbonyl group of the present invention.

[0210] In addition, it is necessary to add a compound that serves as a substrate for the above-mentioned regeneration enzyme, such as glucose when glucose dehydrogenase is used, formic acid when formate dehydrogenase is used, ethanol or isopropanol when alcohol dehydrogenase is used, etc., and the amount thereof to be added is usually 0.1 equivalent to 20 equivalents, preferably 1 equivalent to 10 equivalents, relative to the carbonyl-containing compound represented by the general formula (I") that serves as the reaction substrate.

[0211] In addition, in the above-mentioned method 3), a method of integrating the DNA of the above-mentioned regeneration enzyme into the chromosome together with the DNA encoding the carbonyl reductase of the present invention, a method of introducing two DNAs into one expression vector and transforming the host organism or cell, or a method of transforming the host organism or host cell after introducing the two DNAs into different expression vectors respectively. In the case of the method of transforming the host organism or host cell after introducing the two DNAs into different expression vectors respectively, the incompatibility of the two expression vectors needs to be considered when selecting the expression vector.

[0212] When multiple genes are introduced into one expression vector, regions involved in expression regulation such as promoters and terminators may be linked to each gene, or the genes may be expressed in the form of an operon containing multiple cistrons such as the lactose operon.

[0213] The reaction is carried out in an aqueous medium containing a carbonyl compound represented by the general formula (I") and the above-mentioned enzyme, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell, and various coenzymes contained as needed (more preferably forming a regeneration system thereof, i.e., capable of regenerating the coenzymes), or a mixture of the aqueous medium and an organic solvent. It should be noted that the carbonyl compound represented by the general formula (I") can be produced by the method described in International Publication No. 2015 / 119261, etc.

[0214] Examples of the aqueous medium include water or buffers such as potassium phosphate buffer, sodium citrate buffer, and Tris-hydrochloric acid buffer.

[0215] As the organic solvent, an organic solvent with high solubility for the carbonyl-containing compound represented by the general formula (I"), such as ethyl acetate, isopropyl acetate, butyl acetate, toluene, chloroform, n-hexane, n-heptane, dimethyl sulfoxide, methanol, ethanol, n-propanol, and 2-propanol, can be used. Among them, as the organic solvent, dimethyl sulfoxide, methanol, and ethanol are preferred from the perspective of high solubility of the compound represented by the general formula (I") . In addition, dimethyl sulfoxide is more preferred from the perspective of high conversion rate.

[0216] In addition, the reaction can be carried out in the presence of polyols such as glycerol, ethylene glycol, propylene glycol, erythritol, inositol, sorbitol, and xylitol. The above-mentioned polyols can be polymers or derivatives, and can be used alone or in a mixture of two or more. If the reaction is carried out in the presence of polyols, there is a tendency for the conversion rate to increase. Among them, glycerol is preferred because it is believed that it can maintain the enzyme activity by maintaining the higher structure of the enzyme and is easily available.

[0217] The reaction is usually carried out at a reaction temperature of 4°C to 70°C, preferably 20°C to 60°C, and usually at a pH of 3 to 11, preferably at a pH of 4 to 8. The reaction time is usually 0.5 to 48 hours, preferably 0.5 to 24 hours. Alternatively, the reaction can be carried out using a membrane reactor or the like.

[0218] The optically active compound represented by the obtained general formula (VI") can be purified by adjusting the pH to an appropriate level after separating the bacterial cells, polypeptides, etc. by centrifugation, filtration, etc., and then extracting with an organic solvent such as hexane, ethyl acetate, toluene, purifying by column chromatography, crystallization, etc. in an appropriate combination.

[0219]

[0220] When the optically active compound represented by the general formula (VI") is purified by crystallization, as the organic solvent that can be used, there can be used hydrocarbon solvents such as cyclohexane, n-hexane, n-heptane, toluene, halogen solvents such as chlorobenzene, dichlorobenzene, ether solvents such as tert-butyl methyl ether, tetrahydrofuran (THF), cyclopentyl methyl ether (CPME), alcohol solvents such as methanol, ethanol, n-propanol, isopropanol, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and other polar solvents, etc., which have high solubility for the optically active compound represented by the general formula (VI") . These organic solvents can be used alone or as a mixed solvent of these organic solvents and water.

[0221] When purifying the optically active compound represented by the general formula (VI") by crystallization, it is preferred that: after dissolving the optically active compound represented by (VI") in an organic solvent or a mixed solvent of an organic solvent and water, the solution is cooled at a cooling rate of less than 15°C / hour, thereby precipitating crystals of the optically active compound represented by the general formula (VI") (hereinafter, the step of cooling to precipitate crystals is referred to as the "cooling step").

[0222] In the cooling step, the temperature at the start of cooling is preferably 15°C to 60°C, more preferably 20°C to 55°C.

[0223] The cooling rate in the cooling step is preferably 15°C / hour or less, more preferably 9°C / hour or less, further preferably 6°C / hour or less, and particularly preferably 5°C / hour or less. This is to increase the purity of the optically active compound represented by the general formula (VI") obtained.

[0224] It should be noted that the cooling rate may be changed during the cooling step. In particular, it is preferred to cool slowly within a temperature range of preferably 45°C or less, more preferably 40°C or less. Specifically, it is more preferred to set the cooling rate to 9°C / hour, further preferably to 6°C / hour or less, and particularly preferably to 5°C / hour or less.

[0225] In addition, for example, in the reaction of the following formula, a carbonyl reductase having one or more, preferably two or more, and particularly preferably three or more of the mutations (a), (b), (c), (e) or (f) in the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having carbonyl reductase activity as a homolog of the amino acid sequence can be preferably used. In addition, it can further have a mutation (g). In particular, in the reaction of the following formula, a carbonyl reductase having mutations (b), (e) and (f) is preferred.

[0226]

[0227] Example

[0228] The present invention will be further described in detail with reference to the following examples, but the present invention is not limited to these examples.

[0229] The quantitative analysis in this example was performed using high performance liquid chromatography (HPLC) under the following conditions.

[0230] <Measurement Condition 1>

[0231] Column: Cosmosil 3C18MS-II manufactured by Nacalai Co., Ltd. (4.6×75 mm, 3 μm)

[0232] Mobile phase: methanol / acetonitrile / water / phosphoric acid = 300 / 150 / 450 / 1

[0233] Flow rate: 0.5 mL / min Column temperature: 50°C

[0234] Detection wavelength: UV245nm

[0235] Injection volume: 5 μL

[0236] <Measurement Condition 2>

[0237] Column: Capcell PAK C18 MGIII (4.6×75 mm, 3 μm) manufactured by Shiseido

[0238] Mobile phase: A: 100mM HCOONH4 B: ethanol

[0239] Gradient program (B concentration): 45% (0 min) → 45% (20 min) → 80% (30 min) → 80% (35 min)

[0240] Flow rate: 0.55 mL / min Column temperature: 40°C

[0241] Detection wavelength: UV245nm

[0242] Injection volume: 5 μL

[0243] <Measurement Condition 3>

[0244] Column: ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm)

[0245] Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile

[0246] Gradient program (B concentration): 40% (0 min) → 95% (12 min) → 64% (14 min) → 40% (17 min)

[0247] Flow rate: 0.3 mL / min Column temperature: 40°C

[0248] Detection wavelength: UV245nm

[0249] Injection volume: 5 μL

[0250] <Measurement Condition 4>

[0251] Column: CAPCELL PAK C18 MGS-III (4.6×75 mm, 3 μm) manufactured by Shiseido

[0252] Mobile phase: water / acetic acid / ammonium acetate = 1000 / 100 / 7.7 (volume / volume / weight)

[0253] Flow rate: 1 mL / min Column temperature: 40°C

[0254] Detection wavelength: UV254nm

[0255] Injection volume: 5 μL

[0256] Reference Example 1 (Preparation of bacterial cells)

[0257] (1) Gene cloning

[0258] The gene was cloned with reference to Patent Document 1 (Japanese Patent Gazette No. 4270918). Based on the gene sequence ocrl (sequence number 2) encoding OCR1 (sequence number 1) from methanol-inducible yeast nonfermentative variant (Ogataea minuta var. nonfermentans) NBRC 1473, PCR was performed according to a conventional method to obtain a DNA fragment of about 0.8 kbp in length containing restriction enzyme sites EcoRI upstream and XbaI downstream of the ocrl gene.

[0259] Then, referring to Patent Document 2 (International Publication No. 2015 / 119261), based on the gene sequence of GDH (sequence number 3) in which the 96th amino acid residue, i.e., glutamic acid, in the glucose-1-dehydrogenase encoded by the gene derived from Bacillus subtilis (GeneBank Accession No. AL009126.3) is replaced with alanine (hereinafter referred to as gdh (sequence number 4), PCR was performed according to a conventional method to obtain a DNA fragment with a total length of about 0.8 kbp containing the restriction enzyme site EcoRI upstream and the restriction enzyme site XbaI downstream of the gdh gene.

[0260] (2) Preparation of expression plasmid

[0261] The ocrl DNA fragment obtained in (1) above was digested with restriction enzymes EcoRI and XbaI and introduced downstream of the trc promoter in the plasmid pKV32 described in Japanese Patent Application Laid-Open No. 2005-34025 digested with MunI and XbaI using a Ligation-Convenience Kit (manufactured by Nippondin Corporation) to obtain pKV32OCR1.

[0262] Next, the gdh DNA fragment obtained in (1) above was digested with restriction enzymes EcoRI and XbaI and introduced downstream of the trc promoter in the plasmid pKV32 digested with MunI and XbaI using a Ligation-Convenience Kit (manufactured by Nippondin) to obtain pKV32GDH.

[0263] Furthermore, PCR was performed using pKV32GDH as a template according to a conventional method to obtain a DNA fragment of about 0.8 kbp in length with restriction enzyme sites HindIII added upstream and downstream, and the obtained DNA fragment was digested with restriction enzyme HindIII and then inserted into the downstream of plasmid pKV32OCR1 previously digested with restriction enzyme HindIII to obtain pKV32OCR1-GDH. The orientation of the gdh gene in the obtained plasmid was confirmed by PCR.

[0264] (3) Preparation of plasmid for expression of OCR1 mutants

[0265] Using the plasmid pKV32OCR1-GDH obtained in (2) above as a template, a mutant plasmid capable of expressing the mutants shown in Table 1 below was prepared according to a conventional method using a site-directed mutagenesis method such as QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies).

[0266] It should be noted that in the table shown below, A represents alanine, N represents asparagine, D represents aspartic acid, I represents isoleucine, L represents leucine, M represents methionine, S represents serine, and V represents valine. In addition, for example, I211A represents a mutation in which isoleucine at position 211 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

[0267] [Table 1]

[0268]

[0269] Here, D54V is a (a) mutation, M157V is a (b) mutation, A170S is a (c) mutation, I211A and I211N are (d) mutations, M214L is a (e) mutation, M249L is a (f) mutation, and V166A is a (g) mutation.

[0270] Example 1 (Preparation of expression strain)

[0271] Using the plasmid pKV32OCR1-GDH obtained in (2) and (3) of Reference Example 1 above and its mutant plasmid, Escherichia coli JM109 (manufactured by Takara Bio Co., Ltd.) was transformed according to a conventional method to obtain each transformant.

[0272] Example 2

[0273] DOLE was produced by allowing the transformant obtained in Example 1 to act on 5S-MOLE for reduction. The ratio of syn form to anti form of the obtained DOLE was measured to evaluate the optical selectivity.

[0274] Here, 5S-MOLE, syn-DOLE, and anti-DOLE are compounds having the following structures, respectively.

[0275]

[0276] For OCR1 and the transformants having the mutations shown in Table 2, the following reactions and evaluations were performed.

[0277] The transformant was inoculated into LB liquid medium containing 25 mg / L kanamycin and 0.2 mmol / L isopropyl-β-D-thiogalactoside (hereinafter referred to as "IPTG"), and cultured overnight at 37°C with shaking. The cells were recovered from the obtained culture solution, and 200 μL of 100 mmol / L potassium phosphate buffer (pH 6) and 2 g / L NADP were added. + 30 μL of ethanol and 1 μL of toluene were vigorously shaken for 5 minutes, and then 10 μL of 50 wt% glucose and 10 μL of 5S-MOLE at a concentration of 5 g / L were added, and the mixture was shaken at 45° C. for 1 hour to react.

[0278] To the obtained reaction solution, 750 μL of acetonitrile was added, and after centrifugation, the obtained centrifugation supernatant was subjected to HPLC analysis under measurement condition 2 and measurement condition 1. Table 2 shows the anti-body ratio of each transformant.

[0279] The anti-DOLE ratio was calculated according to the following formula from the ratio of syn-DOLE to anti-DOLE obtained by HPLC analysis.

[0280] Anti-body ratio (%) = anti-DOLE production / syn-DOLE production × 100

[0281] [Table 2]

[0282] Serial Number mutation anti-body ratio 1 None (OCR1) 1.1％ 8 I211A 0.5％ 9 I211N 0.4％

[0283] As can be seen from Table 2, the mutants having the I211A or I211N mutation have a lower anti-body ratio than OCR1. That is, it was found that the optical selectivity was improved by the (d) mutation (I211A or I211N).

[0284] Example 3

[0285] The transformant obtained in Example 1 was allowed to act on DOXE for reduction, and the reaction activity and stereoselectivity were evaluated based on the produced amounts of 3R-MOLE, 5S-MOLE and DOLE.

[0286] Here, DOXE, 3R-MOLE, 5S-MOLE, and DOLE are compounds having the following structures, respectively.

[0287]

[0288] For OCR1 and the transformants having the mutations shown in Table 3, the following reactions and evaluations were performed.

[0289] The transformant was inoculated into LB liquid medium containing 25 mg / L kanamycin and 0.2 mmol / L IPTG, and cultured overnight at 37°C with shaking. The bacterial cells were recovered from the obtained culture solution, suspended in a potassium phosphate buffer solution with a concentration of 100 mmol / L, and the container containing the suspension was immersed in ice water for ultrasonic disruption. The obtained bacterial cell lysate was centrifuged to obtain a supernatant of the bacterial cell disruption.

[0290] To 100 μL of the supernatant of the cell disrupted product, 100 μL of 0.2 mol / L potassium phosphate buffer (pH 7), 100 μL of 20 g / L glucose, and 0.2 g / L NADP were added. + 10 μL, 100 μL of DMSO solution of DOXE with a concentration of 1 g / L and 90 μL of water were reacted at 50 ° C and 1000 rpm for 15 minutes using ThermoMixer R Mixer (manufactured by Eppendorf). After the reaction, 1 mL of acetonitrile was added and mixed, and then centrifuged. The obtained supernatant was subjected to HPLC analysis under measurement condition 3. The reaction activity of each transformant and the 3R-MOLE ratio are shown in Table 3.

[0291] The reaction activity and 3R-MOLE ratio were calculated by the following calculation formula from the peak area values ​​obtained by HPLC analysis.

[0292] Reaction activity = 5S-MOLE peak area + 3R-MOLE peak area

[0293] 3R-MOLE ratio = 3R-MOLE peak area / 5S-MOLE peak area

[0294] The reactivity was evaluated as “-” when OCR1 was used as the standard, 1.0 times as “+” when 1.1 times as “++” when 1.2 times as “+++” when the reactivity was used as the standard.

[0295] In addition, regarding the 3R-MOLE ratio, based on OCR1, 0.9 to 1.0 times was evaluated as "+", 0.7 to 0.8 times was evaluated as "++", and 0.6 times or less was evaluated as "+++".

[0296] [Table 3]

[0297] Serial Number mutation Reactivity 3R-MOLE Ratio 1 None (OCR1) + + 11 A170S / M249L - + 13 M157V / A170S / M249L - ++ 15 V166A / M214L / M249L ++ ++ 17 A170S / M214L / M249L - ++ 18 M157V / V166A / M214L / M249L ++ +++ 19 M157V / A170S / M214L / M249L +++ +++ 20 V166A / A170S / M214L / M249L - ++ 22 M157V / V166A / A170S / M214L / M249L ++ +++

[0298] As shown in Table 3, the mutants with M157V or M214L mutations have a lower 3R-MOLE ratio compared to OCR1. In particular, the mutants with both M157V and M214L mutations not only have a lower 3R-MOLE ratio but also have improved reactivity. That is, it is clear that stereoselectivity and reactivity are improved due to M157V ((b) mutation) and M214L ((e) mutation).

[0299] Example 4

[0300] For OCR1 and the transformants having the mutations shown in Table 4, the following reactions and evaluations were performed.

[0301] The transformant was inoculated into LB liquid medium containing 25 mg / L kanamycin and 0.2 mmol / L IPTG, and cultured overnight at 37°C with shaking. The bacterial cells were recovered from the obtained culture solution, suspended in a potassium phosphate buffer solution with a concentration of 100 mmol / L, and the container containing the suspension was immersed in ice water for ultrasonic disruption. The obtained bacterial cell lysate was centrifuged to obtain a supernatant of the bacterial cell disruption.

[0302] The protein concentration of the obtained supernatant of the cell disruption product was measured according to a conventional method using Quick Start Bradford Protein Assay Kit (manufactured by BIO-RAD Laboratories).

[0303] The supernatant of the obtained bacterial cell disrupted product was divided into two parts, one of which was refrigerated and stored at 4°C to 10°C, and the other was heated at 50°C and 200 rpm for 1 hour using a ThermoMixer R Mixer (manufactured by Eppendorf).

[0304] For each supernatant of the cell disruption product that has been refrigerated or heated, 10 μL of the supernatant of the cell disruption product, 25 μL of potassium phosphate buffer (pH 7) with a concentration of 1 mol / L, 10 μL of NADPH with a concentration of 8 mmol / L, 200 μL of water, and 5 μL of a DMSO solution of 2,2,2-trifluoroacetophenone (TFAP) with a concentration of 0.1 mol / L were mixed. The mixture was added to a 96-well plate (manufactured by Corning), and the absorbance change at 340 nm within 2 minutes was measured using an ELISA reader (manufactured by Meigu Molecular Instruments). The average value of the absorbance change per unit time (mOD / minute) was calculated, and the activity value per unit time / per unit protein was calculated by the following formula from the slope of the absorbance change.

[0305] In addition, the protein concentration of each supernatant of the cell disrupted product subjected to refrigeration or heating treatment was measured according to a conventional method using Quick Start Bradford Protein Assay Kit (manufactured by BIO-RAD Laboratories).

[0306] It should be noted that the calculation was performed with the molar absorption coefficient of NADPH being 6.3 mL / μmol·cm.

[0307] Activity value (μmol / min / mg-protein) = (-1) × slope of absorbance change × 250 μL / 10 μL / 6300 / 0.55 / protein concentration

[0308] The activity values ​​obtained as described above were substituted into the following formula to calculate the activity ratio before and after the heat treatment, thereby evaluating the thermal stability.

[0309] Thermal stability (%) = activity value when heated / activity value when refrigerated × 100

[0310] The case where the thermal stability value was less than 20% was evaluated as "-", the case where the thermal stability value was in the range of 20% to 70% was evaluated as "+", and the case where the thermal stability value was greater than 70% was evaluated as "++".

[0311] [Table 4]

[0312] Serial Number mutation Thermal stability 1 None (OCR1) - 6 V166A + 7 A170S + 10 M249L + 11 A170S / M249L ++ 12 V166A / M249L ++ 14 V166A / A170S / M249L ++

[0313] As can be seen from Table 4, the thermal stability of the mutants having the A170S or M249L mutation is improved. In particular, the thermal stability of the mutants having both the A170S and M249L mutations is further improved. That is, it is clear that the thermal stability is improved by A170S ((c) mutation) and M249L ((f) mutation).

[0314] In addition, it was revealed that mutants having two or more of the A170S, M249L, and V166A mutations had improved thermal stability.

[0315] Example 5

[0316] The enzyme activities of the supernatants of OCR1 obtained in the same manner as in Example 3 and the bacterial cell disruptants having the mutations shown in Table 6 were measured.

[0317] The enzyme activity was confirmed by measuring the amount of NADPH reduction when TFAP was added at a reaction temperature of 30° C. and a wavelength of 340 nm in a reaction solution having a composition shown in Table 5. The unit U of the enzyme activity represents the amount of conversion (μmol) per minute.

[0318] [Table 5]

[0319]

[0320] In the following reaction, the amount of carbonyl reductase was measured based on the above enzyme activity (unit: U).

[0321] In addition, DOXE and DOLE are compounds having the following structures, respectively.

[0322]

[0323] Prepare a solution containing 10 g / L DOXE, 100 mmol / L phosphate buffer (pH 6.0), 120 mmol / L glucose, and 2 mol% NADP. + , 20 wt% DMSO, 10 U of carbonyl reductase and 5 U to 10 U of glucose dehydrogenase 2 mL reaction solution. The reaction solution was shaken and reacted at 50°C for 4 hours. The obtained DOLE was subjected to HLPC analysis under measurement condition 3. The results are shown in Table 6.

[0324] [Table 6]

[0325] Serial Number mutation DOLE conversion rate 1 None (OCR1) 14.6％ 5 D54V 70.1％ 19 M157V / A170S / M214L / M249L 84.3％ 21 D54V / M157V / A170S / M214L / M249L 90.5％

[0326] As can be seen from Table 6, the mutants having the above mutations have significantly improved DOLE conversion rates. In other words, it was found that the mutants having these mutations have significantly improved carbonyl reduction activity and reaction efficiency.

[0327] Reference Example 2

[0328] This reference example is the method described in Example 4-4 of International Publication No. 2015 / 119261.

[0329] In a 1L fermenter (manufactured by Eblu, model BMJ-01), 385.9 mL of ion exchange water, 48.3 g (268.1 mmol) of glucose, 138 mg (0.18 mmol) of NADP+ (manufactured by Oriental Yeast Co., Ltd.), 8.29 g (47.6 mmol) of dipotassium hydrogen phosphate, and 3.97 g (29.2 mmol) of potassium dihydrogen phosphate were added and dissolved. 50.60 g of frozen cells of recombinant Escherichia coli JM109 / pKV32OCR1-GDH and the entire amount of a substrate solution prepared by dissolving 14.4 g (27.7 mmol) of DOXP in 124.20 g (1589.7 mmol) of DMSO were added thereto, and stirred for 5 hours at an internal temperature of 50°C. During the reaction, a 25 wt% aqueous sodium hydroxide solution was added dropwise to keep the pH at 6. The DOLP was obtained as confirmed by the retention time of HPLC, and the conversion rate at this time was 90.25%.

[0330] Here, DOXP and DOLP are compounds having the following structures, respectively.

[0331]

[0332] Example 6

[0333] The transformant JM109 / pKV32OCR1 (mutant having A170S / I211A / M249L mutations (SEQ ID NO: 16))-GDH obtained in Example 1 was inoculated into LB liquid medium containing 25 mg / L kanamycin and 0.2 mmol / L IPTG, and cultured overnight with shaking at 37°C. The bacterial cells were recovered from the obtained culture solution, and reacted in the same manner as in Reference Example 2 to obtain DOLP.

[0334] The obtained DOLP was subjected to HPLC analysis under measurement condition 4. As a result, the conversion rate was 95.98%, which was higher than that of Reference Example 2.

[0335] It was found that the reaction efficiency was improved due to the presence of A170S ((c) mutation), I211A ((d) mutation), and M249L ((f) mutation).

[0336] Industrial Applicability

[0337] According to the present invention, a carbonyl reductase having the activity of reducing a carbonyl-containing compound to convert it into an industrially useful compound as an intermediate raw material for medicines, pesticides, etc., i.e., an optically active compound, a nucleic acid encoding the carbonyl reductase, a recombinant vector containing the nucleic acid, and a transformant containing the recombinant vector can be provided. Furthermore, according to the present invention, a method for producing an industrially useful optically active compound as an intermediate raw material for medicines, pesticides, etc. with high purity and high optical purity and at low price can be provided, which can improve the production efficiency of the medicines, pesticides, etc. and increase their yield.

[0338] This application is based on Japanese Patent Application No. 2019-211797 (filing date: November 22, 2019) filed in Japan, the contents of which are all incorporated herein by reference. Sequence Listing <110> API Co., Ltd. <120> Carbonyl reductase, nucleic acid encoding the enzyme, and method for producing optically active compounds using the same <130> 093099 <150> JP 2019-211797 <151> 2019-11-22 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 1 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 2 <211> 759 <212> DNA <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 2 atggctaaaa ctgtttactt catcgcaggt gcttccagag gtatcggtct cgaggttgct 60 tcccagctga gtgcaaaccc agacaattat gttattgcat cctatagatc tgaaaagtct 120 gcttcaggac ttttggagct ggcaaagaag gataatgtcg acacaattgt gttggatatt 180 gcaagccagg aatcgattga tgctgttcca gcacagattt ccaagctgac tgatggaatc 240 gatgttgcct tgatcaacgc tggaattgcc aacgctatgt gtccgattct cgaatgttct 300 agagagtcct acactgatca ctggacaacc aatgccttgg gtccaatcat gctctaccaa 360 gctattcata agttcatgct ccagagagag accagaaaag tgttctttac cacgagtgct 420 ggtggttcca ttcaggctaa gatacccgtg cctgtgagtg gttacggtat gtccaaggct 480 gcgcttaatt atgctgtgag aaaacttgct gacgagtgct acaaggacaa cttcactatt 540 gtgttgctgc atcctggttt tgttaagacg gacatgggtc aaagcgccat tcagaagatg 600 tcaaatggaa atgctgagct tcttgcttac attgactcaa tgactattga tgttcctacc 660 agtgctggcc aaatcgtcgg tgccattatg accttggaca agcagagcag cggtagattt 720 atcaacgctg ctgaccagtt tgacatgcca ttttagtaa 759 <210> 3 <211> 261 <212> PRT <213> Bacillus subtilis <400> 3 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Ala 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala 145 150 155 160 Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 4 <211> 786 <212> DNA <213> Bacillus subtilis <400> 4 atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60 aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120 aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180 gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240 aaggagtcg gcacactcga tattatgatt aataatgccg gtcttgcaaa tcctgtgcca 300 tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360 ttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420 attaacatgt ccagtgtgca cgaagtgatt ccttggccgt tatttgtcca ctatgcggca 480 agtaaaggcg ggataaagct gatgacagaa acattagcgt tggaatacgc gccgaagggc 540 600 gctgacccta aacagaaagc tgatgtagaa agcatgattc caatgggata tatcggcgaa 660 ccggagaga tcgccgcagt agcagcctgg cttgcttcga aggaagccag ctacgtcaca 720 ggcatcacgt tattcgcgga cggcggtatg acacaatatc cttcattcca ggcaggccgc 780 ggttaa 786 <210> 5 <211> 251 <212> PRT <213> Methanol-inducible yeast (Ogataea minuta var non-fermentans) <400> 5 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Val Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 6 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 6 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 7 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 7 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 8 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 8 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Asn Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 9 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 9 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ala Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Met Pro Phe 245 250 <210> 10 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 10 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 11 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 11 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 12 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 12 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 13 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 13 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Val Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 14 <211> 251 <212> PRT <213> Ogataea minuta var nonfermentans <400> 14 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 15 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 15 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 16 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 16 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ala Asp Ser Met Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 17 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 17 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 18 <211> 251 <212> PRT <213> Ogataea minuta var nonfermentans <400> 18 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Val Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ala Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 19 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 19 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Val Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 20 <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> 20 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Met Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> twenty one <211> 251 <212> PRT <213> Methanol-inducible yeast nonfermenting variant (Ogataea minuta var nonfermentans) <400> twenty one Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Val Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Val Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Val Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250 <210> 22 <211> 251 <212> PRT <213> Ogataea minuta var nonfermentans <400> 22 Met Ala Lys Thr Val Tyr Phe Ile Ala Gly Ala Ser Arg Gly Ile Gly 1 5 10 15 Leu Glu Val Ala Ser Gln Leu Ser Ala Asn Pro Asp Asn Tyr Val Ile 20 25 30 Ala Ser Tyr Arg Ser Glu Lys Ser Ala Ser Gly Leu Leu Glu Leu Ala 35 40 45 Lys Lys Asp Asn Val Asp Thr Ile Val Leu Asp Ile Ala Ser Gln Glu 50 55 60 Ser Ile Asp Ala Val Pro Ala Gln Ile Ser Lys Leu Thr Asp Gly Ile 65 70 75 80 Asp Val Ala Leu Ile Asn Ala Gly Ile Ala Asn Ala Met Cys Pro Ile 85 90 95 Leu Glu Cys Ser Arg Glu Ser Tyr Thr Asp His Trp Thr Thr Asn Ala 100 105 110 Leu Gly Pro Ile Met Leu Tyr Gln Ala Ile His Lys Phe Met Leu Gln 115 120 125 Arg Glu Thr Arg Lys Val Phe Phe Thr Thr Ser Ala Gly Gly Ser Ile 130 135 140 Gln Ala Lys Ile Pro Val Pro Val Ser Gly Tyr Gly Val Ser Lys Ala 145 150 155 160 Ala Leu Asn Tyr Ala Ala Arg Lys Leu Ser Asp Glu Cys Tyr Lys Asp 165 170 175 Asn Phe Thr Ile Val Leu Leu His Pro Gly Phe Val Lys Thr Asp Met 180 185 190 Gly Gln Ser Ala Ile Gln Lys Met Ser Asn Gly Asn Ala Glu Leu Leu 195 200 205 Ala Tyr Ile Asp Ser Leu Thr Ile Asp Val Pro Thr Ser Ala Gly Gln 210 215 220 Ile Val Gly Ala Ile Met Thr Leu Asp Lys Gln Ser Ser Gly Arg Phe 225 230 235 240 Ile Asn Ala Ala Asp Gln Phe Asp Leu Pro Phe 245 250

Claims

1. A carbonyl reductase, which is a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 5 and 7 to 22.

2. A method for producing an optically active compound represented by general formula (IV), characterized in that: Asymmetric reduction of the carbonyl-containing compound is carried out by contacting a carbonyl-containing compound selected from the group consisting of compounds represented by general formula (I), (II') and (III') with the carbonyl reductase according to claim 1, a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell, The processed product of the microorganism or cell refers to a processed product obtained by treating the microorganism or cell with an organic solvent or a surfactant, a processed product obtained by freeze-drying, a product obtained by physical or enzymatic disruption, a processed product obtained by extracting an enzyme fraction in the microorganism or cell in the form of a crude product or a purified product, and a product obtained by immobilizing these substances on a carrier, and the processed product of the microorganism or cell contains the carbonyl reductase according to claim 1, In the formulae (I), (II') and (III'), R represents a hydrogen atom, an alkyl group or an aryl group, express In formula (IV), R and Same meaning as above.

3. The manufacturing method according to claim 2, wherein: The microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding the carbonyl reductase.

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