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

Abstract

The present invention provides a carbonyl reductase that has an activity of reducing a carbonyl group-containing compound and thus converting the compound into an optically active compound, and a method for producing an optically active compound with the use of this enzyme. More specifically, provided are a carbonyl reductase that has, in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence, one or more mutations selected from among the substitutions of 54th aspartic acid, 157th methionine, 170th alanine, 211th isoleucine, 214th methionine and 249th methionine respectively into specific other amino acids, and a method for producing an optically active compound with the use of the same.

Description

[Technical Field]

[0001] The present invention relates to a carbonyl reductase having the activity of reducing a carbonyl group-containing compound and converting it into an optically active compound that is industrially useful as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc.; a nucleic acid encoding the carbonyl reductase; a recombinant vector containing the nucleic acid; and a transformant containing the recombinant vector. The present invention also relates to a method for producing an optically active compound using the carbonyl reductase or the like. [Background technology]

[0002] Statin compounds such as rosuvastatin, pitavastatin, atorvastatin, fluvastatin, and pravastatin, or salts thereof, are HMG-CoA reductase inhibitors and are useful in the treatment of hypercholesterolemia, mixed dyslipidemia, etc. In recent years, generic drugs using these statin compounds or salts thereof have been released, and there is a demand for methods for industrially producing them at lower cost.

[0003] These statin compounds have the following structure:

[0004] [ka]

[0005] or a salt thereof, A structure with consecutive carbonyl groups such as the following:

[0006] [ka]

[0007] A method for producing the compound is known in which a carbonyl group-containing compound having the formula:

[0008] For example, Patent Document 1 describes a method for producing an optically active compound such as an optically active alcohol with high optical purity and high concentration by allowing a specific carbonyl reductase (hereinafter sometimes referred to as "OCR1") to act on a carbonyl group-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.

[0009] Here, since OCR1 has poor thermal stability and the stability of the enzyme decreases during the reaction (heating), it is necessary to use a large amount of OCR1. Therefore, research on mutants aimed at improving the thermal stability of OCR1 has been conducted (Non-Patent Document 1).

[0010] However, in order to industrially produce optically active compounds, particularly statin compounds, which are useful as intermediate raw materials for pharmaceuticals, agricultural chemicals, etc., with high purity, high optical purity, and at low cost, further performance improvement of OCR1 is desired.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0012]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0013] An object of the present invention is to develop a carbonyl reductase that is higher than OCR1 in carbonyl reducing activity, thermostability, stereoselectivity, optical selectivity, etc. Another object of the present invention is to use the carbonyl reductase to industrially obtain optically active compounds with high purity and high optical purity at low cost. [Means for solving the problem]

[0014] As a result of intensive research to solve the above problems, the present inventors have found that by substituting a specific amino acid in OCR1 with another specific amino acid, a carbonyl reductase with high carbonyl reducing activity, thermostability, stereoselectivity, optical selectivity, etc. can be obtained, and have completed the present invention. Furthermore, the present inventors have found that by using this carbonyl reductase, optically active compounds can be obtained industrially at high purity and optical purity at low cost, and have completed the present invention.

[0015] That is, the gist of the present invention is as follows. [1] A carbonyl reductase having a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a homologue of said amino acid sequence, wherein said amino acid sequence has at least one mutation selected from the group consisting of the following (a) to (f): (a) A mutation in which aspartic acid at position 54 in the amino acid sequence of SEQ ID NO: 1 is replaced with valine (b) a mutation in which methionine at position 157 in the amino acid sequence of SEQ ID NO: 1 is replaced with valine (c) A mutation in which alanine at position 170 in the amino acid sequence of SEQ ID NO: 1 is replaced with serine (d) A mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine. (e) A mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine (f) a mutation in which methionine at position 249 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine [2] The carbonyl reductase according to [1], characterized by having at least two mutations selected from the group consisting of (a) to (f). [3] In the amino acid sequence shown in SEQ ID NO: 1 or a homolog of the amino acid sequence, further (g) valine at position 166 in the amino acid sequence of SEQ ID NO: 1 is Alanine The carbonyl reductase according to [1] or [2], which has a polypeptide consisting of an amino acid sequence having a mutation in which it is substituted. [4] A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II), and (III) is

[0016]

Chemical formula

[0017]

Chemical formula

[0018]

Chemical formula

[0019] (In the above formulas (I), (II), and (III), R represents a hydrogen atom, an alkyl group, or an aryl group,

[0020]

Chemical formula

[0021] represents a substituent having an aromatic ring and / or a heterocyclic ring.) [1] to [3] Any of the carbonyl reductases described above, 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 are contacted to asymmetrically reduce a carbonyl group-containing compound, characterized by the following general formula (IV)

[0022] [ka]

[0023] (Wherein R and

[0024] [ka]

[0025] has the same meaning as above.) A method for producing an optically active compound represented by the formula: [5] The carbonyl group-containing compounds represented by the formula (II) and the formula (III) are each represented by the following formula (II'):

[0026] [ka]

[0027] (Wherein R and

[0028] [ka]

[0029] has the same meaning as above.) and the following formula (III'):

[0030] [ka]

[0031] (Wherein R and

[0032] [ka]

[0033] has the same meaning as above.) The method according to [4], wherein the optically active substance is represented by the formula: [6] Substituent

[0034]

Chem.

[0035] is,

[0036]

Chem.

[0037] (wherein,

[0038]

Chem.

[0039] represents a substituent having an aromatic ring and / or a heterocyclic ring.).

[0040]

Chem.

[0041] ,

[0042]

Chem.

[0043] or

[0044]

Chem.

[0045] is the production method according to [4] or [5]. [7] Substituent

[0046]

Chem.

[0047] is

[0048]

Chem.

[0049] or

[0050]

Chem.

[0051] the production method described in [6]. [8] The microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding the carbonyl reductase described in any one of [1] to [3], and the nucleic acid contains a base sequence shown in the following (p), (q), or (r). The production method described in any one of [4] to [7]. (p) A base sequence in which one or more bases are substituted, deleted, and / or added in the base sequence represented by SEQ ID NO: 2, and which encodes a polypeptide having carbonyl reductase activity. (q) A base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 2, and which encodes a polypeptide having carbonyl reductase activity. (r) A base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence represented by SEQ ID NO: 2, and which encodes a polypeptide having carbonyl reductase activity.

[0052] In this specification, the substituent

[0053]

Chem.

[0054] is "substituent A", the substituent

[0055]

Chem.

[0056] may be referred to as "substituent B".

Advantages of the Invention

[0057] According to the present invention, there can be provided a carbonyl reductase having an activity of reducing a carbonyl group-containing compound to convert it into an optically active compound which is an industrially useful compound as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., a nucleic acid encoding the carbonyl reductase, a recombinant vector containing the nucleic acid, and a transformant containing the recombinant vector. Further, according to the present invention, there can be provided a production method by which an optically active compound industrially useful as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc. can be obtained industrially at high purity, high optical purity, and low cost.

Modes for Carrying Out the Invention

[0058] The present invention will be described in detail below.

[0059] 1. Carbonyl Reductase of the Present Invention The carbonyl reductase of the present invention has a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence and having at least one mutation selected from the group consisting of the following (a) to (f), and has carbonyl reductase activity. (a) A mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (b) A mutation in which the 157th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (c) A mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is substituted with serine (d) A mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine (e) A mutation in which the 214th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine (f) A mutation in which the 249th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine

[0060] In the present invention, carbonyl reductase activity refers to the activity of asymmetrically reducing a carbonyl group in a carbonyl group-containing compound to convert it to an optically active compound. The presence or absence of carbonyl reductase activity can be determined by measuring the activity of asymmetrically reducing a carbonyl group in a carbonyl group-containing compound to convert it to an optically active compound using a conventional assay method. For example, carbonyl reductase activity can be confirmed by treating a carbonyl group-containing compound represented by general formula (I), (II), or (III) with the target carbonyl reductase, 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, and directly measuring the amount of the compound represented by general formula (IV) converted from the carbonyl group-containing compound. Furthermore, in an assay system containing NADPH as a coenzyme, carbonyl reductase activity can be confirmed by measuring the initial rate of NADPH reduction.

[0061] In the present invention, the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence (OCR1) derived from Ogataea minuta var. nonfermentans NBRC (formerly IFO) 1473 described in Japanese Patent No. 4270918.

[0062] In the present invention, a homolog of the amino acid sequence represented by SEQ ID NO: 1 is a polypeptide having an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 1, and having carbonyl reductase activity.

[0063] "One to several amino acids" generally refers to 1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5 amino acids.

[0064] In the present invention, a homolog of the amino acid sequence represented by SEQ ID NO: 1 refers to a polypeptide having an amino acid sequence with 90% or more sequence identity to the full-length amino acid sequence represented by SEQ ID NO: 1 and having carbonyl reductase activity. Preferably, the homolog is a polypeptide having an amino acid sequence with 95% or more sequence identity to the full-length amino acid sequence represented by SEQ ID NO: 1, more preferably 98% or more sequence identity, and even more preferably 99% or more sequence identity, and having carbonyl reductase activity.

[0065] The homology (also referred to as identity or similarity) of amino acid sequences in this 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; allowing gaps; matrix = BLOSUM62; filtering = 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 is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], etc., and they can also be preferably used in the same way.

[0066] The amino acid sequence represented by SEQ ID NO: 1 above and homologs of the amino acid sequence can be obtained by the method described in Patent No. 4270918.

[0067] The carbonyl reductase of the present invention has excellent properties by having a mutation of (a), (b), (c), (d), (e) or (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence. Hereinafter, each mutation will be described in the order of (d), (c), (f), (a), (b), (e).

[0068] (d) The mutation is 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 the stereoselectivity and optical selectivity.

[0069] Specifically, the performance of selectively reducing the carbonyl group at the 3-position of the carbonyl group-containing compound represented by the following general formula (I) or (II) is improved. Therefore, an optically active compound represented by the general formula (IV) can be obtained from the carbonyl group-containing compound represented by the general formula (I) or (II) in high yield and high chemical purity. In addition, since the mutation of (d) also improves the optical selectivity, an optically active compound represented by the general formula (IV) can be obtained from the carbonyl group-containing compound represented by the general formula (I), (II) or (III) in high yield and high optical purity.

[0070]

Chemical formula

[0071] In particular, when the substituent A is

[0072]

Chemical formula

[0073] or

[0074]

Chemical formula

[0075] the performance of selectively reducing the carbonyl group at the 3-position is high.

[0076] Therefore, for example, by the following reaction, the optically active compounds shown below can be obtained in high yield, high chemical purity and high optical purity.

[0077]

Chemical formula

[0078]

Chemical formula

[0079] The mutation of (c) is 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 the thermal stability.

[0080] Also, the mutation of (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 the thermal stability.

[0081] Due to these mutations, the activity of the carbonyl reductase does not decrease even in the reduction reaction under heating conditions, and in order to maintain high enzyme activity, an optically active compound represented by the general formula (IV) can be obtained from a carbonyl group-containing compound represented by the general formula (I), (II) or (III) in high yield and high chemical purity. Further, when there are both the mutation of (c) and the mutation of (f), it is preferable because the thermal stability is further improved.

[0082] The mutation of (a) is 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 the stereoselectivity.

[0083] Specifically, since the performance of selectively reducing the carbonyl group at the 3-position of the carbonyl group-containing compound represented by the general formula (I) or (II) is improved, the optically active compound represented by the general formula (IV) can be obtained in high yield and high chemical purity.

[0084] In particular, the substituent A is

[0085] [ka]

[0086] In this case, the reaction rate and reaction efficiency with the carbonyl group-containing compound are improved.

[0087] Therefore, for example, by the reaction shown below, the production rate and production efficiency of the optically active compound shown below can be improved, and the optically active compound can be obtained in high yield, high chemical purity, and high optical purity.

[0088] [ka]

[0089] The mutation (b) is 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.

[0090] Furthermore, the mutation (e) is a mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. This mutation improves stereoselectivity and optical selectivity.

[0091] Here, the carbonyl reductase having the amino acid sequence represented by SEQ ID NO: 1 is represented by the following general formula (I):

[0092] [ka]

[0093] When this compound is reacted with a carbonyl group-containing compound represented by the following general formula (III), the carbonyl group at the 3-position is reduced preferentially over the carbonyl group at the 5-position.

[0094] [ka]

[0095] The compound represented by is more produced than the compound represented by the following general formula (II)

[0096]

Chemical formula

[0097] and is also more produced than the compound represented by the following general formula (III). Further, from the compound represented by the above general formula (III) to the following general formula (IV)

[0098]

Chemical formula

[0099] the reaction rate to the compound represented by is slower than the reaction rate from the compound represented by the above general formula (II) to the optically active compound represented by the above general formula (IV), so there is a possibility that the compound represented by the above general formula (III) remains.

[0100] When a carbonyl reductase having the mutation of (b) and the mutation of (e) is allowed to act on the carbonyl group-containing compound represented by the above general formula (I), the reduction of the carbonyl group at the 3-position is suppressed, and the carbonyl groups at the 3-position and 5-position are reduced in a well-balanced manner. And since the reaction rate and reaction efficiency for converting the compounds represented by the above general formulas (II) and (III) to the optically active compound represented by the above general formula (IV) are improved, the optically active compound represented by the above general formula (IV) can be efficiently obtained in high yield and high chemical purity.

[0101] In particular, when the substituent A is

[0102]

Chemical formula

[0103] the reaction rate and reaction efficiency for converting the carbonyl group-containing compound to the optically active compound are further improved.

[0104] Therefore, for example, by the reaction shown below, the production rate and production efficiency of the optically active compound shown below can be improved, and the optically active compound can be obtained in high yield, high chemical purity, and high optical purity.

[0105] [ka]

[0106] Furthermore, when both the mutation (b) and the mutation (e) are present, the reaction efficiency with carbonyl group-containing compounds is improved, which is more preferable.

[0107] 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) above in the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence homologous to said amino acid sequence and having carbonyl reductase activity. One or more mutations (a), (b), (c), (d), (e), or (f) can be appropriately selected depending on the type of target optically active compound represented by formula (IV) above, but it is preferable to have at least two mutations selected from the group consisting of (a) to (f), and it is even more preferable to have at least three mutations.

[0108] The carbonyl reductase of the present invention may have the mutation (g). The mutation (g) 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 Alanine This mutation improves the thermostability of carbonyl reductase.

[0109] 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 that is a homolog of said amino acid sequence and has carbonyl reductase activity, by methods known to those skilled in the art, such as well-known techniques such as site-directed mutagenesis and PCR.

[0110] The carbonyl reductase of the present invention can also be produced by culturing a transformant containing a nucleic acid encoding it and isolating and purifying the carbonyl reductase from the resulting culture. The nucleic acid encoding the carbonyl reductase of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., the coding strand) or the antisense strand (i.e., the non-coding strand).

[0111] Examples of DNA encoding the carbonyl reductase of the present invention include synthetic DNA. For example, a full-length carbonyl reductase cDNA can be directly amplified by reverse transcriptase-PCR using total RNA or an mRNA fraction prepared from cells or tissues derived from Ogataea minuta var. nonfermentans NBRC1473 strain as a template, and the amplified cDNA can be used in PCR amplification using a known kit, such as Mutan. TM -super Express Km(TAKARA BIO INC.), Mutan TM The cDNA can be obtained by converting the cDNA using a PCR product such as TAKARA BIO INC.-K (TAKARA BIO INC.) according to a method known per se, such as the ODA-LA PCR method, the gapped duplex method, or the Kunkel method, or a method similar thereto. Alternatively, the cDNA can be obtained by converting the cDNA cloned by colony or plaque hybridization or PCR from a cDNA library prepared by inserting the total RNA or mRNA fragments into an appropriate vector according to the above-mentioned method. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid, etc.

[0112] Examples of the nucleic acid encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:1 include, but are not limited to, as long as it encodes a polypeptide having the carbonyl reductase activity of the present invention, those having the base sequences shown in the following (p), (q), or (r). (p) A nucleic acid having a base sequence in which one or more bases are substituted, deleted, and / or added in the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention (q) A nucleic acid having a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention (r) A nucleic acid having a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention

[0113] Examples of the homolog of the nucleic acid shown in (p) above include a nucleic acid containing a base sequence in which one or more bases are deleted, substituted, inserted, and / or added in the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention. In the case of substitution, insertion, or addition, it is preferable that one or more bases are substituted, inserted, or added. Here, "one or more bases" means, for example, 1 to 60 bases, preferably 1 to 30 bases, more preferably 1 to 15 bases, still more preferably 1 to 10 bases, and particularly preferably 1 to 5 bases.

[0114] Examples of the homolog of the nucleic acid shown in (q) above include a nucleic acid having a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention. Preferably, it is a nucleic acid having a base sequence having 95% or more, more preferably 98% or more, and still more preferably 99% or more homology (also referred to as identity) with the base sequence represented by SEQ ID NO:2 and encoding a polypeptide having the carbonyl reductase activity of the present invention.

[0115] The homology (also referred to as identity) of nucleotide sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under, for example, the following conditions (expect value = 10; allowing gaps; filtering = ON; match score = 1; mismatch score = -3). As other algorithms for determining the homology of nucleotide sequences, the above-described homology calculation algorithms for amino acid sequences are similarly preferably exemplified.

[0116] As a homolog of the nucleic acid shown in (r), as long as it encodes a polypeptide having carbonyl reductase activity of the present invention, it may be a nucleic acid that hybridizes with the complementary strand of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions. Here, the "stringent conditions" can be appropriately set with reference to reported conditions (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.16.3.6, 1999). Specifically, for example, the washing conditions for ordinary Southern hybridization, such as 60°C, 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, more preferably 65°C, 0.1 x SSC, 0.1% SDS or 68°C, 0.1 x SSC, 0.1% SDS, etc. (highly stringent conditions), and washing once, more preferably 1 to 3 times, under the salt concentration and temperature corresponding thereto can be mentioned.

[0117] A person skilled in the art can appropriately perform substitution, deletion, insertion and / or addition using site-directed mutagenesis methods for the nucleic acid represented by SEQ ID NO: 2 (Nucleic Acids Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning, PCR A Practical Approach IRL Press pp. 200 (1991)) and introduce desired mutations to obtain homologs of the above-described nucleic acids.

[0118] The nucleic acid of the present invention may encode a polypeptide having the carbonyl reductase activity of the present invention. When the nucleic acid of the present invention has a nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence having a high identity with the nucleotide sequence represented by 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 that containing the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or containing a polypeptide having a homolog of the amino acid sequence, but may differ within an acceptable range (for example, about 0.1 to about 5 times, preferably about 0.3 to about 3 times).

[0119] Also, based on the amino acid sequence or a part thereof in the amino acid sequence of SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 2 or a part thereof, a homology search can be performed against a database such as the DNA Databank of JAPAN (DDBJ) to obtain amino acid sequence information of a polypeptide having carbonyl reductase activity or nucleotide sequence information of DNA encoding the same.

[0120] In the production method of the present invention described later, the above carbonyl reductase may be directly used in the reaction for the carbonyl group-containing compound as the substrate, but it is preferable 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.

[0121] As the microorganism or cell having the ability to produce the carbonyl reductase of the present invention, a microorganism or cell originally having the ability to produce the carbonyl reductase may be used, or a microorganism or cell having the production ability imparted by breeding may also be used. Regarding the microorganism or cell, regardless of its life or death, for example, resting cells can be preferably used. Examples of the type of microorganism or cell having the ability to produce the carbonyl reductase of the present invention include those described later as "host microorganism" or "host cell".

[0122] As a means for imparting the production ability by breeding, known methods such as gene recombination treatment (transformation) and mutation treatment can be employed. Examples of transformation methods include a method of introducing a target DNA and a method of modifying an expression regulatory sequence such as a promoter on a chromosome to enhance the expression of the target DNA.

[0123] Among these, it is preferable to use a microorganism or cell transformed with the DNA encoding the polypeptide of the present invention.

[0124] As described above, the nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention can be cloned by using the chromosomal DNA derived from the Ogataea minuta var. nonfermentans NBRC1473 strain as a template and performing PCR using appropriate primers.

[0125] Also, as described above, the nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention can be cloned by using the total RNA or mRNA derived from the Ogataea minuta var. nonfermentans NBRC1473 strain as a template, preparing a full-length carbonyl reductase cDNA directly amplified by the RT-PCR method, and then performing PCR using appropriate primers.

[0126] For example, by inserting the DNA encoding the polypeptide of the present invention obtained as described above into a known expression vector in an expressible arrangement, a polypeptide gene expression vector of the present invention is provided. Then, by transforming a host cell with the expression vector, a transformant into which the DNA encoding the polypeptide of the present invention has been introduced can be obtained. The transformant can also be obtained by integrally incorporating the DNA encoding the polypeptide of the present invention into the chromosomal DNA of the host in an expressible manner by a technique such as homologous recombination.

[0127] As used herein, the "expression vector" is a genetic factor used to replicate and express a protein having a desired function in a host organism by incorporating a polynucleotide encoding the protein having the desired function into the host organism. Examples include, but are not limited to, plasmids, viruses, phages, cosmids, etc. Preferably, the expression vector is a plasmid.

[0128] As used herein, the "transformant" means a microorganism or cell into which the target gene has been introduced using the above-described expression vector or the like and which can exhibit a desired trait related to the protein having the desired function.

[0129] Specific examples of the method for producing a transformant include introducing DNA encoding the polypeptide of the present invention into a plasmid vector, phage vector, or viral vector stably present in a host cell, introducing the constructed expression vector into the host cell, or directly introducing the DNA into the host genome and transcribing and translating the genetic information. In this case, it is preferable to ligate an appropriate promoter upstream of the 5'-side of the DNA, and more preferably to ligate a terminator downstream of the 3'-side. Such promoters and terminators are not particularly limited as long as they are known to function in the cells used as the host, and for example, vectors, promoters, and terminators detailed in "Microbiology Basic Course 8 Genetic Engineering, Kyoritsu Shuppan" can be used.

[0130] 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 group-containing compound that is the substrate or the optically active compound that is the target product. Examples include the following microorganisms.

[0131] Established bacteria of host-vector systems belonging to the genus Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc.

[0132] Established actinomycetes of host-vector systems belonging to the genus Rhodococcus, Streptomyces, etc.

[0133] Established yeasts of host-vector systems belonging to the genus Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, Candida, etc.

[0134] Established molds of host-vector systems belonging to the genus Neurospora, Aspergillus, Cephalosporium, Trichoderma, etc.

[0135] Procedures for preparing transformants, construction of recombinant vectors compatible with the host, and methods for culturing the host can be carried out according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Green et al., Molecular Cloning: A Laboratory Manual (4 th(Method described in (ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)).

[0136] Hereinafter, specifically, examples of preferred host microorganisms, preferred transformation methods in each microorganism, vectors, promoters, terminators, etc. will be given, but the present invention is not limited to these examples.

[0137] In the genus Escherichia, particularly in Escherichia coli, examples of plasmid vectors include pBR and pUC-based plasmids, and promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (fusion of lac and trp), λ phage PL, PR, etc. Examples of terminators include those derived from trpA, phage, and rrnB ribosomal RNA.

[0138] In the genus Bacillus, examples of vectors include pUB110-based plasmids, pC194-based plasmids, etc., and it can also be integrated into the chromosome. As promoters and terminators, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and α-amylase can be used.

[0139] In the genus Pseudomonas, examples of vectors include general host-vector systems established in Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the degradation of toluene compounds, and broad-host-range vectors based on the TOL plasmid (including genes necessary for autonomous replication derived from RSF1010, etc.) such as pKT240 (Gene, 26, 273-82 (1983)).

[0140] In the genus Brevibacterium, particularly Brevibacterium lactofermentum, examples of vectors include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). As promoters and terminators, various promoters and terminators used in Escherichia coli can be utilized.

[0141] In the genus Corynebacterium, particularly Corynebacterium glutamicum, examples of vectors include plasmid vectors such as pCS11 (Japanese Patent Laid-Open No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)).

[0142] In the genus Saccharomyces, particularly Saccharomyces cerevisiae, examples of vectors include YRp-type, YEp-type, YCp-type, and YIp-type plasmids. 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 utilized.

[0143] In the genus Schizosaccharomyces, examples of vectors include plasmid vectors derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Bio Inc. and can be easily utilized.

[0144] In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. have been most intensively studied among molds. Integration into plasmids and chromosomes is available, and promoters derived from extracellular proteases and amylases are available (Trends in Biotechnology 7, 283-287 (1989)).

[0145] In addition to the above, host-vector systems corresponding to various microorganisms have been established, and they can be used as appropriate.

[0146] In addition to microorganisms, various host-vector systems have been established in 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)), in plants such as rapeseed, corn, and potato, and systems using cell-free protein synthesis systems such as Escherichia coli cell-free extracts and wheat germ have been established and can be preferably used.

[0147] Examples of the processed product of the microorganism or cell having the ability to produce the carbonyl reductase of the present invention include those obtained by treating the microorganism or cell with an organic solvent such as acetone, dimethyl sulfoxide (DMSO), toluene, or a surfactant, freeze-dried products, cell preparations such as those physically or enzymatically disrupted, those obtained by taking out the enzyme fraction in the microorganism or cell as a crude product or a purified product, and further those immobilized on a carrier typified by polyacrylamide gel, carrageenan gel, etc.

[0148] Examples of the culture solution containing the enzyme obtained by culturing the microorganism or cell having the ability to produce the carbonyl reductase of the present invention include a suspension of the cell and a liquid medium, and when the cell is a secretion-expression type cell, the supernatant obtained by removing the cell by centrifugation or the like and its concentrate.

[0149] 2. Composition of the present invention The composition (enzyme preparation) of the present invention comprises 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 enzyme obtained by culturing the microorganism or cell, and is represented by general formula (I), (II), or (III):

[0150] [ka]

[0151] [ka]

[0152] [ka]

[0153] A carbonyl group-containing compound represented by the general formula (IV) is used as a substrate.

[0154] [ka]

[0155] The composition of the present invention is useful because, when used as a catalyst, it enables the industrially useful optically active compound, which is an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., to be industrially produced at high purity and optical purity at low cost.

[0156] The composition of the present invention may contain, in addition to the active ingredient (enzyme, etc.), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc. Examples of excipients that can be used include lactose, sorbitol, D-mannitol, and sucrose. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of stabilizers that can be used include propylene glycol and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0157] 3. Method for producing the optically active compound of the present invention According to the present invention, the carbonyl reductase of the present invention is A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II) and (III):

[0158] [ka]

[0159] [ka]

[0160] [ka]

[0161] to produce an optically active compound represented by the following general formula (IV):

[0162] [ka]

[0163] In addition, the carbonyl group-containing compounds represented by the formula (II) and the formula (III) are respectively represented by the following formula (II') and the following formula (III')[[]] [[]]

[0164] [[]] [[]] [[]]

Chemical formula

[0165] [[]] [[]] [[]]

Chemical formula

[0166] [[]] [[]]and are preferably optically active substances represented by the following formula.[[]] [[]]

[0167] [[]] [[]]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.[[]] [[]]

[0168] [[]] [[]]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 having a fluorine atom as a substituent, a substituent having a heterocyclic ring having a nitrogen atom, and / or a substituent having a naphthalene ring are preferable. Among these,[[]] [[]]

[0169] [[]] [[]] [[]]

Chemical formula

[0170] [[]] [[]],[[]] [[]]

[0171] [[]] [[]] [[]]

Chemical formula

[0172] [[]] [[]],[[]] [[]]

[0173] [[]] [[]] [[]]

Chemical formula

[0174] or

[0175] [ka]

[0176] is preferred, especially

[0177] [ka]

[0178] is preferred. Here, the substituent B represents a substituent having an aromatic ring and / or a heterocyclic ring. Specifically, the substituent B is preferably a substituent having a heterocyclic ring containing a nitrogen atom, and particularly preferably

[0179] [ka]

[0180] or

[0181] [ka]

[0182] is preferred.

[0183] In the present invention, the substituent A is

[0184] [ka] or

[0185] [ka]

[0186] It is particularly preferred that:

[0187] When the carbonyl reductase of the present invention is brought into contact with a carbonyl group-containing compound represented by general formula (I), (II), or (III), the optically active compound represented by general formula (IV) can be produced by contacting a purified or roughly purified carbonyl reductase of the present invention, a microorganism or cell capable of producing the carbonyl reductase of the present invention (e.g., a transformant having 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 with the carbonyl group-containing compound represented by general formula (I), (II), or (III).

[0188] The carbonyl reductase of the present invention may be used directly in the reaction, but it is preferable to use 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. Among these, it is preferable to use a transformant having DNA encoding the polypeptide of the present invention.

[0189] The amounts of microorganisms or cells, processed products of the microorganisms or cells, and / or culture solutions containing the enzymes obtained by culturing the microorganisms or cells to be added to the reaction solution can be appropriately selected depending on the carbonyl group-containing compound used as the substrate. For example, when adding microorganisms or cells, the microorganisms or cells are typically added to the reaction solution so that the concentration of the microorganisms or cells is approximately 0.1 w / v% to 50 w / v%, preferably 1 w / v% to 20 w / v%, based on wet cell weight. When using processed products or culture solutions, the specific activity of the enzyme is determined and an amount is added that will result in the above-mentioned cell concentration upon addition. Here, w / v% stands for weight / volume %.

[0190] Furthermore, the carbonyl reductase, the microorganism or cells capable of producing the enzyme, the processed product of the microorganism or cells, and / or the culture solution containing the enzyme obtained by culturing the microorganism or cells, which are added to the reaction solution, are easy to handle and can be used in a frozen state. When used in a frozen state, their shape is not particularly limited, and can be, for example, a prismatic, cylindrical, blocky, spherical, etc.

[0191] The reaction method is not particularly limited, and a carbonyl group-containing compound serving as a reaction substrate can be added to a liquid containing the carbonyl reductase of the present invention and reacted at an appropriate temperature and pressure (e.g., approximately atmospheric pressure), thereby producing the optically active compound represented by the general formula (IV) above.

[0192] The amount of the carbonyl group-containing compound used as a reaction substrate can be appropriately selected depending on the type of compound. For example, the carbonyl group-containing compound used as a reaction substrate can be used at a substrate concentration of 0.01 w / v% to 90 w / v%, preferably 0.1 w / v% to 30 w / v%.

[0193] The reaction substrate may be added all at once at the start of the reaction, but from the viewpoint of reducing the influence of substrate inhibition of the enzyme and increasing the accumulated concentration of the product, it is desirable to add the reaction substrate continuously or intermittently.

[0194] The reaction medium can be appropriately selected depending on the type of carbonyl group-containing compound used as the reaction substrate. For example, an aqueous medium or a mixture of an aqueous medium and an organic solvent can be used. Examples of aqueous media include water and buffer solutions. Examples of organic solvents that can be used include those that have high solubility for the carbonyl group-containing compound used as the reaction substrate, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, and dimethyl sulfoxide. Furthermore, organic solvents that are effective for removing reaction by-products, such as ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane, can also be used.

[0195] The reaction temperature, pH during the reaction, and reaction time can be appropriately selected depending on the type of carbonyl group-containing compound used 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 3 to 11, preferably 4 to 8, and the reaction time is about 0.5 to 72 hours.

[0196] After the reaction is completed, the optically active compound represented by the general formula (IV) produced by the production method of the present invention can be purified by separating the bacterial cells, proteins, etc. in the reaction solution by a separation or purification method known to those skilled in the art, such as centrifugation or membrane treatment, followed 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 with monohydrochloride, dihydrochloride, calcium salt, etc.

[0197] The present invention further relates to a method for producing an optically active compound by allowing the transformant cells of the present invention, obtained by the above-mentioned method or the like, a culture of the transformant cells, or a treated product of the transformant cells to act on a carbonyl group-containing compound represented by the following general formula (I), (II), or (III), as a reaction substrate, to asymmetrically reduce the carbonyl group of the compound. The transformant cells, the culture of the transformant cells, and the treated product of the transformant cells may be used either alone or in combination.

[0198] In particular, the process of the present invention is particularly advantageous in that the substituent A is

[0199] [ka]

[0200] or

[0201] [ka]

[0202] When it is, that is, it can be suitably used for the production of pitavastatin or rosuvastatin.

[0203] (1) In the case of pitavastatin Examples of the method for producing pitavastatin include the methods described in Japanese Patent No. 4270918, International Publication No. 2017 / 022846, and the like.

[0204] The following general formula (I'), (II''), or (III'') serving as a reaction substrate

[0205]

Chemical formula

[0206]

Chemical formula

[0207]

Chemical formula

[0208] The carbonyl group-containing compound represented by the formula can be arbitrarily produced by combining the methods described in JP-A-1-279866, JP-A-8-127585, JP-A-5-178841, International Publication No. 2017 / 022846, and known methods. These compounds can be used as raw materials alone or in combination of two or more.

[0209] When using the compound represented by the general formula (I') as a reaction substrate, there are cases of passing through the compound represented by the general formula (II'') and the compound represented by the general formula (III'') as production intermediates. Then, from the compound represented by the formula (I'), the compound represented by the formula (II'') and the compound represented by the formula (III'') are produced in advance, isolated, and further the optically active compound represented by the general formula (IV')

[0210] [Chemical]

[0211] It may be induced to, or the compound represented by formula (II'') and the compound represented by formula (III'') may be directly used to produce the compound represented by general formula (IV') without isolation.

[0212] The carbonyl group-containing compound represented by general formula (I'), (II'') or (III''), which is the reaction substrate, is usually used in the range of substrate concentration of 0.01 w / v% to 20 w / v%, preferably 0.1 w / v% to 10 w / v%. The reaction substrate may be present in the reaction system in advance, or may be added all at once at the start of the reaction. Also, when there is substrate inhibition of the enzyme, from the viewpoint of reducing its influence and improving the accumulation concentration of the product, it can also be added continuously or intermittently from the start of the reaction.

[0213] Also, the reaction is preferably carried out in the presence of coenzyme NAD(P)+ or NAD(P)H. In this case, it is preferable to add the above coenzyme so that the concentration is usually 0.001 mmol / L to 100 mmol / L, preferably 0.01 mmol / L to 10 mmol / L.

[0214] When adding the above coenzyme, in order to improve production efficiency, it is preferable to regenerate NAD(P) generated from NAD(P)H to NAD(P)H in the reaction system. As the regeneration method, + is 1) The ability of the microorganism or cell itself of the present invention to generate NAD(P)H from NAD(P), that is, the method using the reducing ability of NAD(P), + from NAD(P), + the method using the reduction ability, 2) NAD(P), +A method of adding one or more enzymes (hereinafter referred to as "regenerating enzymes") that can be used for the regeneration of NAD(P)H, such as microorganisms having the ability to generate NAD(P)H, processed products thereof, or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (such as malate dehydrogenase), etc., into the reaction system. 3) In the production of the microorganism or cell of the present invention, examples include a method of introducing one or more genes of the above regenerating enzyme into a host organism or host cell in combination.

[0215] In the method of 1) above, from the viewpoint of reaction efficiency, it is preferable to add glucose, ethanol, 2-propanol, formic acid, etc. to the reaction system.

[0216] In addition, in the method of 2) above, a microorganism having the ability to produce the above regenerating enzyme, a processed product of the microorganism such as the one treated with acetone, the one treated with glutaraldehyde, the one freeze-dried, the one physically or enzymatically disrupted, etc., the one obtained by taking out the enzyme fraction as a crude product or a purified product, and further, those immobilized on a carrier such as polyacrylamide gel, carrageenan gel, etc. may be used, or a commercially available enzyme may also be used.

[0217] The amount of the above regenerating enzyme used is preferably added so that it is usually 0.01 to 100 times, preferably about 0.5 to 20 times, in terms of enzyme activity, compared with the carbonyl reduction activity of the enzyme having the ability to stereoselectively reduce the carbonyl group of the present invention.

[0218] In addition, the addition of a compound that serves as a substrate for the above regenerating enzyme, for example, glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. is also required, and the addition amount thereof is usually 0.1 mol to 20 mol, preferably 1 mol to 10 mol, per 1 mol of the carbonyl group-containing compound that is the reaction substrate.

[0219] In addition, in the method 3) above, it is possible to use a method in which the DNA of the regenerating enzyme is integrated into a chromosome together with the DNA encoding the enzyme used in step (i), a method in which both DNAs are introduced into a single expression vector and then a host organism or cell is transformed, or a method in which both DNAs are introduced into separate expression vectors and then a host organism or host cell is transformed, etc. In the method in which both DNAs are introduced into separate expression vectors and then a host organism or host cell is transformed, it is necessary to select the expression vectors taking into consideration the incompatibility between the two expression vectors.

[0220] When multiple genes are introduced into a single expression vector, it is possible to link regions involved in expression control, such as a promoter and a terminator, to each gene, or to express the genes as an operon containing multiple cistrons, such as the lactose operon.

[0221] The reaction is carried out in an aqueous medium or 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 the carbonyl group-containing compound as a reaction substrate, as well as the enzyme, 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. Various coenzymes may also be contained as necessary. When a coenzyme is contained, it is more preferable that the coenzyme be regenerated using a regeneration system, i.e., capable of regenerating the coenzyme.

[0222] The carbonyl group-containing compound, which is the reaction substrate, can also be produced by the method described below.

[0223] Examples of aqueous media include water and pH buffer solutions such as potassium phosphate buffer, sodium citrate buffer, and Tris-HCl buffer.

[0224] 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., those with high solubility of the carbonyl group-containing compound as the reaction substrate can be used. Among these, as the organic solvent, dimethyl sulfoxide, methanol, and ethanol are preferred because of their high solubility of the carbonyl group-containing compound as the reaction substrate. Further, dimethyl sulfoxide is more preferred because of its high conversion rate.

[0225] The reaction is usually carried out at a reaction temperature of 4°C to 70°C, preferably 30°C to 60°C, and usually at a pH of 3 to 11, preferably pH 4 to 8. The reaction time is usually 0.5 hours to 48 hours, preferably 0.5 hours to 24 hours.

[0226] The compound represented by the general formula (IV') obtained can be purified by appropriately combining extraction with an organic solvent such as hexane, ethyl acetate, toluene, purification by column chromatography, crystallization, etc. after separating cells, polypeptides, etc. by centrifugation, filtration, etc. and then adjusting to an appropriate pH.

[0227] For example, in the reaction of the following formula, a carbonyl reductase having one or more, preferably two or more mutations of (d), (c), or (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence having carbonyl reductase activity can be preferably used. Further, it may also have a mutation of (g). In particular, for the reaction of the following formula, those having at least the mutation of (d) are preferred.

[0228]

Chemical formula

[0229] In addition, for example, in the reaction of the following formula, a carbonyl reductase having one or more, preferably two or more, particularly preferably three or more mutations of (a), (b), (c), (e) or (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence having carbonyl reductase activity can be preferably used. Further, it may have the mutation of (g). In particular, for the reaction of the following formula, those having the mutations of (b), (e) and (f) are preferred.

[0230] [Chemical formula]

[0231] (2) In the case of rosuvastatin Examples of the method for producing rosuvastatin include the methods described in International Publication No. 2015 / 119261 and the like.

[0232] As the reaction substrate, for example, a carbonyl group-containing compound represented by the following general formula (I'') can be used.

[0233] [Chemical formula]

[0234] The carbonyl group-containing compound represented by the general formula (I'') serving as the reaction substrate is usually used in the range of a substrate concentration of 0.01 w / v% to 20 w / v%, preferably 0.1 w / v% to 10 w / v%. The reaction substrate may be added all at once at the start of the reaction. Further, from the viewpoint of reducing the influence in the case of substrate inhibition of the enzyme and improving the accumulation concentration of the product, it can also be added continuously or intermittently.

[0235] In the reaction, coenzyme NAD(P) +It is preferably carried out in the presence of NAD(P)H. In this case, the above coenzyme is usually added so that the concentration becomes 0.001 mmol / L to 100 mmol / L, preferably 0.01 mmol / L to 10 mmol / L.

[0236] When adding the above coenzyme, in the reaction system, NAD(P) generated from NAD(P)H + is preferably regenerated to NAD(P)H for improving production efficiency. As the regeneration method, 1) The ability of the microorganism or cell itself of the present invention to generate NAD(P)H from NAD(P), that is, the method of utilizing the reducing ability of NAD(P) + from NAD(P), + the method of utilizing the reducing ability, 2) A method of adding one or more microorganisms having the ability to generate NAD(P)H from NAD(P), processed products thereof, or enzymes (hereinafter referred to as "regeneration enzymes") that can be used for the regeneration of NAD(P)H such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (such as malate dehydrogenase), etc. into the reaction system. + from NAD(P), 3) When preparing the microorganism or cell of the present invention, examples include a method of introducing one or more genes of the above regeneration enzyme into a host organism or host cell in combination.

[0237] In the method of 1) above, it is preferable to add glucose, ethanol, 2-propanol, formic acid, etc. to the reaction system.

[0238] In addition, in the method of 2) above, microorganisms having the ability to produce the above regeneration enzyme, processed products of the microorganisms such as those treated with acetone, freeze-dried products, physically or enzymatically disrupted products, etc., those obtained by taking out the enzyme fraction as a crude product or a purified product, and further, those immobilized on carriers such as polyacrylamide gel and carrageenan gel may be used, or commercially available enzymes may also be used.

[0239] In this case, the amount of the above-mentioned regenerating enzyme to be added is such that, compared with the carbonyl reduction activity of the enzyme having the ability to stereoselectively reduce the carbonyl group of the present invention, the enzyme activity is usually 0.01 to 100 times, preferably about 0.5 to 20 times.

[0240] In addition, compounds serving as substrates for the above-mentioned regenerating enzyme, for example, glucose when using glucose dehydrogenase, formic acid when using formic acid dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. also need to be added. The addition amount is usually 0.1 equivalent to 20 equivalents, preferably 1 equivalent to 10 equivalents, relative to the carbonyl group-containing compound represented by the general formula (I'').

[0241] In addition, in the method of 3) above, a method of integrating the DNA of the above-mentioned regenerating enzyme into the chromosome together with the DNA encoding the carbonyl reductase of the present invention, a method of introducing both DNAs into a single expression vector and transforming a host organism or cell, or a method of introducing both DNAs into separate expression vectors and then transforming a host organism or host cell can be used. In the case of the method of introducing both DNAs into separate expression vectors and then transforming a host organism or host cell, it is necessary to select an expression vector in consideration of the incompatibility between the two expression vectors.

[0242] When introducing a plurality of genes into a single expression vector, it is also possible to ligate regions related to expression control such as a promoter and a terminator to each gene, or to express it as an operon containing a plurality of cistrons such as the lactose operon.

[0243] The reaction is carried out in an aqueous medium or a mixture of the aqueous medium and an organic solvent, containing a carbonyl group-containing compound represented by the general formula (I''), the above 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, if necessary, various coenzymes (it is more preferable that a regeneration system thereof, i.e., the coenzyme can be regenerated). The carbonyl group-containing compound represented by the general formula (I'') can be produced by the method described in International Publication No. 2015 / 119261 etc.

[0244] Examples of the aqueous medium include water or buffers such as potassium phosphate buffer, sodium citrate buffer, and tris hydrochloride buffer.

[0245] As the organic solvent, those with high solubility of the carbonyl group-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, 2-propanol, etc., can be used. Among these, as the organic solvent, dimethyl sulfoxide, methanol, and ethanol are preferable because of the high solubility of the compound represented by the general formula (I''). Further, dimethyl sulfoxide is more preferable because of the high conversion rate.

[0246] The reaction can also be carried out in the presence of polyhydric alcohols such as glycerin, ethylene glycol, propylene glycol, erythritol, inositol, sorbitol, and xylitol. The above polyhydric alcohols may be polymers or derivatives, and can be used alone or in combination of two or more. When the reaction is carried out in the presence of polyhydric alcohols, the conversion rate tends to improve. Among them, glycerin is considered to be able to maintain enzyme activity by maintaining the higher-order structure of the enzyme, and is more preferable because it is easily available.

[0247] 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 pH 4 to 8. The reaction time is usually 0.5 hours to 48 hours, preferably 0.5 hours to 24 hours. It is also possible to carry out the reaction using a membrane reactor or the like.

[0248] The optically active compound represented by the general formula (VI'')

[0249]

Chemical formula

[0250] After separating cells, polypeptides, etc. by centrifugation, filtration, etc., the optically active compound represented by the general formula (VI'') is adjusted to an appropriate pH, and then purified by appropriately combining extraction with an organic solvent such as hexane, ethyl acetate, toluene, purification by column chromatography, crystallization, etc.

[0251] When purifying the optically active compound represented by the general formula (VI'') by crystallization, solvents that can be used include 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, polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc., and solvents in which the optically active compound represented by the general formula (VI'') has high solubility can be used. These organic solvents can be used alone, or mixed solvents of these organic solvents and water can also be used.

[0252] When purifying the optically active compound represented by the general formula (VI'') by crystallization, after dissolving the optically active compound represented by (VI'') in an organic solvent or a mixed solvent of an organic solvent and water, it is preferable to cool at a cooling rate of 15 °C / hour or less to precipitate crystals of the optically active compound represented by the general formula (VI'') (the step of cooling to precipitate crystals is hereinafter referred to as the "cooling step").

[0253] In the cooling step, the temperature at which cooling starts is preferably 15 °C to 60 °C, more preferably 20 °C to 55 °C.

[0254] In the cooling step, the cooling rate is preferably 15 °C / hour or less, more preferably 9 °C / hour or less, still more 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'').

[0255] In the cooling step, the cooling rate can also be changed halfway. In particular, in the temperature range of preferably 45 °C or less, more preferably 40 °C or less, it is preferable to cool slowly. Specifically, it is more preferable to set the cooling rate to 9 °C / hour, still more preferably 6 °C / hour or less, and particularly preferably 5 °C / hour or less.

[0256] For example, in the reaction of the following formula, a carbonyl reductase having one or more, preferably two or more, particularly preferably three or more mutations of (a), (b), (c), (e) or (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence having carbonyl reductase activity can be preferably used. Further, it may also have the mutation of (g). In particular, for the reaction of the following formula, those having the mutations of (b), (e) and (f) are preferable.

[0257]

Chemical formula

Examples

[0258] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0259] In the present example, the quantitative analysis was carried out using high performance liquid chromatography (HPLC) under the following conditions.

[0260] <Measurement condition 1> Column: Nacalai Cosmosil 3C18MS-II (4.6 x 75 mm, 3 μm) Mobile phase: methanol / acetonitrile / water / phosphoric acid = 300 / 150 / 450 / 1 Flow rate: 0.5 mL / min Column temperature: 50°C Detection wavelength: UV245nm Injection volume: 5μL

[0261] <Measurement condition 2> Column: SHISEIDO CAPCELL PAK C18 MGIII (4.6 x 75 mm, 3 μm) Mobile phase: A: 100mM HCOONH4 B: Ethanol Gradient program (B concentration): 45% (0 min) → 45% (20 min) → 80% (30 min) → 80% (35 min) Flow rate: 0.55 mL / min Column temperature: 40°C Detection wavelength: UV245nm Injection volume: 5μL

[0262] <Measurement condition 3> Column: ACQUITY UPLC BEH C18 (2.1 x 100 mm, 1.7 μm) Mobile phase: A: 0.1% formic acid aqueous solution B: 0.1% formic acid in acetonitrile Gradient program (B concentration): 40% (0 min) → 95% (12 min) → 64% (14 min) → 40% (17 min) Flow rate: 0.3 mL / min Column temperature: 40°C Detection wavelength: UV245nm Injection volume: 5μL

[0263] <Measurement Condition 4> Column: CAPCELL PAK C18 MGS-III (4.6×75 mm, 3 μm) manufactured by SHISEIDO Mobile phase: water / acetic acid / ammonium acetate = 1000 / 100 / 7.7 (volume / volume / weight) Flow rate: 1 mL / min Column temperature: 40 °C Detection wavelength: UV254 nm Injection volume: 5 μL

[0264] Reference Example 1 (Preparation of Bacterial Cells) (1) Cloning of Genes Gene cloning was performed with reference to Patent Document 1 (Japanese Patent No. 4270918). Based on the gene sequence ocr1 (SEQ ID NO: 2) encoding OCR1 (SEQ ID NO: 1) derived from Ogataea minuta var. nonfermentans NBRC 1473, PCR was carried out according to a conventional method to obtain a DNA fragment with a full length of about 0.8 kbp containing the restriction enzyme site EcoRI upstream of the ocr1 gene and the restriction enzyme site XbaI downstream thereof.

[0265] Next, with reference to Patent Document 2 (International Publication No. 2015 / 119261), based on the gene sequence (hereinafter, gdh (SEQ ID NO: 4)) encoding GDH (SEQ ID NO: 3) in which the 96th amino acid residue glutamate in glucose-1-dehydrogenase encoded by a gene (GeneBank Accession No. AL009126.3) derived from Bacillus subtilis was substituted with alanine, PCR was carried out according to a conventional method to obtain a DNA fragment with a full length of about 0.8 kbp containing the restriction enzyme site EcoRI upstream of the gdh gene and the restriction enzyme site XbaI downstream thereof.

[0266] (2) Preparation of Expression Plasmid The ocr1 DNA fragment obtained in (1) above was digested with the restriction enzymes EcoRI and XbaI, and then introduced downstream of the trc promoter into the plasmid pKV32 described in JP-A-2005-34025, which had been digested with MunI and XbaI, using a Ligation-Convenience Kit (Nippon Gene Co., Ltd.), to obtain pKV32OCR1.

[0267] Next, the gdh DNA fragment obtained in (1) above was digested with the restriction enzymes EcoRI and XbaI, and then inserted downstream of the trc promoter into the plasmid pKV32 digested with MunI and XbaI using a Ligation-Convenience Kit (Nippon Gene Co., Ltd.) to obtain pKV32GDH.

[0268] Furthermore, PCR was performed using pKV32GDH as a template according to standard methods to obtain a DNA fragment of approximately 0.8 kbp in length with HindIII restriction sites added upstream and downstream, and the resulting DNA fragment was digested with the restriction enzyme HindIII and inserted downstream of the plasmid pKV32OCR1, which had previously been digested with the restriction enzyme HindIII, to obtain pKV32OCR1-GDH. The orientation of the gdh gene in the resulting plasmid was confirmed by PCR.

[0269] (3) Preparation of plasmids for expressing OCR1 mutants Using the plasmid pKV32OCR1-GDH obtained in (2) above as a template, mutant plasmids capable of expressing the mutants shown in Table 1 below were prepared according to standard procedures using a site-directed mutagenesis technique such as the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies).

[0270] In the table 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. For example, I211A represents a mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

[0271]

Table 1

[0272] Here, D54V is the mutation of (a), M157V is the mutation of (b), A170S is the mutation of (c), I211A and I211N are the mutations of (d), M214L is the mutation of (e), M249L is the mutation of (f), and V166A is the mutation of (g).

[0273] Example 1 (Preparation of Expression Strain) Using the plasmids pKV32OCR1-GDH obtained in (2) and (3) of the above Reference Example 1 and its mutant plasmids, Escherichia coli JM109 (manufactured by Takara Bio Inc.) was transformed according to a conventional method to obtain respective transformants.

[0274] Example 2 The transformant obtained in Example 1 was allowed to act on 5S-MOLE for reduction to produce DOLE. The ratio of the syn form and anti form of the obtained DOLE was measured to evaluate the optical selectivity. Here, 5S-MOLE, syn-DOLE, and anti-DOLE are compounds having the following structures, respectively.

[0275]

Chemical formula

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

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

[0278] 750 μL of acetonitrile was added to the resulting reaction solution, followed by centrifugation, and the resulting supernatant was subjected to HPLC analysis under measurement conditions 2 and 1. Table 2 shows the anti-antibody ratio of each transformant.

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

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

[0281] [Table 2]

[0282] As is clear from Table 2, those having the I211A or I211N mutation had a reduced anti-isomer ratio compared to OCR1. In other words, it was found that the mutation (d) (I211A or I211N) improved the optical selectivity.

[0283] Example 3 The transformant obtained in Example 1 was allowed to act on DOXE to reduce it, and the reaction activity and stereoselectivity were evaluated based on the amounts of 3R-MOLE, 5S-MOLE, and DOLE produced.

[0284] Here, DOXE, 3R-MOLE, 5S-MOLE and DOLE are compounds having the following structures, respectively.

[0285] [ka]

[0286] For each of the transformants having the mutations shown in OCR1 and Table 3, the following reactions and evaluations were carried out.

[0287] The transformants were inoculated into an LB liquid medium containing 25 mg / L of kanamycin and 0.2 mmol / L of IPTG, and cultured with shaking overnight at 37°C. The cells were recovered from the obtained culture broth, suspended in a 100 mmol / L potassium phosphate buffer, and the container containing the suspension was immersed in ice water for ultrasonic disruption treatment. The obtained cell lysate was centrifuged to obtain a supernatant of disrupted cells.

[0288] To 100 μL of the obtained supernatant of disrupted cells, 100 μL of a 0.2 mol / L potassium phosphate buffer (pH 7), 100 μL of a 20 g / L glucose solution, 10 μL of a 0.2 g / L NADP + 10 μL, 100 μL of a DMSO solution of 1 g / L DOXE, and 90 μL of water were added, and the reaction was carried out at 50°C and 1000 rpm for 15 minutes using a ThermoMixer R Mixer (manufactured by Eppendorf). After the reaction, 1 mL of acetonitrile was added and mixed, followed by centrifugation. The obtained supernatant was subjected to HPLC analysis under measurement condition 3. The reaction activities and 3R-MOLE ratios of each transformant are shown in Table 3.

[0289] The reaction activity and 3R-MOLE ratio were calculated from the peak area values obtained by HPLC analysis using the following calculation formulas. Reaction activity = 5S-MOLE peak area + 3R-MOLE peak area 3R-MOLE ratio = 3R-MOLE peak area / 5S-MOLE peak area Note that the reaction activity was evaluated as "-" when it was 0.9 times or less based on OCR1, "+" when it was 1.0 times, "++" when it was 1.1 times, and "+++" when it was 1.2 times or more. Also, the 3R-MOLE ratio was evaluated as "+" when it was 0.9 to 1.0 times based on OCR1, "++" when it was 0.7 to 0.8 times, and "+++" when it was 0.6 times or less.

[0290]

Table 3

[0291] As is clear from Table 3, those having the M157V or M214L mutation had a reduced 3R-MOLE ratio compared to OCR1. In particular, those having both the M157V and M214L mutations not only had a reduced 3R-MOLE ratio but also had improved reaction activity. That is, it was found that the stereoselectivity and reaction activity were improved by M157V (the mutation in (b)) and M214L (the mutation in (e)).

[0292] Example 4 For each of OCR1 and the transformants having the mutations shown in Table 4, the following reactions and evaluations were carried out.

[0293] The transformants were inoculated into an LB liquid medium containing 25 mg / L of kanamycin and 0.2 mmol / L of IPTG and cultured with shaking overnight at 37°C. The cells were recovered from the obtained culture solution, suspended in a 100 mmol / L potassium phosphate buffer, and the container containing the suspension was immersed in ice water for ultrasonic disruption treatment. The obtained cell lysate was centrifuged to obtain a supernatant of the disrupted cells.

[0294] For the obtained supernatant of the disrupted cells, the protein concentration was measured according to a conventional method using a Quick Start Bradford protein assay kit (manufactured by BIO-RAD Laboratories).

[0295] Also, the obtained supernatant of the disrupted cells was divided into two. One was stored refrigerated at 4°C to 10°C, and the other was heat-treated at 50°C and 200 rpm for 1 hour using a ThermoMixer R Mixer (manufactured by Eppendorf).

[0296] For each cell lysate supernatant that had been stored refrigerated or heat-treated, 10 μL of the cell lysate supernatant, 25 μL of a 1 mol / L potassium phosphate buffer (pH 7), 10 μL of an 8 mmol / L NADPH, 200 μL of water, and 5 μL of a DMSO solution of 0.1 mol / L 2,2,2-trifluoroacetophenone (TFAP) were mixed. The mixture was placed in a 96-well plate (manufactured by Corning), and using a microplate reader (manufactured by Molecular Bio), the change in absorbance at 340 nm was measured for 2 minutes. The average value of the change in absorbance per unit time (mOD / min) was calculated, and from the slope of the change in absorbance, the activity value per unit time and per unit protein was calculated using the following formula.

[0297] Also, for each cell lysate supernatant that had been stored refrigerated or heat-treated, the protein concentration was measured according to a conventional method using a Quick Start Bradford Protein Assay Kit (manufactured by BIO-RAD Laboratories).

[0298] Note that the molar extinction coefficient of NADPH was calculated as 6.3 mL / μmol·cm. Activity value (μmol / min / mg-protein) = (-1) × slope of absorbance change × 250 μL / 10 μL / 6300 / 0.55 / protein concentration

[0299] By applying the activity value obtained as described above to the following formula, the activity ratio before and after heat treatment was calculated to evaluate the thermal stability. The thermal stability of each transformant is shown in Table 4. Thermal stability (%) = activity value during heat treatment / activity value during refrigerated storage × 100

[0300] When the thermal stability value was less than 20%, it was evaluated as "-", when it was in the range of 20% to 70%, it was evaluated as "+", and when it was more than 70%, it was evaluated as "++".

[0301]

Table 4

[0302] As is clear from Table 4, those having the A170S or M249L mutation had improved thermal stability. In particular, those having both the A170S and M249L mutations had further improved thermal stability. That is, it was found that the thermal stability was improved by A170S (the mutation in (c)) and M249L (the mutation in (f)).

[0303] In addition, among the mutations of A170S, M249L, and V166A, it was found that those having two or more of them had improved thermal stability.

[0304] Example 5 The enzyme activity was measured for the cell lysate supernatant of OCR1 obtained in the same manner as in Example 3 and having the mutations shown in Table 6.

[0305] The enzyme activity was confirmed by measuring the decrease in the amount of NADPH at the time of TFAP addition at a reaction temperature of 30°C at a wavelength of 340 nm in a reaction solution having the composition shown in Table 5. The unit U of the enzyme activity indicates the conversion amount (μmol) per minute.

[0306] [Table 5]

[0307] In the following reaction, the amount of carbonyl reductase was weighed based on the above enzyme activity (unit: U).

[0308] In addition, DOXE and DOLE are compounds having the following structures, respectively.

[0309] [Chemical formula]

[0310] DOXE at a concentration of 10 g / L, phosphate buffer (pH 6.0) at a concentration of 100 mmol / L, glucose at a concentration of 120 mmol / L, NADP at a concentration of 2 mol% +A 2 mL reaction solution containing 20 wt% DMSO, 10 U of carbonyl reductase, and 5 U to 10 U of glucose dehydrogenase was prepared. The reaction solution was reacted at 50 °C for 4 hours while shaking. The obtained DOLE was analyzed by HLPC under measurement condition 3. The results are shown in Table 6.

[0311]

Table 6

[0312] As is clear from Table 6, those having the above mutations had a significantly improved DOLE conversion rate. That is, it was found that those having these mutations had a greatly improved carbonyl reduction activity and reaction efficiency.

[0313] Reference Example 2 This reference example is the method described in Example 4-4 of International Publication No. 2015 / 119261.

[0314] To a 1 L jar fermenter (manufactured by Able Corporation, model BMJ-01), 385.9 mL of ion-exchanged 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 charged and dissolved. Then, a total amount of a substrate solution prepared by dissolving 14.4 g (27.7 mmol) of DOXP in 50.60 g of frozen cells of recombinant Escherichia coli JM109 / pKV32OCR1-GDH and 124.20 g (1589.7 mmol) of DMSO was added, and the internal temperature was stirred at 50 °C for 5 hours. During the reaction, a 25 wt% aqueous sodium hydroxide solution was added dropwise to maintain the pH at 6. The presence of DOLP was confirmed by the retention time of HPLC, and the conversion rate at this time was 90.25%.

[0315] Here, DOXP and DOLP are compounds having the following structures, respectively.

[0316]

Chemical formula

[0317] Example 6 The transformant JM109 / pKV32OCR1 (having the mutations of A170S / I211A / M249L (SEQ ID NO: 16))-GDH obtained in Example 1 was inoculated into an LB liquid medium containing 25 mg / L kanamycin and 0.2 mmol / L IPTG, and cultured with shaking overnight at 37°C. Cells were collected from the obtained culture solution, and the reaction was carried out in the same manner as in Reference Example 2 to obtain DOLP.

[0318] When the obtained DOLP was analyzed by HPLC under measurement condition 4, the conversion rate was 95.98%, and the conversion rate was improved as compared with Reference Example 2.

[0319] It was found that the reaction efficiency was improved by having the mutations of A170S ((c) mutation), I211A ((d) mutation) and M249L ((f) mutation).

Industrial Applicability

[0320] According to the present invention, there can be provided a carbonyl reductase having an activity of reducing a carbonyl group-containing compound and converting it into an optically active compound which is an industrially useful compound as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., a nucleic acid encoding the carbonyl reductase, a recombinant vector containing the nucleic acid, and a transformant containing the recombinant vector. Furthermore, according to the present invention, there is provided a production method by which an optically active compound industrially useful as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc. can be obtained industrially at high purity, high optical purity and low cost, and the production efficiency of the pharmaceuticals, agricultural chemicals, etc. can be improved and the production can be increased.

[0321] This application is based on Japanese Patent Application No. 2019-211797 (filing date: November 22, 2019) filed in Japan, the content of which is incorporated herein in its entirety.

Claims

1. A carbonyl reductase having a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence represented by SEQ ID NO: 1 having 90% or more sequence identity to the full length of the amino acid sequence represented by SEQ ID NO: 1, and having at least one mutation selected from the group consisting of the following (a), (c), (d), and (f). (a) A mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (c) A mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is substituted with serine (d) A mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine (f) A mutation in which the 249th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine

2. The carbonyl reductase according to claim 1, characterized by having at least two mutations selected from the group consisting of (a), (c), (d), and (f).

3. The carbonyl reductase according to claim 1 or 2, further characterized by having the following mutations (b) and / or (e). (b) A mutation in which the 157th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (e) A mutation in which the 214th methionine in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine

4. The carbonyl reductase according to any one of claims 1 to 3, having a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a homolog of the amino acid sequence, and further having a mutation in which the 166th valine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (g).

5. A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II), and (III). 【Chemical 1】 【Chemical 2】 【Chemical Formula 3】 (In the above formulas (I), (II), and (III), R represents a hydrogen atom, an alkyl group, or an aryl group, and represents a substituent selected from.) 【Chemical 4】 is 【Chemical Formula 5】 represents a substituent selected from.) The carbonyl reductase according to any one of claims 1 to 4, a microorganism or cell having the ability to produce the enzyme, a microorganism or cell treated with an organic solvent or a surfactant, a freeze-dried product, a physically or enzymatically disrupted product, or an enzyme fraction in a microorganism or cell taken out as a crude product or a purified product, which is selected from those containing the carbonyl reductase of the present invention or having the ability to produce the carbonyl reductase of the present invention, and / or contacting a culture solution containing the enzyme obtained by culturing the microorganism or cell to asymmetrically reduce a carbonyl group-containing compound, characterized by the following general formula (IV) 【Chemical Formula 6】 (wherein R and 【Chemical Formula 7】 are as defined above.) A method for producing an optically active compound represented by the formula:

6. The carbonyl group-containing compounds represented by the formula (II) and the formula (III) are respectively the following formula (II')[ [Chemical Formula 8] (wherein R and 【Chemical Formula 9】 are as defined above.) And the following formula (III')[ 【Chemical 10】 (wherein R and 【Chemical 11】 are as defined above.) The production method according to claim 5, characterized in that it is an optically active substance represented by the formula:

7. Substituent 【Chemical Formula 12】 is 【Chemical 13】 The production method according to claim 5 or 6

8. The microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding the carbonyl reductase according to any one of claims 1 to 4 and containing a nucleotide sequence shown in the following (p), (q) or (r), The production method according to any one of claims 5 to 7 (p) A nucleotide sequence having one or more bases substituted, deleted and / or added in the nucleotide sequence represented by SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity (q) A nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity (r) A nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence represented by SEQ ID NO: 2 and encodes a polypeptide having carbonyl reductase activity

9. The carbonyl reductase according to any one of claims 1 to 4, a microorganism or cell having the ability to produce the enzyme, a product obtained by treating the microorganism or cell with an organic solvent or a surfactant, a lyophilized product, a physically or enzymatically disrupted product, a product obtained by taking out the enzyme fraction in the microorganism or cell as a crude product or a purified product, and being selected from those containing the carbonyl reductase of the present invention or having the ability to produce the carbonyl reductase of the present invention, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell, represented by the following general formulas (I), (II) and (III) 【Chemical Formula 14】 【Chemical 15】 【Chemical Formula 16】 (In the above formulas (I), (II) and (III), R represents a hydrogen atom, an alkyl group or an aryl group, 【Chemical 17】 represents 【Chemical Formula 18】 a substituent selected from.) A composition that catalyzes a reaction to produce an optically active compound represented by the following general formula (IV) 【Chemical Formula 19】 (wherein R and 【Chemical 20】 are as defined above.) from a carbonyl group-containing compound selected from the group consisting of compounds represented by

10. The composition according to claim 9, wherein the substituent 【Chemical Formula 21】 is 【Chemical 22】 .

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