Process for producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and intermediate thereof

文档序号：1894716 发布日期：2021-11-26 浏览：17次中文

阅读说明：本技术 (1r,3r)-3-(三氟甲基)环己烷-1-醇及其中间体的生产方法 (Process for producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and intermediate thereof ) 是由佐佐野晴花井浦崇敦大木健二于 2020-04-17 设计创作，主要内容包括：本发明要解决的问题是提供一种以高光学纯度、高选择性、高效率和低成本工业化生产(1R,3R)-3-(三氟甲基)环己烷-1-醇及其中间体的新方法。本发明涉及一种由式(3)表示的化合物的生产方法,所述方法的特征在于包括将碳-碳双键还原酶、能够产生所述酶的微生物或细胞、所述微生物或细胞的加工产物和/或通过培养所述微生物或细胞获得的含有所述酶的培养液和羰基还原酶、能够产生所述酶的微生物或细胞、所述微生物或细胞的加工产物和/或通过培养所述微生物或细胞获得的含有所述酶的培养液与由式(1)表示的化合物相接触,从而得到由式(3)表示的化合物。(The invention aims to provide a new method for industrially producing (1R,3R) -3- (trifluoromethyl) cyclohexane-1-alcohol and an intermediate thereof with high optical purity, high selectivity, high efficiency and low cost. The present invention relates to a method for producing a compound represented by formula (3), which is characterized by comprising contacting a carbon-carbon double bond 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 with a 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 with a compound represented by formula (1) to obtainTo a compound represented by the formula (3).)

1. A process for producing a compound represented by the formula (3),

the method comprising contacting a carbon-carbon double bond reductase, 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 a carbonyl reductase, 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 with a compound represented by formula (1),

to obtain the compound represented by formula (3).

2. The production method according to claim 1, wherein the carbon-carbon double bond reductase comprises a protein shown in the following (a), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 1;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 1, and has an activity of catalyzing a reaction represented by formula (I) and/or formula (II):

(C) has a sequence similar to SEQ ID NO: 1, wherein at least one amino acid substitution selected from the group consisting of the following groups (I) to (II) is introduced, and has an activity of catalyzing a reaction shown in formula (I) and/or formula (II):

(i) converting SEQ ID NO: 1 with an amino acid other than aspartic acid, phenylalanine, tryptophan and tyrosine,

(ii) converting SEQ ID NO: 1 by substituting alanine at position 104 with an amino acid other than alanine.

3. The production method according to claim 2, wherein the amino acid substitution in (i) is the following (i'):

(i') converting SEQ ID NO: 1 by substituting the cysteine at position 26 in the amino acid sequence shown in figure 1 with alanine.

4. The production method according to claim 2 or 3, wherein the amino acid substitution in (ii) is the following (ii'):

(ii') converting SEQ ID NO: 1 by substituting alanine at position 104 with histidine, phenylalanine, tryptophan or tyrosine.

5. The production method according to claim 1 or 2, wherein the carbonyl reductase comprises a protein shown in the following (a), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 2.3 or 4;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 2.3 or 4, and has an activity of catalyzing a reaction shown in formula (III) and/or formula (IV):

(C) has a sequence similar to SEQ ID NO: 2.3 or 4 has an amino acid sequence of not less than 80% identity, and has an activity of catalyzing a reaction shown in formula (III) and/or formula (IV).

6. The production method according to any one of claims 1 to 5, wherein the carbon-carbon double bond reductase, a microorganism or cell having an 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 is contacted with the compound represented by formula (1),

to obtain a compound represented by the formula (2)

And in addition thereto

Contacting the carbonyl reductase, a microorganism or cell having an 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 with the compound represented by formula (2) to obtain the compound represented by formula (3)

7. The production method according to any one of claims 1 to 5, wherein the carbonyl reductase, a microorganism or cell having an 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 is contacted with the compound represented by formula (1),

to obtain a compound represented by the formula (4)

And in addition thereto

Contacting a carbon-carbon double bond reductase, 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 with the compound represented by formula (4) to obtain the compound represented by formula (3)

8. The production method according to any one of claims 1 to 7, wherein the compound represented by formula (5) contained in compound (3)

And/or a compound represented by the formula (6)

The content of (B) is not more than 8 mol%.

9. A process for producing a compound represented by the formula (2),

to obtain the compound represented by formula (2).

10. The production method according to claim 9, wherein the carbon-carbon double bond reductase comprises a protein shown in the following (a), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 1;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 1, and has an activity of catalyzing a reaction shown in formula (I):

(i) converting SEQ ID NO: 1 with an amino acid other than aspartic acid, phenylalanine, tryptophan and tyrosine,

(ii) converting SEQ ID NO: 1 by substituting alanine at position 104 with an amino acid other than alanine.

11. A process for producing a compound represented by the formula (4),

the method comprising contacting a carbonyl reductase, 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 with a compound represented by formula (1),

to obtain the compound represented by formula (4).

12. The production method according to claim 11, wherein the carbonyl reductase comprises a protein shown in the following (a), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 2.3 or 4;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 2.3 or 4, and has an activity of catalyzing a reaction shown in formula (IV):

(C) has a sequence similar to SEQ ID NO: 2.3 or 4 has an amino acid sequence of not less than 80% identity, and has an activity of catalyzing the reaction shown in formula (IV).

Technical Field

The present invention relates to a process for producing a cyclohexane derivative (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and intermediates thereof, which are useful as starting materials and intermediates for the synthesis of pharmaceutical products.

Background

The (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol, its intermediates and its derivatives are useful as starting materials and intermediates for the synthesis of various pharmaceutical products.

As a method for producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol, a biological method for producing them from racemic 3-trifluoromethylcyclohexanone is known (non-patent document 4).

However, with the microorganism used in non-patent document 4, the reaction did not proceed sufficiently, the yield was as low as about 34%, and the optical purity of the obtained (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol was also as low as about 39% compared with 4 isomers, thus confirming low optical selectivity (run 14). Therefore, since enantiomer (1S,3S) -3- (trifluoromethyl) cyclohexan-1-ol and two diastereomers (1R,3S) -3- (trifluoromethyl) cyclohexan-1-ol and (1S,3R) -3- (trifluoromethyl) cyclohexan-1-ol are produced in large amounts as by-products in addition to desired (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol, it is difficult to produce the desired products with high efficiency. In addition, in the case of industrial production, large separation equipment and large purification equipment are required, which increases the cost, and the method is not suitable for an industrial production method as an intermediate of a pharmaceutical product.

Although patent documents 1, 2 and non-patent document 3 describe enzymes that convert carbonyl groups into hydroxyl groups, they do not describe the application of the enzymes to the compounds of the present invention.

Although patent document 3 and non-patent document 1 describe a carbon-carbon double bond reductase, they do not describe the application of the reductase to the compound of the present invention.

Non-patent document 2 describes a method for obtaining a compound analogous to (3R) -3- (trifluoromethyl) cyclohexan-1-one (hereinafter sometimes referred to as compound (2)), which comprises contacting a carbonyl group and a compound analogous to 3-trifluoromethyl-2-cyclohexen-1-one (hereinafter sometimes referred to as compound (1)) but in which the trifluoromethyl group at the 3-position has been replaced by a methyl group, with a specific carbon-carbon double bond reductase. However, the application of specific enzymes to the compounds of the present invention is not described. As is clear from comparative examples 1, 2 and 6 mentioned below, when an enzyme (YqjM variants 1, 2 and 15) described in non-patent document 2 and superior to a compound similar to compound (1) in reactivity and optical selectivity is brought into contact with compound (1), the reaction hardly proceeds.

Therefore, there is a need for a method for industrially producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol (hereinafter sometimes referred to as compound (3)) useful as an intermediate for pharmaceutical products at low cost, high purity and high efficiency.

[ List of documents ]

[ patent document ]

Patent document 1: WO 2008/042876

Patent document 2: WO 2004/027055

Patent document 3: WO 2011/052718

[ non-patent document ]

Non-patent document 1: journal of Biological Chemistry, volume 278, No. 22, month 5, 30, pages 19891-19897

Non-patent document 2: adv. Synth. Catal.2009,351, page 3287-

Non-patent document 3: org. chem.,1992,57(5), pages 1532-1536

Non-patent document 4: tetrahedron (1998),54(12), page 809 + 2818

Disclosure of Invention

[ problem ] to

The present invention aims to provide a novel method for industrially producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and its intermediate at low cost, high optical purity, high selectivity and high efficiency.

[ technical solution ]

In order to solve the above problems, the present inventors have intensively studied and found that a carbon-carbon double bond reductase (hereinafter sometimes referred to as C ═ C reductase) derived from Bacillus subtilis reduces 3-trifluoromethyl-2-cyclohexen-1-one with high selectivity and can produce an intermediate (3R) -3- (trifluoromethyl) cyclohexan-1-one with high efficiency.

In addition, they found that carbonyl reductase (hereinafter sometimes referred to as C ═ O reductase) derived from Lactobacillus kefir (Lactobacillus kefir), Pichia finlandica, and navelbia riboflavin (hereinafter sometimes referred to as compound (4)) reduced 3-trifluoromethyl-2-cyclohexene-1-one with high selectivity and could produce (1R) -3-trifluoromethyl-2-cyclohexene-1-ol (hereinafter sometimes referred to as compound (4)) as an intermediate with high efficiency.

Further, they found that (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol can be obtained at a low cost in high optical purity and high concentration by contacting the enzyme, a microorganism or cell having an ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture broth containing the enzyme obtained by culturing the microorganism or cell (these are sometimes collectively referred to as "enzyme or the like" hereinafter) with 3-trifluoromethyl-2-cyclohexen-1-one, (3R) -3- (trifluoromethyl) cyclohexan-1-one or (1R) -3-trifluoromethyl-2-cyclohexen-1-ol. The present invention has been made on the basis of these findings.

That is, the gist of the present invention is as follows:

[1] a process for producing a compound represented by the formula (3),

((1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol (hereinafter sometimes referred to as compound (3))),

(3-trifluoromethyl-2-cyclohexen-1-one (hereinafter sometimes referred to as Compound (1)))

To obtain the compound represented by formula (3).

[2] The production process according to [1] above, wherein the above-mentioned carbon-carbon double bond reductase comprises a protein represented by the following (A), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 1;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 1, and has an activity of catalyzing a reaction represented by formula (I) and/or formula (II):

(i) converting SEQ ID NO: 1 with an amino acid other than aspartic acid, phenylalanine, tryptophan and tyrosine,

(ii) converting SEQ ID NO: 1 by substituting alanine at position 104 with an amino acid other than alanine.

[3] The production process according to the above [2], wherein the amino acid substitution in the (i) is the following (i'):

(i') converting SEQ ID NO: 1 by substituting the cysteine at position 26 in the amino acid sequence shown in figure 1 with alanine.

[4] The production process according to the above [2] or [3], wherein the amino acid substitution in (ii) is the following (ii'):

(ii') converting SEQ ID NO: 1 by substituting alanine at position 104 with histidine, phenylalanine, tryptophan or tyrosine.

[5] The production method according to [1] or [2] above, wherein the carbonyl reductase comprises a protein represented by the following (A), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 2.3 or 4;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 2.3 or 4, and has an activity of catalyzing a reaction shown in formula (III) and/or formula (IV):

(C) has a sequence similar to SEQ ID NO: 2.3 or 4 has an amino acid sequence of not less than 80% identity, and has an activity of catalyzing a reaction shown in formula (III) and/or formula (IV).

[6] The production process according to any one of the above [1] to [5], wherein the carbon-carbon double bond reductase, a microorganism or cell having an 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 is contacted with the compound represented by the formula (1),

to obtain a compound represented by the formula (2)((3R) -3- (trifluoromethyl) cyclohexan-1-one (hereinafter sometimes referred to as Compound (2))), and further

[7] The production process according to any one of the above [1] to [5], wherein the carbonyl reductase, a microorganism or cell having an 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 is contacted with the compound represented by the formula (1),

to obtain a compound represented by the formula (4)((1R) -3-trifluoromethyl-2-cyclohexen-1-ol (hereinafter sometimes referred to as Compound (4))), and further

[8] The production process described in any one of the above [1] to [7], wherein the compound represented by the formula (5) contained in the compound (3)

(hereinafter sometimes referred to as Compound (5))

And/or a compound represented by the formula (6)(hereinafter sometimes referred to as Compound (6))

The content of (B) is not more than 8 mol%.

[9] A process for producing a compound represented by the formula (2),

to obtain the compound represented by formula (2).

[10] The production method according to the above [9], wherein the above-mentioned carbon-carbon double bond reductase comprises a protein represented by the following (A), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 1;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 1, and has an activity of catalyzing a reaction shown in formula (I):

(i) converting SEQ ID NO: 1 with an amino acid other than aspartic acid, phenylalanine, tryptophan and tyrosine,

(ii) converting SEQ ID NO: 1 by substituting alanine at position 104 with an amino acid other than alanine.

[11] A process for producing a compound represented by the formula (4),

to obtain the compound represented by formula (4).

[12] The production method according to [11] above, wherein the carbonyl reductase comprises a protein represented by the following (A), (B) or (C):

(A) has the sequence shown in SEQ ID NO: 2.3 or 4;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 2.3 or 4, and has an activity of catalyzing a reaction shown in formula (IV):

(C) has a sequence similar to SEQ ID NO: 2.3 or 4 has an amino acid sequence of not less than 80% identity, and has an activity of catalyzing the reaction shown in formula (IV).

[ advantageous effects ]

According to the present invention, a novel method for industrially producing (1R) -3-trifluoromethyl-2-cyclohexen-1-ol or (3R) -3- (trifluoromethyl) cyclohexan-1-one, which is useful as a starting material and an intermediate for synthesizing various pharmaceutical products, at low cost, high optical purity, high selectivity and high efficiency, can be provided. Further, using the thus obtained (1R) -3-trifluoromethyl-2-cyclohexen-1-ol or (3R) -3- (trifluoromethyl) cyclohexan-1-one, (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol having high optical purity can be produced efficiently at low cost.

The (1R) -3-trifluoromethyl-2-cyclohexen-1-ol or (3R) -3- (trifluoromethyl) cyclohexan-1-one produced by the process of the present invention, the compound (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol produced using them, can be used as starting materials and intermediates for the synthesis of various pharmaceutical products.

Detailed Description

The present invention is explained in detail below.

< production method of the present invention >

In the present specification, production methods 1 to 3 mean the following production methods, respectively.

The production method 1: process for producing Compound (3) comprising step 1 and step 2

The production method 2 comprises the following steps: process for producing Compound (3) comprising step 3 and step 4

The production method 3: process for producing Compound (3) comprising step 5

In the present specification, steps 1 to 5 mean the following steps, respectively.

Step 1: a step of contacting C ═ C reductase or the like with compound (1) to obtain compound (2)

Step 2: a step of oxidizing compound (3) by contacting C ═ O reductase or the like with compound (2)

And step 3: a step of contacting C ═ O reductase or the like with compound (1) to obtain compound (4)

And 4, step 4: a step of contacting C ═ C reductase or the like with compound (4) to obtain compound (3)

And 5: a step of contacting C ═ C reductase or the like and C ═ O reductase or the like with compound (1) to obtain compound (3)

The present invention is explained in detail below.

1. Enzyme used in the production method of the present invention

[ C ═ C reductase ]

The production method of the present invention is characterized in that a carbon-carbon double bond reductase (C ═ C reductase), 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 a carbonyl reductase (C ═ O reductase), 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 with the compound represented by formula (1) to obtain the compound represented by formula (3). In the present specification, the enzyme, the microorganism or cell having an ability to produce the enzyme, the processed product of the microorganism or cell, and/or the culture solution containing the enzyme obtained by culturing the microorganism or cell are sometimes referred to as "enzyme or the like". The C ═ C reductase is explained below.

In the production method of the present invention, the C ═ C reductase is not particularly limited. The C ═ C reductase can also be obtained, for example, by purification and isolation from bacillus subtilis using a known method. As used herein, purification methods include, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, combinations of these methods, and the like.

The C ═ C reductase can also be obtained by culturing transformants containing nucleic acids encoding them, and isolating and purifying the C ═ C reductase from the obtained culture. The nucleic acid encoding C ═ C reductase may be DNA or RNA or a DNA/RNA chimera. Preferred is DNA. The nucleic acid may be double-stranded or single-stranded. When it is double-stranded, double-stranded DNA, double-stranded RNA, or DNA: RNA hybrid can be used. When it is single stranded, either the sense strand (i.e., the coding strand) or the antisense strand (i.e., the non-coding strand) may be used.

The DNA encoding C ═ C reductase includes, for example, the base sequence (SEQ ID NO: 5) of the yqjm gene derived from Bacillus subtilis (GeneBank accession number P54550).

The DNA encoding C ═ C reductase also includes synthetic DNA and the like. For example, a DNA encoding C ═ C reductase derived from bacillus subtilis can be obtained by converting full-length C ═ C reductase cDNA by reverse transcriptase-PCR using total RNA or mRNA fraction derived from bacillus subtilis as a template according to a method known per se such as ODA-LA PCR method, gapped duplex method, Kunkel method, etc. or a method similar thereto, and by using a known kit such as Mutan^TM-super Express Km(TAKARA BIO INC.)、Mutan^TM-K (TAKARA BIO INC.) and the like. Alternatively, it can be obtained by converting cDNA cloned by colony or phage hybridization or PCR or the like from a cDNA library obtained by inserting the above-mentioned total RNA or mRNA fragment into a suitable vectorThe carrier of (1). The vector for the library may be any vector such as a phage, plasmid, cosmid, phagemid, etc.

A nucleic acid (DNA) encoding C ═ C reductase can be cloned by PCR, for example, using chromosomal DNA derived from bacillus subtilis as a template and appropriate primers. For example, a C ═ C reductase gene expression vector is provided by inserting the DNA encoding C ═ C reductase obtained as described above into a known expression vector in a configuration allowing expression. By transforming a host cell with the expression vector, a transformant into which a DNA encoding C ═ C reductase is introduced can be obtained. The transformant can also be obtained by incorporating a DNA encoding C ═ C reductase into the chromosomal DNA of the host in an expressible manner using a method such as homologous recombination.

In the present specification, an "expression vector" is a genetic element for replicating and expressing a protein having a desired function in a host organism by incorporating a polynucleotide encoding the protein having the desired function into the vector and introducing the vector into the host organism. Examples of expression vectors include, but are not limited to, plasmids, viruses, phages, cosmids, and the like. Preferred expression vectors are plasmids.

In the present specification, "transformant" means a microorganism or cell which has become capable of expressing a desired property related to a protein having a desired function by introducing a target gene using the above-mentioned expression vector or the like.

As a method for producing a transformant, specifically, the following methods can be mentioned as examples: a method comprising introducing a DNA encoding C ═ C reductase into a plasmid vector, a phage vector or a viral vector stably present in a host cell and introducing the constructed expression vector into the host cell, and a method comprising directly introducing the DNA into a host genome and transcribing or translating genetic information thereof. In this case, it is preferable to ligate a suitable promoter to the 5 '-side upstream of the DNA in the host, and it is further more preferable to ligate a terminator to the 3' -side downstream. Such promoters and terminators are not particularly limited as long as they are known to function in a cell used as a host, and, for example, those described in "microbiological fundamentals 8: genetic Engineering (Microbiological Basic feature 8Genetic Engineering) (BISEIUTSUGAKU KISO-KOHZA 8IDENSHI-KOHGAKU), KYORITSU SHIPPPAN CO., LTD, vectors, promoters and terminators as described in detail in LTD.

The host microorganism which is a target of transformation to express C ═ C reductase is not particularly limited as long as the host itself does not adversely affect the compound (1), the compound (2), the compound (4), or the compound (3), and, for example, microorganisms shown below can be mentioned.

Having an established host vector system, and belonging to the genus Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc.

Actinomycetes belonging to the genus Rhodococcus, Streptomyces or the like having an established host vector system.

Yeasts having an established host vector system and belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, Candida, etc.

Fungi having an established host vector system and belonging to the genus Neurospora, Aspergillus, Cephalosporium, Trichoderma and the like.

The procedures for producing the transformant, the construction of a recombinant vector suitable for the host, and the methods for culturing the host can be carried out according to techniques conventionally used in the fields of molecular biology, biotechnology, and genetic engineering (for example, the methods described in "molecular cloning").

Specific examples of preferred host microorganisms, preferred transformation methods in each microorganism, vectors, promoters, terminators, and the like are given below, but the present invention is not limited to these examples.

In the genus Escherichia, particularly Escherichia coli, examples of plasmid vectors include pKV32, pKW32, pBR, pUC plasmids and the like, and promoters such as those derived from lac (. beta. -galactosidase), trp (tryptophan operon), tac, trc (fusion of lac, trp), lambda phage PL, PR and the like. Examples of the terminator include terminators derived from trpA, phage, rrnB ribosomal RNA and the like.

In the genus Bacillus, examples of the vector include pUB 110-based plasmids, pC 194-based plasmids, and the like, and the vector may also be integrated with a chromosome. As the promoter and terminator, promoters and terminators of enzyme genes such as alkaline protease, alpha-amylase, and the like can be used.

In the genus Pseudomonas, examples of the vector include a general-purpose host vector system established in Pseudomonas putida (Pseudomonas putida), Pseudomonas cepacia (Pseudomonas cepacia), etc., a plasmid associated with the decomposition of toluene compound, a broad-host range vector based on TOL plasmid (including a Gene required for autonomous replication derived from RSF1010, etc.) pKT240(Gene,26,273-82(1983)), etc.

In the genus Brevibacterium, particularly Brevibacterium lactofermentum (Brevibacterium lactofermentum), examples of vectors include plasmid vectors such as pAJ43(Gene 39,281(1985)) and the like. As the promoter and terminator, various promoters and terminators for Escherichia coli can be used.

In the genus Corynebacterium, particularly Corynebacterium glutamicum, examples of vectors include plasmid vectors such as pCS11(JP-A-57-183799), pCB101(mol.Gen.Genet.196,175(1984)), and the like.

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

In the genus Schizosaccharomyces, examples of the vector include a plasmid vector derived from Schizosaccharomyces pombe (Schizosaccharomyces pombe) described in mol.cell.biol.6,80(1986), and the like. Specifically, pAUR224 is commercially available from Takara Bio inc.

Among the genus Aspergillus, Aspergillus niger (Aspergillus niger), Aspergillus oryzae (Aspergillus oryzae) and the like are the most well studied fungi, are available for integration with plasmids and chromosomes, and promoters derived from extracellular proteases and amylases can be used (Trends in Biotechnology 7,283-287 (1989)).

In addition to this, host vector systems corresponding to various microorganisms have been established, and they may be used as appropriate.

In addition to microorganisms, a variety of different host/vector systems have been established in plant cells and animal cells. Specifically, systems for expressing a large amount of heterologous proteins in animal cells such as insects (e.g., Bombyx mori) and the like (Nature 315,592-594(1985)) and plant cells such as rapeseed, corn, potato and the like, and systems using cell-free protein synthesis systems such as Escherichia coli cell-free extracts, wheat germs and the like have been established and can be preferably used.

In the production method of the present invention, the "microorganism or cell having an ability to produce the enzyme" is not particularly limited as long as it is a microorganism or cell having an ability to produce "C ═ C reductase (activity capable of stereoselectively reducing a carbon-carbon double bond)" or a microorganism or cell inherently having the ability or a microorganism or cell to which the ability is imparted by breeding. As a means for imparting such ability by breeding, known methods such as gene recombination treatment (transformation), mutation treatment, and the like can be employed. Among them, microorganisms or cells transformed with a DNA encoding C ═ C reductase are preferable. As a transformation method, for example, a method of introducing a C ═ C reductase gene, enhancing the expression of the C ═ C reductase gene in the biosynthetic pathway of an organic compound, reducing the expression of the C ═ C reductase gene in the byproduct biosynthetic pathway, or the like can be used. As a specific method for producing the transformant, the explanation above for C ═ C reductase can be transferred.

In the production method of the present invention, the "microorganism or cell having an ability to produce the enzyme" is not limited to a living microorganism or cell, but also includes a microorganism or cell which has died as a living body but has an enzymatic activity. It also includes frozen microorganisms or cells.

In the production method of the present invention, "the processed product of the microorganism or the cell" means a product containing a protein having a desired function and obtained by the following method: culturing the microorganism or cell, and 1) disrupting the microorganism or cell with an organic solvent (acetone, dimethyl sulfoxide (DMSO), toluene, etc.), a surfactant, etc., 2) freeze-drying it, 3) immobilizing it on a carrier, etc., 4) physically or enzymatically disrupting it, or 5) extracting an enzyme fraction as a crude product or a purified product from the above-mentioned treated products of 1) to 4), or further immobilizing them on a carrier (polyacrylamide gel, carrageenan gel, etc.), etc. The treated product may be, for example, a protein obtained by disrupting the cultured microorganism or cell. The protein is obtained, for example, using a commercially available kit such as a Bugbuster master mix (manufactured by Merck). The treated product is sometimes referred to hereinafter as an "enzyme solution".

In the production method of the present invention, the "culture solution containing the enzyme obtained by culturing the microorganism or the cell" may be 1) a medium of the microorganism or the cell (for example, a supernatant or a concentrate thereof obtained by removing the cell with centrifugation or the like when it is a suspension of the cell and the liquid medium, or when the cell is a cell expressing a secretory protein), 2) a medium of the microorganism or the cell treated with an organic solvent or the like, 3) a cell membrane of the microorganism or the cell physically or enzymatically disrupted, or further an enzyme obtained by subjecting 2) or 3) to crude purification or purification.

Examples of the C ═ C reductase include those comprising a polypeptide having the amino acid sequence of SEQ ID NO: 1 (protein of (a)), or an enzyme comprising a protein having an amino acid sequence identical to that shown in SEQ ID NO: 1 (hereinafter, sometimes referred to as "homologue of amino acid sequence") and the like, and a protein having an activity of reducing compound (1) and/or compound (4) (the protein of (B) or (C)) (hereinafter, sometimes referred to as "homologue of C reductase"). Specifically, the C ═ C reductase includes enzymes containing the following proteins (a), (B), or (C).

(A) Has the sequence shown in SEQ ID NO: 1;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 1, and has an activity of catalyzing a reaction represented by formula (I) and/or formula (II):

(i) converting SEQ ID NO: 1 with an amino acid other than aspartic acid, phenylalanine, tryptophan, and tyrosine;

(ii) converting SEQ ID NO: 1 by substituting alanine at position 104 with an amino acid other than alanine.

In the protein of (a), the sequence of SEQ ID NO: 1 is an amino acid sequence of a carbon-carbon double bond reductase YqjM derived from bacillus subtilis.

In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO: 1, which comprises the protein of (B) or (C) above.

In the protein shown in said (B), said "one to more amino acids" means generally 1 to 100, preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or 2 amino acids. In the case of a substitution, the amino acid is preferably conservatively substituted.

In the protein shown in (C), the amino acid substitution of (i) is preferably an amino acid substitution of (i ') below, and the amino acid substitution of (ii) is preferably an amino acid substitution of (ii '), more preferably an amino acid substitution of (ii "), and particularly preferably an amino acid substitution of (ii '").

(i') converting SEQ ID NO: 1 by substituting the cysteine at position 26 in the amino acid sequence shown in figure 1 with alanine.

(ii') converting SEQ ID NO: 1 by substituting alanine at position 104 with histidine, phenylalanine, tryptophan or tyrosine.

(ii ") converting SEQ ID NO: 1 by substituting alanine at position 104 with histidine, tryptophan or tyrosine.

(ii' ") converting SEQ ID NO: 1 by substituting tryptophan for alanine at position 104 in the amino acid sequence shown in figure 1.

In the protein shown in (C), the amino acid substitution may be, for example, substitution of SEQ ID NO: 1 with alanine and phenylalanine, tyrosine, tryptophan or histidine at position 104 (C26A and a104F, C26A and a104Y, C26A and a104W or C26A and a 104H). Furthermore, mention may be made of SEQ ID NO: 1 by substituting the cysteine at position 26 with an alanine (C26A). Furthermore, mention may be made of SEQ ID NO: 1 with phenylalanine, tyrosine, tryptophan, or histidine (a104F, a104Y, a104W, or a 104H). Among these amino acid substitutions, C26A and a104F, C26A and a104Y, C26A and a104W, C26A and a104H, C26A, a104F, a104Y, a104W or a104H are preferable, C26A and a104Y, C26A and a104W, C26A and a104H, a104Y, a104W, or a104H are more preferable, and C26A and a104W or a104W are particularly preferable.

In the protein shown in (C), the relative activity, conversion rate and optical purity can be improved relative to the wild type by performing the above amino acid substitution.

In the proteins shown in (C), a protein having a sequence similar to SEQ ID NO: 1, preferably not less than 85%, more preferably not less than 90%, more preferably not less than 95%, more preferably not less than 98%, and particularly preferably not less than 99%.

The identity of amino acid sequences in the present description, expressed, where appropriate, by homology or similarity, can be calculated, for example, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information based Local Alignment Search Tool), under the following conditions (expectation value 10; allowance for gaps; matrix BLOSUM 62; filtration OFF). Examples of other algorithms for determining amino acid sequence homology include the algorithms described in Karlin et al, Proc. Natl. Acad. Sci. USA,90: 5873-.

In the protein shown in the (B) or (C), the activity of catalyzing the reaction shown in the formula (I) means an activity of catalyzing the reaction to produce the compound (2) by reducing the compound (1).

The selectivity in the reaction shown in formula (I) can be confirmed using the ratio of the (3R) -form to the (3S) -form of compound (2) produced by C ═ C reduction as an index. The lower the amount of the (3S) -form produced as compared with the amount of the (3R) -form produced, the more the yield of the compound (2) is improved, which is advantageous in industrialization.

In the protein shown in the (B) or (C), the activity of catalyzing the reaction shown in the formula (II) means an activity of catalyzing the reaction to produce the compound (3) by reducing the compound (4).

The selectivity in the reaction shown in formula (II) can be confirmed using, as an index, the ratio of the (1R,3R) -form to the (1R,3S) -form of the compound (3) produced by reduction. The lower the amount of the (1R,3S) -form produced as compared with the amount of the (1R,3R) -form produced, the more the yield of the compound (3) is improved, which is advantageous in industrialization.

The production ratio of the (1R,3R) -form to the (1R,3S) -form can be compared using the ratio of the produced trans form and cis form as an index. The trans-and cis-forms mean that the geometric isomers, (1R,3R) -and (1S,3S) -are trans forms, (1R,3S) -and (1S,3R) -are cis forms. In the present invention, since the product obtained from C ═ C reduction contains the (1R,3R) -form as a main component, the obtained product can be quantified not only by using an evaluation system that can quantify the individual (1R,3R) -form but also by using an evaluation system that can separate the trans form and cis form. That is, the quantification of the (1R,3R) -form can be replaced by the quantification of the amount of the trans-form produced. Likewise, quantification of the (1R,3S) -form can be replaced by quantification of the amount of the cis-form produced. The lower the amount of the cis form produced, the more the yield of the compound (3) is improved, which is advantageous in industrialization.

Their selectivity can be confirmed by contacting compound (4) with a target C ═ C reductase or the like to give compound (3), and measuring the amounts of the (1R,3R) -form and the (1R,3S) -form of the produced compound (3) or the amounts of the trans form and the cis form produced.

The contacting method is not particularly limited. For example, the compound (1) or the compound (4) is added to a liquid containing the C ═ C reductase as a target, and the reaction is carried out at an appropriate temperature (e.g., about 10 ℃ to 45 ℃) and under an appropriate pressure (e.g., around atmospheric pressure) or the like.

As described above, an example of C ═ C reductase is a reductase containing a polypeptide having the sequence of SEQ ID NO: 1 (the protein shown in (a)), or a protein having C ═ C reduction activity of compound (1) and/or compound (4) (the protein shown in (B) or (C)) which is a homologue of the above amino acid sequence. The extent of C ═ C reduction activity of compound (1) and/or compound (4) by C ═ C reductase of a protein containing a homologue having the amino acid sequence described above may be quantitatively equivalent to a protein containing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, but may differ within an acceptable range (e.g., from about 0.1-fold to about 20-fold, preferably from about 0.3-fold to about 15-fold, more preferably from about 0.5-fold to about 10-fold).

As a method for obtaining a C ═ C reductase containing a protein shown in (a), (B), or (C), the above explanations for the method for obtaining a C ═ C reductase, the method for producing a transformant containing a C ═ C reductase, and the like can be transferred.

The C ═ C reductase ((protein shown in B) or (C)) has a high affinity for a protein comprising SEQ ID NO: 1, (a) has an activity to catalyze the reaction shown in the above formula (I) and/or formula (II) with higher selectivity than that of a conventional known protein sequence, namely C ═ C reductase (a) shown in formula (a).

In the production method of the present invention described below, the C ═ C reductase described above may be directly reacted with compound (1) or compound (4). It is preferable to use a microorganism or a cell having an ability to produce the enzyme, a processed product of the microorganism or the cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or the cell.

[ C ═ O reductase ]

The production method of the present invention is characterized in that a carbon-carbon double bond reductase (C ═ C reductase), 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 a carbonyl reductase (C ═ O reductase), 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 with the compound represented by formula (1) to obtain the compound represented by formula (3). The enzyme, the microorganism or cell having the ability to produce the enzyme, the processed product of the microorganism or cell, and/or the culture solution containing the enzyme obtained by culturing the microorganism or cell are sometimes referred to as "enzyme or the like". The C ═ O reductase is explained below.

In the production method of the present invention, the C ═ O reductase is not particularly limited. C ═ O reductase can also be obtained by purification and isolation according to known methods from, for example, lactobacillus kefir, pichia finnishis, or dvis riboflavin. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination of these methods, and the like.

In addition, the C ═ O reductase can also be produced by culturing transformants containing nucleic acids encoding them, and isolating and purifying the C ═ O reductase from the obtained culture. The nucleic acid encoding C ═ O reductase may be DNA or RNA, or may be a DNA/RNA chimera. Preferred is DNA. Furthermore, the nucleic acid may be double-stranded or single-stranded. When it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or DNA: RNA hybrid. When it is single stranded, it may be the sense strand (i.e., the coding strand) or the antisense strand (i.e., the non-coding strand).

As the nucleic acid (DNA) encoding C ═ O reductase, there can be mentioned, for example, the nucleotide sequence of the lkadh gene derived from Lactobacillus kefir (SEQ ID NO: 6) (GeneBank accession No. AY267012), the nucleotide sequence of the pfodh1 gene derived from Pichia finnishensis (SEQ ID NO: 7) (GeneBank accession No. AB259114.1), the nucleotide sequence of the drcr1 gene derived from Devorax riboflavin (SEQ ID NO: 8) (GeneBank accession No. BD 450088.1).

As the DNA encoding C ═ O reductase, synthetic DNA and the like can be mentioned. For example, in the case of a DNA encoding C ═ O reductase derived from lactobacillus kefir, pichia finnishensis or dvis riboflavin, it can be obtained by converting full-length C ═ C reductase cDNA by reverse transcriptase-PCR by using total RNA or mRNA fractions derived from lactobacillus kefir, pichia finnishensis or dvis riboflavin as a template according to a method known per se as exemplified in the method known per seSuch as the ODA-LA PCR method, the gapped duplex method, the Kunkel method, etc., or a method similar thereto, by using a known kit such as Mutan^TM-super Express Km(TAKARA BIO INC.)、Mutan^TM-K (TAKARA BIO INC.) and the like. Alternatively, it can be obtained by converting cDNA cloned by colony or phage hybridization or PCR or the like from a cDNA library prepared by inserting the above-mentioned total RNA or mRNA into an appropriate vector, according to the above-mentioned method. The vector for the library may be any vector such as a phage, plasmid, cosmid, phagemid, etc.

A nucleic acid (DNA) encoding C ═ O reductase can be cloned by PCR using, for example, chromosomal DNA derived from lactobacillus kefir, pichia finnishensis, or dverskola riboflavin as a template and appropriate primers.

For a method of producing an expression vector, a method of producing a transformant, a host microorganism as a target of transformation, and the like, explanations regarding C ═ C reductase may be transferred.

In the production method of the present invention, the "microorganism or cell having an ability to produce the enzyme" is not particularly limited as long as it is a microorganism or cell having an ability to produce "C ═ O reductase (activity capable of stereoselectively reducing carbonyl group)", a microorganism or cell inherently having the ability, or a microorganism or cell to which the ability is imparted by breeding. As a means for imparting such ability by breeding, known methods such as gene recombination treatment (transformation), mutation treatment, and the like can be employed. Among them, microorganisms or cells transformed with a DNA encoding C ═ O reductase are preferable. As a transformation method, for example, a method of introducing a C ═ O reductase gene, enhancing the expression of the C ═ O reductase gene in the biosynthetic pathway of organic compounds, and reducing the expression of the C ═ O reductase gene in the byproduct biosynthetic pathway can be used. As a specific method for producing the transformant, the contents described in C ═ C reductase can be transferred.

For the "microorganism or cell having the ability to produce the enzyme", "processed product of the microorganism or cell" and "culture solution containing the enzyme obtained by culturing the microorganism or cell" in the production method of the present invention, explanations regarding C ═ C reductase may be transferred.

Examples of the C ═ O reductase include those comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2.3 or 4 (protein of (a)), or an enzyme comprising a protein having an amino acid sequence identical to that shown in SEQ ID NO: 2.3 or 4 (hereinafter sometimes referred to as "homologues of the amino acid sequence") and has an activity to reduce compound (1) and/or compound (2) (the protein of (B) or (C) (hereinafter sometimes referred to as "homologues of the C ═ O reductase"), and the like. Specifically, C ═ O reductase includes enzymes containing the following proteins (a), (B), or (C).

(A) Has the sequence shown in SEQ ID NO: 2.3 or 4;

(B) has a sequence defined by the sequence set forth in SEQ ID NO: 2.3 or 4, and has an activity of catalyzing a reaction shown in formula (III) and/or formula (IV):

(C) has a sequence similar to SEQ ID NO: 2.3 or 4 has an amino acid sequence of not less than 80% identity, and has an activity of catalyzing the reaction shown in the above formula (III) and/or formula (IV).

Among the proteins shown in said (a), SEQ ID NOs: 2 is the amino acid sequence of carbonyl reductase Lkadh derived from lactobacillus kefir, SEQ ID NO: 3 is the amino acid sequence of carbonyl reductase PfODH derived from pichia finnishica, SEQ ID NO: 4 is the amino acid sequence of the carbonyl reductase DrCR derived from moraxella riboflavin. These amino acid sequences were identified by the present inventors as C ═ O reductase for compound (1) or compound (2).

In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO: 2.3 or 4 contains the protein of (B) or (C).

In the proteins shown in (C), a protein having a sequence similar to SEQ ID NO: 2.3 or 4 is not less than 80%, preferably not less than 85%, more preferably not less than 90%, more preferably not less than 95%, more preferably not less than 98%, and particularly preferably not less than 99%.

For the identity of amino acid sequences (which may be rewritten as homology or similarity where appropriate), explanations regarding C ═ C reductase may be transferred.

In the protein shown in the above (B) or (C), the activity of catalyzing the reaction shown in the formula (III) refers to an activity of catalyzing the reaction to produce the compound (3) by C ═ O reduction of the compound (2).

In the present invention, the selectivity in the reaction shown in formula (III) can be confirmed using, as an index, the ratio of the (1R,3R) -form to the (1S,3R) -form of the compound (3) produced by C ═ O reduction. The lower the amount of the (1S,3R) -form produced as compared with the amount of the (1R,3R) -form produced, the more the yield of the compound (3) is improved, which is advantageous in industrialization.

The production ratio of the (1R,3R) -form to the (1S,3R) -form of the compound (3) can be compared using the ratio of the produced trans form to cis form as an index. The trans-and cis-forms mean that the geometric isomers, (1R,3R) -and (1S,3S) -are trans forms, (1R,3S) -and (1S,3R) -are cis forms. In the present invention, since the product obtained from C ═ O reduction contains the (1R,3R) -form as a main component, the obtained product can be quantified not only by using an evaluation system that can quantify the individual (1R,3R) -form but also by using an evaluation system that can separate the trans form and cis form. That is, the quantification of the (1R,3R) -form can be replaced by the quantification of the amount of the trans-form produced. Likewise, the quantification of the (1S,3R) -form can be replaced by the quantification of the amount of the cis-form produced. The lower the amount of the cis form produced, the more the yield of the compound (3) is improved, which is advantageous in industrialization.

Their selectivity can be confirmed by contacting compound (2) with a target C ═ O reductase or the like to give compound (3), and measuring the amounts of the (1R,3R) -form and the (1R,3S) -form of the produced compound (3) or the amounts of the trans form and the cis form produced.

In the protein shown in the (B) or (C), the activity of catalyzing the reaction shown in the formula (IV) means an activity of catalyzing the reaction to produce the compound (4) by reducing the compound (1).

The selectivity in the reaction shown in formula (IV) can be confirmed using, as an index, the ratio of the (1R) -form to the (1S) -form of the compound (4) produced by reduction. The lower the amount of the (1S) -form produced as compared with the amount of the (1R) -form produced, the more the yield of the compound (4) is improved, which is advantageous in industrialization.

The contacting method is not particularly limited. For example, the compound (1) or the compound (2) is added to a liquid containing the C ═ O reductase as a target, and the reaction is performed at an appropriate temperature (e.g., about 10 ℃ to 45 ℃) and under an appropriate pressure (e.g., around atmospheric pressure).

As described above, an example of C ═ O reductase is a reductase containing a polypeptide having the sequence of SEQ ID NO: 2.3 or 4 (the protein shown in (a)), or a protein that is a homologue of the above amino acid sequence and has C ═ O reduction activity of compound (1) and/or compound (2) (the protein shown in (B) or (C)). The degree of carbonyl reduction activity of compound (1) and/or compound (2) by C ═ O reductase of a protein containing a homologue having the amino acid sequence described above may be quantitatively equivalent to a protein containing a protein having an amino acid sequence of SEQ ID NO: 2.3 or 4, but may differ within an acceptable range (e.g., from about 0.1 to about 20 times, preferably from about 0.3 to about 15 times, more preferably from about 0.5 to about 10 times).

For the method of obtaining a C ═ O reductase containing the protein shown in (a), (B), or (C), explanations for C ═ C reductase may be transferred.

The C ═ O reductase ((B) or (C) shown protein) was found to react with a protein containing SEQ ID NO: 2.3 or 4 has an activity to catalyze the reaction shown in the above formula (III) or (IV) with higher selectivity than C ═ C reductase of the conventionally known protein shown in (a).

In the production method of the present invention described below, the C ═ O reductase described above may be directly reacted with compound (1) or compound (2). It is preferable to use a microorganism or a cell having an ability to produce the enzyme, a processed product of the microorganism or the cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or the cell.

2. Compositions of the invention

The composition (enzyme) of the present invention comprises a C ═ C reductase, a microorganism or cell having an 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 catalyzes a reaction for producing the compound (2) using the compound (1) as a substrate and a reaction for producing the compound (3) using the compound (4) as a substrate. The composition of the present invention is useful because it can be used as a catalyst for industrially producing the compound (2) or the compound (3) having high optical purity at low cost with high efficiency.

Further, the composition (enzyme) of the present invention comprises C ═ O reductase, a microorganism or cell having an ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture broth containing the enzyme obtained by culturing the microorganism or cell, and catalyzes a reaction for producing compound (4) using compound (1) as a substrate and a reaction for producing compound (3) using compound (2) as a substrate. The composition of the present invention is useful because it can be used as a catalyst for industrially producing the compound (3) or the compound (4) having high optical purity at low cost with high efficiency.

The composition of the present invention may contain, in addition to the active ingredient (enzyme, etc.), excipients, buffers, suspensions, stabilizers, preservatives, antibacterial agents, saline, and the like. As the excipient, lactose, sorbitol, D-mannitol, sucrose, etc. can be used. As the buffer, phosphate, citrate, acetate, or the like can be used. As the stabilizer, propylene glycol, ascorbic acid, or the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben, and the like can be used. As the antibacterial agent, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

3. Production method of the present invention

[ step 1]

Compound (2) is produced by a reaction shown in the following formula (I) using C ═ C reductase or the like

According to the present invention, there is provided a method for producing compound (2) by the reaction shown in formula (I), i.e., reacting C ═ C reductase or the like with compound (1).

When the C ═ C reductase is contacted with compound (1), purified or roughly purified C ═ C reductase, a microorganism or cell having an ability to produce C ═ C reductase (e.g., a transformant or the like having a DNA encoding a protein), a processed product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell is contacted with compound (1), whereby compound (2) can be produced.

The C ═ C reductase may be used directly in the reaction, 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. Among them, a transformant having a DNA encoding a protein is preferably used.

With respect to the amount of the microorganism or cell to be added to the reaction mixture, the processed product of the microorganism or cell, and/or the culture solution containing the enzyme obtained by culturing the microorganism or cell, when the microorganism or cell is added, it is added to the reaction mixture so that the concentration of the microorganism or cell is generally about 0.1 w/v% to 50 w/v%, preferably 1 w/v% to 20 w/v%, based on the body weight of wet bacteria. When using processed products or culture media, the specific activity of the enzyme is determined and added in an amount that provides the cell concentration described above after addition. In the present specification, w/v% shows weight/volume%.

The method of the reaction is not particularly limited, and the compound (1) as a substrate is added to a liquid containing C ═ C reductase, and the reaction may be carried out at a suitable temperature (e.g., about 10 ℃ to 45 ℃) and a suitable pressure (e.g., around atmospheric pressure). In this way, compound (2) can be produced.

The compound (1) as a reaction substrate is usually used at a substrate concentration in the range of 0.01 w/v% to 90 w/v%, preferably 0.1 w/v% to 30 w/v%. The reaction substrate may be added all at once at the beginning of the reaction. However, from the viewpoint of reducing the influence of enzyme substrate inhibition and increasing the accumulation concentration of the resulting product, continuous or intermittent addition is desirable.

The reaction is usually carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. As the aqueous medium, for example, water or a buffer solution may be mentioned. As the organic solvent, a solvent having high solubility of the compound (1) as a reaction substrate, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, or the like can be used. As the organic solvent, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and the like, which are effective in removing reaction by-products, can also be used.

The reaction is carried out at a reaction temperature of usually 4 ℃ to 60 ℃, preferably 10 ℃ to 45 ℃ and at a pH of usually 3 to 11, preferably 5 to 8. The reaction time is generally about 1hr to 72 hr.

The compound (2) produced by the production method of the present invention can be purified as follows: after completion of the reaction, bacteria, proteins, etc. in the reaction mixture are separated by separation or purification methods known to those of ordinary skill in the art, such as centrifugation, membrane treatment, etc., and then extraction using an organic solvent, such as 1-butanol, t-butanol, etc., distillation, column chromatography using an ion exchange resin, silica gel, etc., crystallization at the isoelectric point, crystallization using monohydrochloride, dihydrochloride, calcium salt, etc., and the like are performed in a suitable combination.

Using the compound (2) obtained in the present invention, the compound (3) having high optical purity, which compound (3) is a starting material for synthesizing various different pharmaceutical products, and an intermediate useful for producing intermediates for synthesizing various different pharmaceutical products, can be produced efficiently at low cost.

[ step 2]

Compound (3) is produced by a reaction shown in the following formula (III) using C ═ O reductase or the like

According to the present invention, there is provided a method for producing compound (3) by the reaction shown in formula (III), that is, by reacting C ═ O reductase or the like with compound (2).

When the C ═ O reductase is contacted with compound (2), purified or roughly purified C ═ O reductase, a microorganism or cell having the ability to produce the C ═ O reductase of the present invention (for example, a transformant having a DNA encoding a protein, or the like), a processed product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell is contacted with compound (2), whereby compound (3) can be produced.

The C ═ O reductase may be used directly in the reaction, 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. Among them, a transformant having a DNA encoding a protein is preferably used.

For 3- (trifluoromethyl) cyclohexan-1-one, which is a reaction substrate for C ═ O reductase, the (3R) -form is generally used.

The method of the reaction is not particularly limited, and the compound (2) as a substrate is added to a liquid containing C ═ O reductase, and the reaction may be carried out at a suitable temperature (e.g., about 10 ℃ to 45 ℃) and a suitable pressure (e.g., around atmospheric pressure). In this way, compound (3) can be produced.

The compound (2) as a reaction substrate is usually used at a substrate concentration in the range of 0.01 w/v% to 90 w/v%, preferably 0.1 w/v% to 30 w/v%. The reaction substrate may be added all at once at the beginning of the reaction. However, from the viewpoint of reducing the influence of enzyme substrate inhibition and increasing the accumulation concentration of the resulting product, continuous or intermittent addition is desirable.

The reaction is usually carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. As the aqueous medium, for example, water or a buffer solution may be mentioned. As the organic solvent, a solvent having high solubility of the compound (2) as a reaction substrate, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, or the like can be used. As the organic solvent, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and the like, which are effective in removing reaction by-products, can also be used.

The compound (3) produced by the production method of the present invention can be purified as follows: after completion of the reaction, bacteria, proteins, etc. in the reaction mixture are separated by separation or purification methods known to those of ordinary skill in the art, such as centrifugation, membrane treatment, etc., and then extraction using an organic solvent, such as 1-butanol, t-butanol, etc., distillation, column chromatography using an ion exchange resin, silica gel, etc., crystallization at the isoelectric point, crystallization using monohydrochloride, dihydrochloride, calcium salt, etc., and the like are performed in a suitable combination.

The obtained compound (3) includes a compound represented by the following formula (5) as a diastereomer thereof

(hereinafter sometimes referred to as compound (5)) or a compound represented by the following formula (6)

(hereinafter sometimes referred to as compound (6)). When the compound (3) is used as a starting material or an intermediate for synthesizing a pharmaceutical product, these compounds are preferably used in a small amount. In the compound (3) obtained by the above production method, the content of the compound (5) and/or the compound (6) is preferably not more than 8 mol%, more preferably not more than 6 mol%, more preferably not more than 4 mol%, particularly preferably not more than 2 mol%.

Using the compound (3) obtained in the present invention, a compound having high optical purity, i.e., a starting material for synthesizing various different pharmaceutical products, and an intermediate for synthesizing various different pharmaceutical products can be produced efficiently at low cost. The compound (3) obtained in the present invention can be used for the production of starting materials for the synthesis of various pharmaceutical products and intermediates for the synthesis of various pharmaceutical products without purification and the like, because it contains only a small amount of impurities. Therefore, the production cost becomes low, and the compound (3) is preferable for industrial production.

[ step 3]

Compound (4) is produced by a reaction shown in the following formula (IV) using C ═ O reductase or the like

According to the present invention, there is provided a method for producing compound (4) by the reaction shown in formula (IV), i.e., reacting C ═ O reductase or the like with compound (1).

When C ═ O reductase is contacted with compound (1), purified or roughly purified C ═ O reductase, a microorganism or cell having an ability to produce C ═ O reductase (e.g., a transformant or the like having a DNA encoding a protein), a processed product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell is contacted with compound (1), whereby compound (4) can be produced.

For compound (1) which is a reaction substrate for C ═ O reductase, commercially available products are generally used.

The method of the reaction is not particularly limited, and the compound (1) as a substrate is added to a liquid containing C ═ O reductase, and the reaction may be carried out at a suitable temperature (e.g., about 10 ℃ to 45 ℃) under a suitable pressure (e.g., around atmospheric pressure). In this way, compound (4) can be produced.

The compound (4) produced by the production method of the present invention can be purified as follows: after completion of the reaction, bacteria, proteins, etc. in the reaction mixture are separated by separation or purification methods known to those of ordinary skill in the art, such as centrifugation, membrane treatment, etc., and then extraction using an organic solvent, such as 1-butanol, t-butanol, etc., distillation, column chromatography using an ion exchange resin, silica gel, etc., crystallization at the isoelectric point, crystallization using monohydrochloride, dihydrochloride, calcium salt, etc., and the like are performed in a suitable combination.

Using the compound (4) obtained in the present invention, the compound (3) having high optical purity, which compound (3) is a starting material for synthesizing various pharmaceutical products, and an intermediate useful for producing synthetic intermediates, can be produced efficiently at low cost.

[ step 4]

Compound (3) is produced by a reaction shown in the following formula (II) using C ═ C reductase or the like

According to the present invention, there is provided a method for producing compound (3) by the reaction shown in formula (II), i.e., reacting C ═ C reductase or the like with compound (4).

When the C ═ C reductase is contacted with compound (4), purified or roughly purified C ═ C reductase, a microorganism or cell having the ability to produce C ═ C reductase of the present invention (for example, a transformant or the like having a DNA encoding a protein), a processed product of the microorganism or cell, and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell is contacted with compound (4), whereby compound (3) can be produced.

For 3-trifluoromethyl-2-cyclohexen-1-ol, which is a reaction substrate for C ═ C reductase, the (1R) -form is generally used.

The method of the reaction is not particularly limited, and the compound (4) as a substrate is added to a liquid containing C ═ C reductase, and the reaction may be carried out at a suitable temperature (e.g., about 10 ℃ to 45 ℃) under a suitable pressure (e.g., around atmospheric pressure). In this way, compound (3) can be produced.

The compound (4) as a reaction substrate is usually used at a substrate concentration in the range of 0.01 w/v% to 90 w/v%, preferably 0.1 w/v% to 30 w/v%. The reaction substrate may be added all at once at the beginning of the reaction. However, from the viewpoint of reducing the influence of enzyme substrate inhibition and increasing the accumulation concentration of the resulting product, continuous or intermittent addition is desirable.

The reaction is usually carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent. As the aqueous medium, for example, water or a buffer solution may be mentioned. As the organic solvent, a solvent having high solubility of the compound (4) as a reaction substrate, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, or the like can be used. As the organic solvent, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and the like, which are effective in removing reaction by-products, can also be used.

The obtained compound (3) includes the compound (5) or the compound (6) as a diastereomer thereof. When the compound (3) is used as a starting material or an intermediate for synthesizing a pharmaceutical product, these compounds are preferably used in a small amount. In the compound (3) obtained by the above production method, the content of the compound (5) and/or the compound (6) is preferably not more than 8 mol%, more preferably not more than 6 mol%, more preferably not more than 4 mol%, particularly preferably not more than 2 mol%.

[ step 5]

Compound (3) is produced by a reaction shown in the following formula (V) using C ═ C reductase or the like

According to the present invention, there is provided a method for producing compound (3) by the reaction shown in formula (V), i.e., reacting C ═ C reductase or the like with compound (1).

When C ═ C reductase and C ═ O reductase are contacted with compound (1), purified or roughly purified C ═ C reductase, a microorganism or cell having an ability to produce C ═ C reductase (e.g., a transformant having a DNA encoding a protein, etc.), a processed product of the microorganism or cell and/or a culture solution containing the enzyme obtained by culturing the microorganism or cell, and C ═ O reductase, a microorganism or cell having an ability to produce C ═ O reductase (e.g., a transformant having a DNA encoding a protein, etc.), 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 with compound (1), whereby compound (3) can be produced.

The C ═ C reductase and C ═ O reductase may be used directly in the reaction, 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. Among them, a transformant having a DNA encoding a protein is preferably used.

The method of the reaction is not particularly limited, and the compound (1) as a substrate is added to a liquid containing C ═ C reductase and C ═ O reductase, and the reaction may be carried out at a suitable temperature (e.g., about 10 ℃ to 45 ℃) under a suitable pressure (e.g., around atmospheric pressure). In this way, compound (3) can be produced.

The production methods 1 to 3 of the present invention may be carried out in the presence of a coenzyme. As the coenzyme, reduced Nicotinamide Adenine Dinucleotide (NADH), reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), oxidized Nicotinamide Adenine Dinucleotide (NAD) can be used⁺) Or oxidized form of Nicotinamide Adenine Dinucleotide Phosphate (NADP)⁺) And NADPH or NADP can be preferably used⁺。

The amount of the coenzyme to be used is not particularly limited as long as it functions as a coenzyme. It is preferably added to the reaction system to a concentration of usually 0.001mmol/L to 100mmol/L, preferably 0.01mmol/L to 10 mmol/L.

When a coenzyme is added, NAD (P) produced from NAD (P) H is preferably used⁺NAD (P) H is regenerated to improve the production efficiency. Examples of regeneration methods include:<1>using host microorganism's own NAD (P)⁺A method for the reduction of a protein in a protein,<2>adding a reagent selected from NAD (P)⁺A microorganism having an ability to produce NAD (P) H or a processed product thereof or an enzyme (regenerating enzyme) useful for NAD (P) H regeneration such as glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.),<3>a method of introducing the above-mentioned gene of a regenerating enzyme, which is an enzyme useful for NAD (P) H regeneration, simultaneously with the introduction of the DNA of the present invention at the time of producing a transformant, and the like.

[ examples ]

The present invention is more specifically described with reference to the following examples; however, the present invention is not limited by these examples as long as it does not depart from the gist thereof.

In the tables shown below, a is alanine, C is cysteine, D is aspartic acid, F is phenylalanine, H is histidine, W is tryptophan, and Y is tyrosine. Further, for example, a104W shows a mutation in which alanine at position 104 in the amino acid sequence is replaced with tryptophan.

In the examples, each abbreviation shows the following compound.

(R) -TFCH: (3R) -3- (trifluoromethyl) cyclohexan-1-one

(S) -TFCH: (3S) -3- (trifluoromethyl) cyclohexan-1-one

TFCL: 3- (trifluoromethyl) cyclohexan-1-ol

(1R,3R) -TFCL: (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol

(1S,3S) -TFCL: (1S,3S) -3- (trifluoromethyl) cyclohexan-1-ol

The production amount, purity, and the like of the target product were measured by gas chromatography under GC analysis conditions 1 and 2 shown below. The peaks obtained from the reaction intermediates as example 1 mentioned below and reference example 8 described later are assigned the elution times of (R) -TFCH and (S) -TFCH, respectively. The elution times of (1R,3R) -TFCL and (1S,3S) -TFCL are the elution times of (1R,3R) -TFCL of example 1 and (1S,3S) -TFCL of reference example 8 described later, respectively. Of the 4 peaks detected when a mixture of 4 isomers of TFCL (TFC isomers 1-4) was analyzed, TFCL isomer 1 was (1R,3R) -TFCL (compound (3)), TFCL isomer 2 was (1S,3S) -TFCL, and their diastereomer TFCL isomer 3 or TFCL isomer 4 was two different diastereomers shown by compound (5) and compound (6).

TFCL-RPPA ester means the (R) - (-) -2-phenylpropionate of TFCL.

[ Table 1]

[ GC analysis Condition 1]

[ Table 2]

[ GC analysis Condition 2]

Reference proportion 1 (preparation of expression vector)

[ preparation of a carbon-carbon double bond reductase (hereinafter referred to as "YqjM") expression plasmid ]

(1) Cloning of genes

PCR was carried out according to a conventional method to obtain a DNA fragment of about 1kbp encoding YqjM gene derived from YqjM (GeneBank accession No. P54550) of Bacillus subtilis. PCR was performed according to a conventional method using the DNA fragment as a template to obtain a DNA fragment in which the restriction enzyme cleavage site of the restriction enzyme EcoRI was added to the 5 'end and the restriction enzyme cleavage site of the restriction enzyme XbaI was added to the 3' end.

(2) Preparation of expression plasmid

The DNA fragment of YqjM obtained in the above (1) was introduced into the downstream of trc promoter of plasmid pKV32 (described in japanese patent No. 4270918) obtained by digestion with restriction enzymes EcoRI and XbaI using T4 DNA ligase (manufactured by TAKARA BIO inc., to obtain pKV 32-YqjM.

Reference ratio 2 (preparation of expression plasmid)

[ preparation of carbonyl reductase (hereinafter referred to as "Lkadh") expression plasmid ]

(1) Cloning of genes

PCR was carried out according to a conventional method to obtain a DNA fragment of about 0.75kbp encoding the lkadh gene derived from Lactobacillus kefir (GeneBank accession No. AY 267012). Using this DNA fragment, PCR was performed according to a conventional method to obtain a DNA fragment in which the restriction enzyme cleavage site of the restriction enzyme EcoRI was added to the 5 'end and the restriction enzyme cleavage site of the restriction enzyme XbaI was added to the 3' end.

(2) Preparation of expression plasmid

The DNA fragment of Ikadh obtained in the above (1) was introduced into the downstream of the trc promoter of plasmid pKV32 in the same manner as in reference example 1(2) to give pKV 32-Lkadh.

Reference ratio 3 (preparation of expression plasmid)

[ preparation of expression plasmid for glucose dehydrogenase (hereinafter referred to as "BsGDH") ]

(1) Cloning of genes

According to the method described in JP-B-6476110, a DNA fragment of about 0.8kbp encoding the gene sequence of the BsGDH gene derived from the BsGDH (GenBank accession No. NP-388275) protein of Bacillus subtilis in which the amino acid at position 96 was replaced with alanine and the amino acid at position 252 was replaced with leucine was obtained. Using this DNA fragment, PCR was performed according to a conventional method to obtain a DNA fragment in which the restriction enzyme cleavage site of the restriction enzyme EcoRI was added to the 5 'end and the restriction enzyme cleavage site of the restriction enzyme XbaI was added to the 3' end.

(2) Preparation of expression plasmid

The DNA fragment of BsGDH obtained in the above (1) was introduced into the downstream of trc promoter of plasmid pKW32 (described in JP-B-5613660) digested with restriction enzymes EcoRI and XbaI using T4 DNA ligase (manufactured by TAKARA BIO INC.) to obtain pKW 32-BsGDH.

Reference ratio 4 (preparation of expression plasmid)

Using the plasmid pKV32-YqjM obtained in reference example 1 as a template, a plasmid having SEQ ID NO: 1, wherein the amino acid C at position 26 is replaced with any one of A, D, F, W, Y, and the amino acid a at position 104 is replaced with any one of F, H, W, Y. The resulting plasmids with introduced mutations are shown in table 3.

[ Table 3]

Reference ratio 5 (preparation of expression plasmid)

[ preparation of expression plasmid for carbon-carbon double bond reductase (hereinafter referred to as "NEMA") ]

(1) Cloning of genes

PCR was carried out according to a conventional method to obtain a DNA fragment of about 1kbp encoding the nemA gene of NEMA derived from Escherichia coli (described in biol. pharm. Bull.20:110-112 (1997)). Using this DNA fragment as a template, PCR was performed according to a conventional method to obtain a DNA fragment in which the restriction enzyme cleavage site of the restriction enzyme EcoRI was added to the 5 'end and the restriction enzyme cleavage site of the restriction enzyme XbaI was added to the 3' end.

(2) Preparation of expression plasmid

The DNA fragment of nemA obtained in the above (1) was introduced into the downstream of the trc promoter of plasmid pKV32 in the same manner as in reference example 1(2) to obtain pKV 32-NEMA.

Reference ratio 6 (preparation of expression plasmid)

[ preparation of carbonyl reductase (hereinafter referred to as "IsADH") expression plasmid ]

(1) Cloning of genes

PCR was carried out according to the conventional method to obtain a DNA fragment of about 1kbp encoding the isadh gene derived from Issatchankia spatula var. sutula JCM1828 strain (described in JP-B-4205496). Using this DNA fragment, PCR was performed according to a conventional method to obtain a DNA fragment in which the restriction enzyme cleavage site of the restriction enzyme EcoRI was added to the 5 'end and the restriction enzyme cleavage site of the restriction enzyme XbaI was added to the 3' end.

(2) Preparation of expression plasmid

The DNA fragment of IsADH obtained in the above (1) was introduced downstream of the trc promoter of plasmid pKV32 in the same manner as in reference example 1(2) to give pKV 32-IsADH.

Reference ratio 7 (preparation of bacterial cells)

[ preparation of Escherichia coli JM109 expressing various enzymes ]

(1) Preparation of expression Strain

Using the plasmids obtained in reference examples 1 to 6, competent cells of Escherichia coli JM109 (manufactured by TAKARA BIO INC.) were transformed according to a conventional method to obtain a recombinant Escherichia coli into which the plasmids were introduced.

(2) Preparation of enzyme solutions

The recombinant Escherichia coli obtained in the above (1) was grown in LB medium containing kanamycin (25. mu.g/mL), 0.2mmol/L isopropyl beta-D-thiogalactopyranoside (IPTG) at 30 ℃ for 18 hr. After 2mL of the resulting bacterial cell culture solution was collected by centrifugation, an enzyme solution was obtained according to a conventional method using a Bugbuster master mix (manufactured by Merck) to which totipotent nuclease and recombinant lysozyme were added.

Example 1: preparation of TFCL (TFCL isomer 1) (Compound (3))

(1) Preparation of TFCL isomer 1

7mL of 1mol/L potassium phosphate buffer (pH7.0) and 5.6mL of 50mmol/L NADP⁺10.5mL of A1 mol/L glucose solution, 718mg of the compound (1), the enzyme solution of Escherichia coli JM109/pKV32-YqjM (A104W (YqjM variant 13)) obtained in referential examples 4 and 7, the enzyme solution of Escherichia coli JM109/pKV32-Lkadh obtained in referential examples 2 and 7, and the enzyme solution of Escherichia coli JM109/pKW32-BsGDH (E96A, Q252L) obtained in referential examples 3 and 7 were mixed in a 200mL fermenter, and pure water was added to prepare 70mL of a reaction mixture. The reaction mixture is reacted at a reaction temperature of 28 ℃ to 30 ℃ overnight with sufficient stirring speed, during which the pH is 7.0.

After the completion of the reaction, methyl tert-butyl ether (hereinafter referred to as MTBE) was added to the reaction mixture, the mixture was stirred at room temperature and an MTBE layer was obtained. MTBE was evaporated from the extract using a rotary evaporator to give TFCL isomer 1.

(2) Preparation of TFCL isomer 1-RPPA ester by modified Moscher's method

A30 mL test tube was charged with (R) - (-) -2-phenylpropionic acid (RPPA) (15mg, 0.1mmol), N-dimethylaminopyridine (1.2mg, 0.01mmol) and TFCL isomer 1(20.2mg, 0.12mmol) obtained in (1) above, dichloromethane solvent (200. mu.L) was added, and the mixture was stirred at room temperature for reaction. The reaction mixture was analyzed by GC analysis condition 1 and the production of the target substance was confirmed. The resulting reaction mixture was cooled to 0 ℃, N' -dicyclohexylcarbodiimide (24.7mg, 0.12mmol) was added, and the mixture was reacted at room temperature for 3 hr. After the reaction, water (200 μ L) was added for partitioning, and the organic layer was recovered. The organic layer was filtered and the resulting filtrate was concentrated under reduced pressure on a rotary evaporator. The concentrated residue was purified by silica gel column to give the desired TFCL isomer 1-RPPA ester as an oily substance.

TFCL isomer 1-RPPA ester

GC-MS(CI:CH₄Gas): 301(M + H) with M/z, theoretical formula C₁₆H₁₉F₃O₂Calculated value is 300.1337.

¹H-NMR(CDCl₃,400MHz)：

[ Table 4]

eq: the equator; ax: shaft

The reference ratio is 8: preparation of TFCL (TFCL isomer 2)

(1) Preparation of TFCL isomer 2

The reaction was carried out in the same manner as in example 1 except for replacing the enzyme solution of Escherichia coli JM109/pKV32-YqjM with the enzyme solution of Escherichia coli JM109/pKV32-NEMA obtained in referential examples 5 and 7 and replacing the enzyme solution of Escherichia coli JM109/pKV32-Lkadh with the enzyme solution of Escherichia coli JM109/pKV32-IsADH obtained in referential examples 6 and 7.

After completion of the reaction, MTBE was added to the reaction mixture, the mixture was stirred at room temperature and an MTBE layer (extract) was obtained. MTBE was evaporated from the extract using a rotary evaporator to give TFCL isomer 2.

(2) Preparation of TFCL isomer 2-RPPA ester by modified Moscher's method

A30 mL test tube was charged with (R) - (-) -2-phenylpropionic acid (RPPA) (15mg, 0.1mmol), N-dimethylaminopyridine (1.2mg, 0.01mmol) and TFCL isomer 2(20.2mg, 0.12mmol) obtained in (1) above, dichloromethane solvent (200. mu.L) was added, and the mixture was stirred at room temperature for reaction. The reaction mixture was analyzed by GC analysis condition 1 and the production of the target substance was confirmed. The resulting reaction mixture was cooled to 0 ℃, N' -dicyclohexylcarbodiimide (24.7mg, 0.12mmol) was added, and the mixture was reacted at room temperature for 3 hr. After the reaction, water (200 μ L) was added for partitioning, and the organic layer was recovered. The organic layer was filtered and the resulting filtrate was concentrated under reduced pressure on a rotary evaporator. The concentrated residue was purified by silica gel column to give the desired TFCL isomer 2-RPPA ester as an oily substance.

TFCL isomer 2-RPPA ester

GC-MS(CI:CH₄Gas): 301(M + H) with M/z, theoretical formula C₁₆H₁₉F₃O₂Calculated value is 300.1337.

¹H-NMR(CDCl₃,400MHz)：

[ Table 5]

eq: the equator; ax: shaft

Preparation of TFCL isoform 1-RPPA ester from example 1(2) and reference 8(2)¹The following structure is supported by the analytical data of H-NMR and GC-MS.

[ Table 6]

Preparation of TFCL isoform 1-RPPA ester from example 1(2) and reference 8(2)¹H-NMR confirmed that the hydrogen at the 1-position is in the equatorial position because the J value of the proton at the 1-position is 2.7Hz or broad and the value is small.

In general, the modified Mocsher method can determine spatial configuration by confirming the phenomenon that protons near the benzene ring migrate to high magnetic fields.

Comparing the chemical shifts of the 3-position protons, it was confirmed that TFCL isoform 1-RPPA ester migrated to a higher magnetic field from 0.4ppm to 0.5ppm than TFCL isoform 2-RPPA ester, and that the benzene ring of TFCL isoform 1-RPPA ester was closer to the 3-position protons (TFCL isoform 1-RPPA ester: 1.8ppm, TFCL isoform 2-RPPA ester: 2.2ppm-2.3 ppm).

From the above results, it was confirmed that TFCL isomer 1 obtained in example 1(1) was in the (1R,3R) -form, and TFCL isomer 2 obtained in reference example 8(1) was in the (1S,3S) -form.

Examples 2 to 11 and comparative examples 1 to 8: measurement of initial Activity of YqjM variants

Measurement of initial activity of YqjM variant activity was confirmed by measuring the decrease in NADPH upon addition of compound (1) at a wavelength of 340 nm. mu.L of the enzyme solution (example 2: use of the enzyme solution obtained in reference examples 1 and 7, examples 3 to 11 and comparative examples 1 to 8: use of the enzyme solution obtained in reference examples 4 and 7), 25. mu.L of 1mol/L potassium phosphate buffer (pH7), 10. mu.L of 8mmol/L NADPH, 200. mu.L of water, and 5. mu.L of a DMSO solution of compound (1) (0.1mol/L) were mixed. The mixture was placed in a 96-well plate (manufactured by Corning), and the change in absorbance at 340nm was measured for 2min using a microplate reader (manufactured by Molecular Bio) warmed to 30 ℃. The average value of the change in absorbance per unit time (mOD/min) was calculated, and the activity value per unit time/unit protein was calculated from the slope of the change in absorbance by the following formula. The molar absorptivity of NADPH was calculated to be 6.3 mL/. mu.mol. cm.

Activity value (μmol/min/mg protein) — 1 × slope of absorbance change × 250 μ L/10 μ L/6300/0.55/protein concentration

The relative activity of each variant relative to the wild type is shown in table 7. The case where the relative activity is less than 15% is shown by "-".

[ Table 7]

As is clear from Table 2, it was found that the replacement of C at position 26 with A (example 7: YqjM variant 10) or the replacement of A at position 104 with F, Y, W or H (examples 8-11: YqjM variant 11-14) showed a significant increase in relative activity compared with the wild type. Furthermore, it was found that the replacement of C at position 26 with A and the replacement of A at position 104 with F, Y, W or H (examples 3-6: YqjM variant 6-9) also showed a significant increase in relative activity compared to the wild type.

On the other hand, it was found that the replacement of C at position 26 with W, D, F or Y (comparative examples 1-5: YqjM variants 1-5, comparative examples 6-8: YqjM variants 15-17) showed a significant decrease in relative activity compared to the wild type. Specifically, it was found that in C26W/A104F (comparative example 1: YqjM variant 1), C26W/A104Y (comparative example 2: YqjM variant 2) and C26D/A104W (comparative example 6: YqjM variant 15), the reactivity to the compound described in non-patent document 2 and the reactivity to the compound (1) of the present invention were greatly different.

Examples 12 to 21: production of (R) -TFCH (Compound (2))

The enzyme solution of recombinant Escherichia coli introduced with pKV32-YqjM (100. mu.L) prepared by the methods of reference examples 1 and 7 or the enzyme solution of recombinant Escherichia coli introduced with pKV32-YqjM (100. mu.L) prepared by the methods of reference examples 4 and 7, 50g/L of KRED mixture N (manufactured by Codexis) (400. mu.L), and compound (1) (4.1mg) were added to a 2 mL-micro tube, and the mixture was reacted by stirring at 30 ℃ for 1 hr. Heptane (1mL) was added to the resulting reaction mixture, and the mixture was stirred at room temperature. The upper layer obtained after standing was analyzed under GC analysis condition 2. The conversion rate of compound (1) to TFCH and the optical purity of the obtained (R) -TFCH (compound (2)) are shown in table 8. The conversion and optical purity were calculated by the following formulas.

[ equation 1]

C1: concentration of Compound (1) [ mmol/L ]

C2 (R): (R) -TFCH concentration [ mmol/L ]

C2 (S): (S) -TFCH concentration [ mmol/L ]

[ Table 8]

As is clear from table 8, YqjM variants 6-10 (examples 13-17) in which C at position 26 was replaced with a were found to show significantly improved conversion and optical purity compared to the wild type. In addition, YqjM variants 6-9 (examples 13-16) in which a at position 104 was replaced with F, Y, W or H were found to show further improved conversion and optical purity.

Example 22: production of (1R,3R) -TFCL (Compound (3))

7mL of 1mol/L potassium phosphate buffer (pH7.0), 0.7mL of 50mmol/L NADP⁺10.5mL of a 4mol/L glucose solution, 33mL of pure water, 2873mg of the compound (1), 8.4mL of an enzyme solution of Escherichia coli JM109/pKV32-YqjM (A104W (YqjM variant 13)) obtained in referential examples 4 and 7 (equivalent to 0.84g of wet bacteria), 4.2mL of an enzyme solution of Escherichia coli JM109/pKV32-Lkadh obtained in referential examples 2 and 7 (equivalent to 0.42g of wet bacteria), and 6.1mL of an enzyme solution of Escherichia coli JM109/pKW32-BsGDH (E96A, Q252L) obtained in referential examples 3 and 7The solutions (equivalent to 0.61g of wet bacteria) were mixed in a 200mL fermentor and the mixture was reacted at a reaction temperature of 28 ℃ to 30 ℃ for 17hr with sufficient stirring speed and pH was maintained at 7.0 during the reaction using 25 wt% aqueous NaOH.

After the completion of the reaction, 35mL of methyl tert-butyl ether (hereinafter referred to as MTBE) was added to the reaction mixture, the mixture was stirred at room temperature, and an MTBE layer containing (1R,3R) -TFCL was obtained. To the resulting aqueous layer was again added MTBE (35mL), the mixture was stirred at room temperature, and an MTBE layer containing (1R,3R) -TFCL was obtained. As a result of purity analysis and optical purity analysis of the obtained MTBE layer under GC analysis condition 2, the pure content of (1R,3R) -TFCL was 2348mg, and the yield was 81.8%. The optical purity was 99.8% e.e. The diastereomer was 1.6%. The optical purity and the existence ratio of diastereoisomers were calculated by the following formulas.

[ formula 2]

C3 (RR): (1R,3R) -TFCL concentration [ mmol/L ]

C3 (SS): (1S,3S) -TFCL concentration [ mmol/L ]

C3 (RS): (1R,3S) -TFCL concentration [ mmol/L ]

C3 (SR): (1S,3R) -TFCL concentration [ mmol/L ]

Example 23: production of (1R,3R) -TFCL (Compound (3))

(1) Production of (R) -TFCH (Compound (2))

After the reaction, heptane (1mL) was added to reaction mixture B, and the mixture was stirred at room temperature for 1 min. The upper layer obtained after standing was analyzed under GC analysis condition 2. The conversion of compound (1) to compound (2) was 43.1%, and the optical purity of the obtained compound (2) was 67.8% ee. The conversion and the optical purity were calculated by the following formulas.

[ formula 3]

C1: concentration of Compound (1) [ mmol/L ]

C2 (R): (R) -TFCH concentration [ mmol/L ]

C2 (S): (S) -TFCH concentration [ mmol/L ]

(2) Production of (1R,3R) -TFCL (Compound (3))

The reaction mixture A obtained in the above (1) was further reacted at a reaction temperature of 30 ℃ and a sufficient stirring rate for 8 hr. To reaction mixture A was added 20. mu.L of 50mmol/L NAD⁺、20μL 50mmol/L NADP⁺75. mu.L of a 1mol/L glucose solution and 100. mu.L of the enzyme solution of Escherichia coli JM109/pKV32-Lkadh obtained in reference examples 2 and 7, and the mixture was further reacted for 16 hr. Heptane (1mL) was added to the resulting reaction mixture, and the mixture was stirred at room temperature for 1 min. The upper layer obtained after standing was analyzed under GC analysis condition 2. The optical purity of the obtained (1R,3R) -TFCL (compound (3)) was 75.8% ee. The diastereomer was present in a ratio of 23.8%. Optical purity and diastereomer abundance ratioCalculated by the following formula.

[ formula 4]

C3 (RR): (1R,3R) -TFCL concentration [ mmol/L ]

C3 (SS): (1S,3S) -TFCL concentration [ mmol/L ]

C3 (RS): (1R,3S) -TFCL concentration [ mmol/L ]

C3 (SR): (1S,3R) -TFCL concentration [ mmol/L ]

Example 24: production of (1R) -3-trifluoromethyl-2-cyclohexen-1-ol (Compound (4))

50. mu.L of 1mol/L potassium phosphate buffer (pH7.0), 20. mu.L of 50mmol/L NAD⁺、20μL 50mmol/L NADP⁺75 μ L of a 1mol/L glucose solution, 8.2mg of the compound (1), the enzyme solution of Escherichia coli JM109/pKV32-Lkadh obtained in referential examples 2 and 7, and the enzyme solution of Escherichia coli JM109/pKW32-BsGDH (E96A, Q252L) obtained in referential examples 3 and 7 were mixed in a 2mL microtube, and pure water was added to prepare 500 μ L of a reaction mixture. The mixture was reacted at a reaction temperature of 30 ℃ and sufficient stirring speed for 23 hr. Heptane (1mL) was added to the resulting reaction mixture, and the mixture was stirred at room temperature for 1 min. The upper layer obtained after standing was subjected to GC-MS analysis and the production of (1R) -3-trifluoromethyl-2-cyclohexen-1-ol (compound (4)) was confirmed. The product obtained is presumed to be in the (1R) -form from the results of examples 1, 22 and 23 (2). The conversion of compound (1) to compound (4) was 43.6%. The conversion is calculated from the following formula.

[ formula 5]

C1: concentration of Compound (1) [ mmol/L ]

C4 (R): concentration of Compound (4) [ mmol/L ]

C4 (S): compound (4) (1S) -form concentration [ mmol/L ]

[ Industrial Applicability ]

Sequence listing

<110> API from Kabushiki Kaisha

<120> (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and process for producing intermediate thereof

<130> 092995

<150> US 62/836324

<151> 2019-04-19

<160> 10

<170> PatentIn 3.5 edition

<210> 1

<211> 338

<212> PRT

<213> Bacillus subtilis

<400> 1

Met Ala Arg Lys Leu Phe Thr Pro Ile Thr Ile Lys Asp Met Thr Leu

1 5 10 15

Lys Asn Arg Ile Val Met Ser Pro Met Cys Met Tyr Ser Ser His Glu

20 25 30

Lys Asp Gly Lys Leu Thr Pro Phe His Met Ala His Tyr Ile Ser Arg

35 40 45

Ala Ile Gly Gln Val Gly Leu Ile Ile Val Glu Ala Ser Ala Val Asn

50 55 60

Pro Gln Gly Arg Ile Thr Asp Gln Asp Leu Gly Ile Trp Ser Asp Glu

65 70 75 80

His Ile Glu Gly Phe Ala Lys Leu Thr Glu Gln Val Lys Glu Gln Gly

85 90 95

Ser Lys Ile Gly Ile Gln Leu Ala His Ala Gly Arg Lys Ala Glu Leu

100 105 110

Glu Gly Asp Ile Phe Ala Pro Ser Ala Ile Ala Phe Asp Glu Gln Ser

115 120 125

Ala Thr Pro Val Glu Met Ser Ala Glu Lys Val Lys Glu Thr Val Gln

130 135 140

Glu Phe Lys Gln Ala Ala Ala Arg Ala Lys Glu Ala Gly Phe Asp Val

145 150 155 160

Ile Glu Ile His Ala Ala His Gly Tyr Leu Ile His Glu Phe Leu Ser

165 170 175

Pro Leu Ser Asn His Arg Thr Asp Glu Tyr Gly Gly Ser Pro Glu Asn

180 185 190

Arg Tyr Arg Phe Leu Arg Glu Ile Ile Asp Glu Val Lys Gln Val Trp

195 200 205

Asp Gly Pro Leu Phe Val Arg Val Ser Ala Ser Asp Tyr Thr Asp Lys

210 215 220

Gly Leu Asp Ile Ala Asp His Ile Gly Phe Ala Lys Trp Met Lys Glu

225 230 235 240

Gln Gly Val Asp Leu Ile Asp Cys Ser Ser Gly Ala Leu Val His Ala

245 250 255

Asp Ile Asn Val Phe Pro Gly Tyr Gln Val Ser Phe Ala Glu Lys Ile

260 265 270

Arg Glu Gln Ala Asp Met Ala Thr Gly Ala Val Gly Met Ile Thr Asp

275 280 285

Gly Ser Met Ala Glu Glu Ile Leu Gln Asn Gly Arg Ala Asp Leu Ile

290 295 300

Phe Ile Gly Arg Glu Leu Leu Arg Asp Pro Phe Phe Ala Arg Thr Ala

305 310 315 320

Ala Lys Gln Leu Asn Thr Glu Ile Pro Ala Pro Val Gln Tyr Glu Arg

325 330 335

Gly Trp

<210> 2

<211> 252

<212> PRT

<213> Lactobacillus kefir (Lactobacillus kefir)

<400> 2

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 3

<211> 234

<212> PRT

<213> Pichia Finnishis (Pichia finlandica)

<400> 3

Leu Ser Val Ala Lys Lys Phe Leu Gln Leu Gly Ala Lys Val Thr Ile

1 5 10 15

Ser Asp Val Ser Gly Glu Lys Lys Tyr His Glu Thr Val Val Ala Leu

20 25 30

Lys Ala Gln Asn Leu Asn Thr Asp Asn Leu His Tyr Val Gln Ala Asp

35 40 45

Ser Ser Lys Glu Glu Asp Asn Lys Lys Leu Ile Ser Glu Thr Leu Ala

50 55 60

Thr Phe Gly Gly Leu Asp Ile Val Cys Ala Asn Ala Gly Ile Gly Lys

65 70 75 80

Phe Ala Pro Thr His Glu Thr Pro Phe Asp Val Trp Lys Lys Val Ile

85 90 95

Ala Val Asn Leu Asn Gly Val Phe Leu Leu Asp Lys Leu Ala Ile Asn

100 105 110

Tyr Trp Leu Glu Lys Ser Lys Pro Gly Val Ile Val Asn Met Gly Ser

115 120 125

Val His Ser Phe Val Ala Ala Pro Gly Leu Ala His Tyr Gly Ala Ala

130 135 140

Lys Gly Gly Val Lys Leu Leu Thr Gln Thr Leu Ala Leu Glu Tyr Ala

145 150 155 160

Ser His Gly Ile Arg Val Asn Ser Val Asn Pro Gly Tyr Ile Ser Thr

165 170 175

Pro Leu Ile Asp Glu Val Pro Lys Glu Arg Leu Asp Lys Leu Val Ser

180 185 190

Leu His Pro Ile Gly Arg Leu Gly Arg Pro Glu Glu Val Ala Asp Ala

195 200 205

Val Ala Phe Leu Cys Ser Gln Glu Ala Thr Phe Ile Asn Gly Val Ser

210 215 220

Leu Pro Val Asp Gly Gly Tyr Thr Ala Gln

225 230

<210> 4

<211> 210

<212> PRT

<213> Devorax riboflavin (Devosia riboflavina)

<400> 4

Leu Glu Gly Ala Gln Ala Val Ala Asp Ala Val Lys Ala Ala Gly Gly

1 5 10 15

Glu Ala Ala Ala Val Ala Val Asp Val Ala Lys Ala Asp Gln Val Glu

20 25 30

Lys Ala Val Gln Phe Ala Val Asp Thr Phe Gly Ala Leu His Leu Ala

35 40 45

Val Asn Asn Ala Gly Ile Gly Gly Ala Ser Ala Pro Leu Gly Asp Tyr

50 55 60

Ser Phe Asp Asp Trp His Arg Val Ile Asp Val Asn Leu Asn Ser Val

65 70 75 80

Phe Tyr Ser Met Lys Tyr Glu Ile Val Ala Met Leu Arg Ala Gly Gly

85 90 95

Gly Ala Ile Val Asn Met Ala Ser Ile Leu Gly Ser Val Thr Phe Pro

100 105 110

Asn Ala Pro Ala Tyr Val Thr Ala Lys His Gly Val Val Gly Met Thr

115 120 125

Lys Ser Ala Ala Val Asp Tyr Ala Lys Lys Gly Ile Arg Val Thr Ala

130 135 140

Val Gly Pro Gly Phe Ile Asp Thr Pro Leu Leu Ser Ala Leu Pro Lys

145 150 155 160

Glu Thr Leu Asp Tyr Leu Lys Ser Val His Pro Ile Gly Arg Leu Gly

165 170 175

Thr Ser Asp Glu Val Ala Ala Leu Thr Ala Phe Leu Leu Ser Asp Ala

180 185 190

Ala Ser Asn Ile Thr Gly Ser Tyr His Leu Val Asp Gly Gly Tyr Val

195 200 205

Ala Gln

210

<210> 5

<211> 1017

<212> DNA

<213> Bacillus subtilis

<400> 5

atggccagaa aattatttac acctattaca attaaagata tgacgttaaa aaaccgcatt 60

gtcatgtcgc caatgtgcat gtattcttct catgaaaagg acggaaaatt aacaccgttc 120

cacatggcac attacatatc gcgcgcaatc ggccaggtcg gactgattat tgtagaggcg 180

tcagcggtta accctcaagg acgaatcact gaccaagact taggcatttg gagcgacgag 240

catattgaag gctttgcaaa actgactgag caggtcaaag aacaaggttc aaaaatcggc 300

attcagcttg cccatgccgg acgtaaagct gagcttgaag gagatatctt cgctccatcg 360

gcgattgcgt ttgacgaaca atcagcaaca cctgtagaaa tgtcagcaga aaaagtaaaa 420

gaaacggtcc aggagttcaa gcaagcggct gcccgcgcaa aagaagccgg ctttgatgtg 480

attgaaattc atgcggcgca cggatattta attcatgaat ttttgtctcc gctttccaac 540

catcgaacag atgaatatgg cggctcacct gaaaaccgct atcgtttctt gagagagatc 600

attgatgaag tcaaacaagt atgggacggt cctttatttg tccgtgtatc tgcttctgac 660

tacactgata aaggcttaga cattgccgat cacatcggtt ttgcaaaatg gatgaaggag 720

cagggtgttg acttaattga ctgcagctca ggcgcccttg ttcacgcaga cattaacgta 780

ttccctggct atcaggtcag cttcgctgag aaaatccgtg aacaggcgga catggctact 840

ggtgccgtcg gcatgattac agacggttca atggctgaag aaattctgca aaacggacgt 900

gccgacctca tctttatcgg cagagagctt ttgcgggatc cattttttgc aagaactgct 960

gcgaaacagc tcaatacaga gattccggcc cctgttcaat acgaaagagg ctggtaa 1017

<210> 6

<211> 759

<212> DNA

<213> Lactobacillus kefir (Lactobacillus kefir)

<400> 6

atgactgatc gtttaaaagg caaagtagca attgtaactg gcggtacctt gggaattggc 60

ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120

gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180

caacacgatg cttctgatga agccggctgg actaagttgt ttgatacgac tgaagaagca 240

tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300

gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgtcttcttc 360

ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420

atgtcatcta tcgaaggttt tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480

ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540

gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600

ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660

aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720

gcagaattcg ttgtcgatgg tggatacact gctcaataa 759

<210> 7

<211> 765

<212> DNA

<213> Pichia Finnishis (Pichia finlandica)

<400> 7

atgtcttata acttccataa caaggttgca gttgttactg gagctctatc aggaatcggc 60

ttaagcgtcg caaaaaagtt ccttcagctc ggcgccaaag taacgatctc tgatgtcagt 120

ggagagaaaa aatatcacga gactgttgtt gctctgaaag cccaaaatct caacactgac 180

aacctccatt atgtacaggc agattccagc aaagaagaag ataacaagaa attgatttcg 240

gaaactctgg caacctttgg gggcctggat attgtttgtg ctaatgcagg aattggaaag 300

ttcgctccca cccatgaaac acccttcgac gtatggaaga aggtgattgc tgtgaatttg 360

aatggagtat tcttactgga taagctagcc atcaattact ggctagagaa aagcaaaccc 420

ggcgtaattg tcaacatggg atcagtccac tcttttgtag cagctcctgg ccttgcgcat 480

tatggagctg caaaaggcgg tgtcaaactg ttaacacaaa cattggctct agagtacgca 540

tctcatggta ttagagtaaa ttctgtcaat ccggggtaca tttcgactcc tttgatagat 600

gaggttccga aagagcggtt ggataaactt gtaagcttgc accctattgg gagactaggt 660

cgtccagagg aagttgctga tgcagtcgca tttctgtgtt cccaggaggc cactttcatc 720

aacggcgttt ctttgccggt tgacgggggg tacacagccc agtaa 765

<210> 8

<211> 753

<212> DNA

<213> Devorax riboflavin (Devosia riboflavina)

<400> 8

atgtcccagg atttttcagg caaggtcgca ttcgtaacgg gtggtgcctc gggcatcggt 60

gaggcggtcg tcaagcagct tgccgcgcgc ggcgccaagg ttgtggttgc cgatctcaag 120

ctcgaaggcg cgcaggcggt tgccgatgcg gtcaaggccg ccggcggcga agcggccgcg 180

gtagctgtcg atgtcgccaa ggccgatcag gtggagaagg ctgtccagtt cgccgtcgac 240

acctttggcg ccctgcatct ggcggtcaat aatgccggca ttggcggcgc ttccgctccc 300

ctcggcgatt attccttcga cgactggcat agggttatcg acgtcaatct caattccgtc 360

ttctattcga tgaagtacga gatcgtcgcc atgctcaggg caggcggtgg cgccatcgtc 420

aacatggcct ccatcctcgg ctcggtgacc tttcccaatg caccggccta tgtcaccgcc 480

aagcacggcg tggtcggcat gaccaagtcg gccgcggtgg actatgccaa aaagggcatt 540

cgcgtcacgg ccgtcgggcc cggtttcatc gacacgccgc tcctatccgc cttgcccaag 600

gaaaccctgg actacctcaa atccgtccat ccgatcggac ggctgggtac ctcggatgaa 660

gtcgcagcgc tgaccgcgtt cctgctctcc gatgcagcgt cgaacatcac cggctcctat 720

cacctggtcg atggcggcta cgtcgcccaa tag 753

<210> 9

<211> 261

<212> PRT

<213> Bacillus subtilis

<400> 9

Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala

1 5 10 15

Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala

20 25 30

Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val

35 40 45

Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly

50 55 60

Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile

65 70 75 80

Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Ala

85 90 95

Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val

100 105 110

Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

<210> 10

<211> 786

<212> DNA

<213> Bacillus subtilis

<400> 10

atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60

aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120

aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180

gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240

aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgcaaa tcctgtgcca 300

tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360

tttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420

attaacatgt ccagtgtgca cgaagtgatt ccttggccgt tatttgtcca ctatgcggca 480

agtaaaggcg ggataaagct gatgacagaa acattagcgt tggaatacgc gccgaagggc 540

attcgcgtca ataatattgg gccaggtgcg atcaacacgc caatcaatgc tgaaaaattc 600

gctgacccta aacagaaagc tgatgtagaa agcatgattc caatgggata tatcggcgaa 660

ccggaggaga tcgccgcagt agcagcctgg cttgcttcga aggaagccag ctacgtcaca 720

ggcatcacgt tattcgcgga cggcggtatg acactgtatc cttcattcca ggcaggccgc 780

ggttaa 786

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Process for producing (1R,3R) -3- (trifluoromethyl) cyclohexan-1-ol and intermediate thereof

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