JP2007274901A - Method for producing optically active propargyl alcohol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active propargyl alcohol useful as an intermediate for the synthesis of a medicine. <P>SOLUTION: This optically active propargyl alcohol is produced by asymmetrically reducing propargyl ketone by using a secondary alcohol dehydrogenase or carbonylreductase. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing optically active propargyl alcohol useful as an optically active raw material for various pharmaceuticals, particularly as a raw material for the ω side chain essential for prostaglandin compounds.

As a method for producing optically active propargyl alcohol represented by Formula 2 or Formula 5 as defined in the present specification,
(1) A method in which a racemic mixture of propargyl alcohol represented by Formula 2 or Formula 5 is used as a phthalic acid monoester and an optically active amine is used as an optical resolution agent (J. Am. Chem. Soc., 94, 7827 (1972) ),
(2) A method of obtaining an optically active propargyl alcohol by subjecting an ester of a racemic mixture of propargyl alcohol represented by formula 2 or formula 5 to a reaction kinetic resolution by a hydrolysis reaction using lipase, baker's yeast or the like 3-259094),
(3) A method of dividing a racemic mixture of propargyl alcohol represented by formula 2 or formula 5 with an optical resolution column to obtain optically active propargyl alcohol (Japanese Patent Laid-Open No. 2003-64009),
(4) A method of asymmetric reduction of propargyl ketone represented by Formula 1 defined in the present specification using an asymmetric reduction catalyst (J. Am. Chem. Soc., 101, 5843 (1979)),
(5) A method for asymmetrically reducing propargyl ketone represented by formula 1 by a microorganism (J. Am. Chem. Soc., 97, 865 (1975)),
(6) A method for obtaining an optically active α-halogen-substituted propargyl alcohol by asymmetric reduction of an α-halogen-substituted product of propargyl ketone represented by Formula 1 using an alcohol dehydrogenase derived from Lactobacillus (WO02 / 64579),
(7) A method of obtaining optically active γ-iodoallylic alcohol from γ-allyl iodide alcohol by the sharpless optical resolution method and obtaining optically active propargyl alcohol via dehydroiodination (Tetrahedron Lett., 30, 7083 (1989) )),
Etc. are known.

  However, the method (1) requires a low yield and an expensive resolving agent, the method (2) has low optical purity, and it is difficult to separate the unreacted ester and the hydrolyzed alcohol. The method (3) requires an expensive optical resolution column, the method (4) requires an expensive asymmetric reduction catalyst, requires extremely low temperature, and the method (5) has low yield and optical purity. The method (6) has a low productivity, and the method (7) has a problem as an industrial production method such that an expensive resolving agent is required and the process is complicated.

J. Am. Chem. Soc., 94, 7827 (1972) J. Am. Chem. Soc., 101, 5843 (1979) J. Am. Chem. Soc., 97, 865 (1975) Tetrahedron Lett., 30, 7083 (1989) Japanese Patent Laid-Open No. 3-259094 JP 2003-64009 A WO02 / 64579

  The present invention relates to an optically active propargyl alcohol represented by formula 2 or formula 5, especially (S) -1-cyclohexyl-2-propyn-1-ol represented by formula 4 or (R) -1 represented by formula 6 An object of the present invention is to provide a production method capable of giving a product with high optical purity in a high yield in the production of -cyclohexyl-2-propyn-1-ol.

The inventors of the present invention are not a resolution method in which the theoretical yield exceeds 50%, but can use 100% of the raw material, and propargyl ketone has high optical purity by asymmetric reduction reaction using an enzyme having high stereoselectivity. A method for producing optically active propargyl alcohol was investigated.
1-Cyclohexyl-2-propyn-1-ol is used by using an enzyme that asymmetrically reduces many carbonyls using 1-cyclohexylpropynone (hereinafter abbreviated as CHPN) in which R in Formula 1 is a cyclohexyl group as a substrate. (1-cyclohexyl-2-propyn-1-ol, hereinafter abbreviated as CHPO) As a result of the synthesis, (R) -2-octanol dehydrogenase derived from Pichia finlandica (described in SEQ ID NO: 2) (S) -CHPO produces (S) -CHPO, and Saccharomyces cerevisiae (
Saccharomyces cerevisiae) -derived carbonyl reductase ScGRE2 (protein consisting of the amino acid sequence shown in SEQ ID NO: 4) and ScYGD9 (protein consisting of the amino acid sequence shown in SEQ ID NO: 6), and carbonyl derived from Torulaspora delbrueckii (Torulaspora delbrueckii) It was found that all of the reductases TdCR1 (protein consisting of the amino acid sequence shown in SEQ ID NO: 8) produce (R) -CHPO with extremely high stereoselectivity of 99% ee or more, and the present invention has been completed. .

  (R) -2-octanol dehydrogenase is known to asymmetrically reduce ethyl acetoacetate to produce ethyl (R) -3-hydroxybutyrate (WO01 / 61014), but CHPN It is unpredictable whether it has activity against it, how much stereoselectivity it will reduce, and which stereo form it produces, with a high selectivity of 99% ee or higher (S) -CHPO It was a surprising result.

  Similarly, ScGRE2 (J. Org. Chem. 63, 4996-5000 (1998)), ScYGD9 and TdCR1 (all of Japanese Patent Application No. 2003-113402) asymmetrically reduce ethyl acetoacetate, and (S) -3 -It has been reported to produce ethyl hydroxybutyrate, but as in the case of (R) -2-octanol dehydrogenase, whether or not it has activity against CHPN, how much stereoselectivity is It was unpredictable whether to reduce or produce any stereo form, and it was a surprising result that (R) -CHPO was produced with high selectivity of 99% ee or higher. For example, (S) -specific secondary alcohol dehydrogenase (JP 07-231785 A) derived from Candida parapsilosis asymmetrically reduces ethyl acetoacetate to produce ethyl (S) -3-hydroxybutyrate Although it has been reported, it showed virtually no activity against CHPN. Further, ScYDR1 (Japanese Patent Application No. 2003-113402) having 61% homology to TdCR1 also had substantially no activity against CHPN.

In addition, regeneration of NAD ⁺ produced from NADH to NADH accompanying the above reaction, and regeneration of NADPH produced from NADPH to NADP ⁺ include, for example, glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase. , Amino acid dehydrogenase, organic acid dehydrogenase, etc., the present inventors introduced these genes into the host simultaneously with the DNA encoding the alcohol dehydrogenase or carbonyl reductase used in the present invention. By simultaneously expressing alcohol dehydrogenase or carbonyl enzyme and NAD (P) H regenerating enzyme, and performing a reduction reaction using the transformed strain or its treated product, optically active propargyl alcohol is more efficiently produced. It has been found that derivatives can be produced.

（式中、Ｒは、Ｃ１−Ｃ１０の置換若しくは未置換の直鎖、分岐鎖又は環状のアルキル基、アルケニル基又はアルキニル基、あるいはＣ６−Ｃ２０のアリール基を表す）
で示されるプロパルギルケトン誘導体に作用させ、該ケトンを還元して式２：

（式中、Ｒは、式１の場合と同義である）
で示される光学活性プロパルギルアルコール誘導体を製造することを含む、光学活性プロパルギルアルコール誘導体の製造方法。 That is, according to the present invention, the following inventions are provided.
(1) From (a) to (e) below;
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
(C) a protein composed of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having an activity to produce an optically active propargyl alcohol derivative represented by Formula 2:
(D) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, wherein the propargyl ketone derivative represented by Formula 1 is reduced, and the optical activity represented by Formula 2 A polynucleotide encoding a protein having an activity of producing a propargyl alcohol derivative; or (e) a propargyl ketone represented by formula 1 having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2 A polynucleotide encoding a protein having an activity of reducing the derivative to produce an optically active propargyl alcohol derivative represented by formula 2:
A protein encoded by the polynucleotide according to any one of the above, a microorganism or a transformant functionally expressing the protein, or a processed product thereof, is represented by the formula 1:

Wherein R represents a C1-C10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group or alkynyl group, or a C6-C20 aryl group.
Is reacted with a propargyl ketone derivative represented by formula (2), and the ketone is reduced to give a compound of formula 2:

(Wherein R has the same meaning as in formula 1)
A process for producing an optically active propargyl alcohol derivative, comprising producing an optically active propargyl alcohol derivative represented by the formula:

で示される１−シクロヘキシル−２−プロピン−１−オンであり、式２で示される光学活性プロパルギルアルコール誘導体が式４：

で示される（Ｓ）−１−シクロヘキシル−２−プロピン−１−オールである、（１）に記載の光学活性プロパルギルアルコール誘導体の製造方法。 (2) The propargyl ketone derivative represented by Formula 1 is represented by Formula 3:

Wherein the optically active propargyl alcohol derivative represented by formula 2 is represented by formula 4:

The manufacturing method of the optically active propargyl alcohol derivative as described in (1) which is (S) -1-cyclohexyl-2-propyn-1-ol shown by these.

(3) A protein encoded by the polynucleotide according to any one of (a) to (e), a corresponding coenzyme, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate An optical activity represented by Formula 2 is obtained by reducing propargyl ketone represented by Formula 1 using a transformant that expresses a dehydrogenase having an activity of regenerating (NADPH), or a processed product of the transformant. The method according to (1) or (2), wherein propargyl alcohol is produced.

(4) The method according to (3), wherein the dehydrogenase having NADH or NADPH regeneration activity is glucose dehydrogenase or formate dehydrogenase.

（式中、Ｒは、Ｃ１−Ｃ１０の置換若しくは未置換の直鎖、分岐鎖又は環状のアルキル基、アルケニル基又はアルキニル基、あるいはＣ６−Ｃ２０のアリール基を表す）
で示されるプロパルギルケトン誘導体に作用させ、該ケトンを還元して式５：

（式中、Ｒは、式１の場合と同義である）
で示される光学活性プロパルギルアルコール誘導体を製造することを含む、光学活性プロパルギルアルコール誘導体の製造方法。 (5) The following (a) to (e);
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3, 5 or 7;
(B) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8;
(C) a protein comprising amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 4, 6 or 8, and a propargyl ketone derivative represented by formula 1 A polynucleotide that encodes a protein having an activity to produce an optically active propargyl alcohol derivative represented by formula 5;
(D) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, 5 or 7, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having activity to produce the optically active propargyl alcohol derivative shown; or (e) having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 4, 6 or 8 A polynucleotide encoding a protein having an activity of reducing the propargyl ketone derivative represented by formula 1 to produce an optically active propargyl alcohol derivative represented by formula 5:
A protein encoded by the polynucleotide according to any one of the above, a microorganism or a transformant functionally expressing the protein, or a processed product thereof, is represented by the formula 1:

(Wherein R represents a C1-C10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group, alkynyl group, or C6-C20 aryl group)
And reacting with a propargyl ketone derivative represented by

(Wherein R has the same meaning as in formula 1)
A process for producing an optically active propargyl alcohol derivative, comprising producing an optically active propargyl alcohol derivative represented by the formula:

で示される１−シクロヘキシル−２−プロピン−１−オンであり、式５で示される光学活性プロパルギルアルコール誘導体が式６：

で示される（Ｒ）−１−シクロヘキシル−２−プロピン−１−オールである、（５）に記載の光学活性プロパルギルアルコール誘導体の製造方法。 (6) The propargyl ketone derivative represented by formula 1 is represented by formula 3:

1-cyclohexyl-2-propyn-1-one represented by formula (5), and the optically active propargyl alcohol derivative represented by formula (5)

(R) -1-cyclohexyl-2-propyn-1-ol represented by the method for producing an optically active propargyl alcohol derivative according to (5).

(7) A protein encoded by the polynucleotide according to any one of (a) to (e), a corresponding coenzyme, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate An optical activity represented by Formula 5 is obtained by reducing propargyl ketone represented by Formula 1 using a transformant that coexpresses a dehydrogenase having an activity to regenerate (NADPH), or a processed product of the transformant. The method according to (5) or (6), wherein propargyl alcohol is produced.

(8) The method according to (7), wherein the dehydrogenase having NADH or NADPH regeneration activity is glucose dehydrogenase or formate dehydrogenase.

  The present invention has made it possible to provide a method for efficiently producing optically active propargyl alcohol having high optical purity.

The present invention provides the following (a) to (e);
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
(C) a protein composed of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having an activity to produce an optically active propargyl alcohol derivative represented by Formula 2:
(D) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, wherein the propargyl ketone derivative represented by Formula 1 is reduced, and the optical activity represented by Formula 2 A polynucleotide encoding a protein having an activity of producing a propargyl alcohol derivative; or (e) a propargyl ketone represented by formula 1 having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2 A polynucleotide encoding a protein having an activity of reducing the derivative to produce an optically active propargyl alcohol derivative represented by formula 2:
A propargyl ketone derivative represented by Formula 1 is reduced using a protein encoded by the polynucleotide according to any one of the above, a microorganism or transformant functionally expressing the protein, or a processed product thereof, and Formula 2 It relates to a method for producing an optically active propargyl alcohol derivative represented by the formula:

  Examples of the protein described in SEQ ID NO: 2 include (R) -2-octanol dehydrogenase derived from Pichia finlandica. The enzyme can be prepared from Pichia finlandica cells by the method described in WO01 / 61014. As the Pichia finlandica, the Pichia finlandica DSM 70280 strain can be preferably used. In addition, DNA encoding the protein of SEQ ID NO: 2 which is the amino acid sequence of (R) -2-octanol dehydrogenase derived from Pichia finlandica DSM 70280 is cloned from Pichia finlandica DSM 70280. Alternatively, (R) -2-octanol dehydrogenase can be obtained by chemically synthesizing a DNA encoding the amino acid sequence shown in SEQ ID NO: 2 and introducing it in a form that can be expressed in a heterologous host such as E. coli. It can also be expressed and the enzyme can be obtained from the recombinant.

  The measurement of the enzyme activity of (R) -2-octanol dehydrogenase used in the present invention can be confirmed as follows. Derived from an increase in NADH in a reaction solution containing 100 mM Tris-HCl buffer (pH 9.0), 2.5 mM oxidized nicotinamide adenine dinucleotide, 5 mM (R) -2-octanol and enzyme at 30 ° C. Measure the increase in absorbance at 340 nm. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute.

Furthermore, the present invention provides the following (a) to (e);
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3, 5 or 7;
(B) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8;
(C) a protein comprising amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 4, 6 or 8, and a propargyl ketone derivative represented by formula 1 A polynucleotide that encodes a protein having an activity to produce an optically active propargyl alcohol derivative represented by formula 5;
(D) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, 5 or 7, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having activity to produce the optically active propargyl alcohol derivative shown; or (e) having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 4, 6 or 8 A polynucleotide encoding a protein having an activity of reducing the propargyl ketone derivative represented by formula 1 to produce an optically active propargyl alcohol derivative represented by formula 5:
A propargyl ketone derivative represented by formula 1 is reduced using a protein encoded by the polynucleotide according to any one of the above, a microorganism or a transformed strain that functionally expresses the protein, or a processed product thereof; It relates to a method for producing an optically active propargyl alcohol derivative represented by the formula:

  The carbonyl reductase ScGRE2 utilized in the present invention can be prepared from baker's yeast by the method described in J. Org. Chem. 63, 4996-5000 (1998). In addition, the DNA described in SEQ ID NO: 3 encoding ScGRE2 derived from Saccharomyces cerevisiae is cloned by PCR or the like from Saccharomyces cerevisiae (for example, X2180-1B strain) (Yeast Genetic Stock Center), etc. ScGRE2 can be expressed using a recombinant introduced in an expressible form, and the enzyme can be obtained from the recombinant.

  The measurement of the enzyme activity of ScGRE2 used in the present invention can be confirmed as follows. In a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM reduced nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH), 20 mM ethyl acetoacetate and an enzyme, the reaction is performed at 30 ° C. The decrease in absorbance at 340 nm resulting from the decrease in NADPH is measured. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADPH per minute.

  The carbonyl reductase ScYGD9 used in the present invention is a Saccharomyces cerevisiae (for example, Saccharomyces cerevisiae (for example, Saccharomyces cerevisiae), for example, by the method described in Japanese Patent Application No. 2003-113402. X2180-1B) (Yeast Genetic Stock Center), etc., and cloned by PCR, etc., and expressed ScYGD9 using a recombinant introduced in a form that can be expressed in a heterogeneous host such as E. coli. It can be prepared by obtaining. The enzyme activity of ScYGD9 used in the present invention can be confirmed by the same method as ScGRE2.

  The carbonyl reductase TdCR1 used in the present invention can be prepared from Torulaspora delbrueckii by the method described in Japanese Patent Application No. 2003-113402. In addition, the DNA described in SEQ ID NO: 7 that encodes TdCR1 derived from Torlas pora delbrecki is cloned from Torlas pora delbrecki (eg, IFO 0381 strain) by PCR or the like, and can be expressed in heterologous hosts such as E. coli. TdCR1 can be expressed using the introduced recombinant and the enzyme can be obtained from the recombinant.

  The measurement of the enzyme activity of TdCR1 according to the present invention can be confirmed as follows. Decrease in absorbance at 340 nm due to reduction of NADPH by reaction at 30 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADPH, 5 mM 3,4-dimethoxyphenylacetone and enzyme Measure. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol of NADPH per minute.

  The yeast (Saccharomyces cerevisiae, Torrus pora delbrecky) is cultured in a general medium used for yeast culture such as YM medium. After sufficiently growing, the cells are collected and crushed in a buffer solution containing a reducing agent such as 2-mercaptoethanol or phenylmethanesulfonyl fluoride or a protease inhibitor to obtain a cell-free extract. From cell-free extracts, fractionation by protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.), cation exchange, anion exchange, gel filtration, hydrophobic chromatography, chelates, dyes, antibodies, etc. It can be purified by appropriately combining affinity chromatography using For example, electrophoretic single band through hydrophobic chromatography using phenyl-sepharose, anion exchange chromatography using MonoQ, hydrophobic chromatography using butyl-sepharose, adsorption chromatography using hydroxyapatite, etc. Can be purified.

  The amino acid sequence in which one or more (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5) amino acids are deleted, substituted, inserted and / or added in the present invention is: Site-directed mutagenesis method (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2ndEdt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991)), etc., by appropriately introducing mutations such as substitution, deletion, insertion, and / or addition. It is possible to obtain.

  The polynucleotide homologue in the present invention is a polynucleotide encoding a protein having at least 70%, preferably at least 80%, more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. Contains nucleotides. Protein homology searches include, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, databases related to predicted amino acid sequences based on DNA sequences, etc. For example, it can be performed through the Internet using programs such as BLAST and FASTA. The homology of the present invention refers to a numerical value indicated by identity using, for example, the BLAST program.

  The polynucleotide capable of hybridizing under stringent conditions with the polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, 3, 5 or 7 in the present invention is any at least 20 in any polynucleotide SEQ ID NO: DNA, preferably at least 30, for example, 40, 60 or 100, one or a plurality of contiguous sequences is used as a probe DNA, for example, using ECL direct nucleic acid labeling and detection system (Amersham Pharmaica Biotech) The polynucleotide that hybridizes under the conditions described in the manual (for example, wash: 42 ° C., primary wash buffer containing 0.5 × SSC) is shown. More specific “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions, The conditions are preferably 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  An expression vector for each enzyme is provided by inserting a polynucleotide encoding an alcohol dehydrogenase or carbonyl reductase that can be used in the present invention into a known expression vector. Further, by culturing a transformant transformed with this expression vector, an alcohol dehydrogenase and a carbonyl reductase that can be used in the present invention can be obtained from the recombinant. In order to express alcohol dehydrogenase and carbonyl reductase in the present invention, a microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding each enzyme and expresses each enzyme activity. There is no particular limitation as long as it is an organism that can be used. Examples of the microorganisms that can be used include the following microorganisms.

Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, and Lactobacillus Bacteria for which host vector systems such as (Lactobacillus) are developed;
Actinomycetes for which host vector systems have been developed, such as the genus Rhodococcus and the genus Streptomyces;
The genus Saccharomyces, the genus Kluyveromyces, the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Yeasts for which host vector systems such as the genus Pichia and the genus Candida have been developed;
Molds for which host vector systems such as the genus Neurospora, Aspergillus, Cephalosporium, and Trichoderma have been developed:

  The procedure for producing transformants and the construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al., Molecular cloning, Cold Spring Harbor Laboratories). In order to express a carbonyl reductase gene using NADPH used in the present invention as an electron donor in microorganisms, the DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, It is necessary to transcribe and translate information. For that purpose, a promoter corresponding to a unit controlling transcription / translation may be incorporated upstream of the 5′-side of the DNA strand of the present invention, more preferably a terminator downstream of the 3′-side. As the promoter and terminator, it is necessary to use a promoter and terminator known to function in a microorganism used as a host. Regarding the vectors, promoters, and terminators that can be used in these various microorganisms, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”, especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992), etc.

  In addition to microorganisms, various host and vector systems have been developed in plants and animals, especially in insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system for expressing a large amount of heterologous protein has been developed and can be suitably used.

  In a preferred embodiment of the production of the optically active propargyl alcohol derivative represented by Formula 2 or Formula 5 described in the present specification, the characteristics of enzyme molecules, processed products thereof, cultures containing the enzyme molecules, microorganisms that produce the enzymes, etc. The optically active propargyl alcohol derivative can be produced by bringing the converted body into contact with the reaction solution to cause the target enzyme reaction. The contact form between the enzyme and the reaction solution is not limited to these specific examples.

  Specifically, the microorganism treated product containing alcohol dehydrogenase or carbonyl reductase in the present invention is a microorganism whose glass membrane permeability has been changed by treatment with an organic solvent such as a surfactant or toluene, or a glass bead or enzyme. A cell-free extract obtained by crushing bacterial cells by treatment or a partially purified product thereof is included.

  In the propargyl ketone derivative represented by the formula 1 in the present invention, R represents a C1-C10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group, alkynyl group, or C6-C20 aryl group. To express. Examples of R include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl, isobutyl, t-butyl, 2-methylpentyl, 3-methylhexyl, cyclopentyl, cyclohexyl, cyclopentylmethyl, Examples thereof include cyclohexylmethyl and phenyl, and these substituents may be further substituted with chlorine, bromine, iodine, trialkylsilyl, trialkyltin or the like.

  In a preferred embodiment of the present invention, a protein having (R) -2-octanol dehydrogenase activity, a microorganism producing the enzyme or protein, or a processed product of the microorganism is represented by 1-cyclohexylpropinone represented by Formula 3 ( (S) -1-cyclohexyl-, characterized in that (S) -1-cyclohexyl-2-propyn-1-ol ((S) -CHPO) represented by formula 4 is produced by acting on (CHPN) A method for producing 2-propyn-1-ol is provided. In addition, a protein having a carbonyl reductase activity selected from ScGRE2, ScYGD9, TdCR1, or a variant thereof, a microorganism that produces the enzyme or protein, or a processed product of the microorganism, is represented by 1-cyclohexylpropinone represented by Formula 3. (R) -1-cyclohexyl characterized by producing (R) -1-cyclohexyl-2-propyn-1-ol ((R) -CHPO) represented by formula 6 by acting on (CHPN) A process for producing 2-propyn-1-ol is provided.

The regeneration of NAD ⁺ or NADP ⁺ produced from NADH or NADPH in association with the above reduction reaction to NADH or NADPH is due to NAD ⁺ , NADP ⁺ reducing ability of the microorganism (glycolytic system, C1 compound utilization pathway of methylotroph) Etc.). These NAD ⁺ and NADP ⁺ reducing ability can be enhanced by adding glucose or ethanol to the reaction system. It can also be carried out by adding a microorganism having the ability to produce NADH and NADPH from NAD ⁺ and NADP ⁺ , a treated product thereof, and an enzyme to the reaction system. For example, microorganisms including glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.), processed products thereof, and partially purified or purified enzymes Can be used to regenerate NADH or NADPH. The components constituting the reaction necessary for the regeneration of NADH and NADPH can be added to the reaction system for the production of the optically active alcohol according to the present invention, can be added, or can be exchanged for NADH and NADPH. It can be contacted through a membrane.

  Moreover, this invention can also include the process of culture | cultivating the transformant transformed by the recombinant vector containing the polynucleotide which codes the polypeptide which can be used for this invention. In this method, when a living cell of a microorganism transformed with a recombinant vector containing the polynucleotide of the present invention is used in the method for producing an optically active alcohol, an additional reaction system for regeneration of NADH and NADPH May be unnecessary. That is, by using a microorganism having high NADH and NADPH regeneration activity as a host, an efficient reaction can be performed in the reduction reaction using the transformant without adding an enzyme for NADH and NADPH regeneration. In addition, genes such as NADH, glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc.) that can be used for regeneration of NADP and the like are disclosed in the present invention. More efficient NADH, NADPH regenerating enzyme and NADPH-dependent carbonyl reductase expression and reductive reaction by introducing into the host simultaneously with DNA encoding NADH or NADPH-dependent alcohol dehydrogenase or carbonyl reductase It is also possible. In order to introduce these two or more genes into the host, a method of transforming the host with a recombinant vector in which genes are separately introduced into a plurality of vectors different from the origin of replication in order to avoid incompatibility, A method of introducing both genes into a single vector, a method of introducing both genes into a chromosome, or the like can be used.

  When introducing multiple genes into a single vector, methods such as linking promoter- and terminator-related regions related to expression control to each gene, or expressing as an operon containing multiple cistrons such as the lactose operon. Is possible.

  For example, glucose dehydrogenase derived from Bacillus subtilis or Thermoplasma acidophilum can be used as an enzyme for regeneration of NADH and NADPH. As the NADH regenerating enzyme, formate dehydrogenase derived from Mycobacterium baccae and its mutants can be suitably used.

  Specifically, the (R) -2-octanol dehydrogenase gene derived from Pichia finlandica and the formate dehydrogenase gene derived from Mycobacterium baccae were introduced into the (S) -CHPO synthesis. Co-expression plasmids, pSF-PFO2 (WO01 / 61014), co-expression plasmids pSG-PFO1 (WO01 / 61014) and pSG-PFO2 (WO01 / 61014) into which a glucose dehydrogenase gene derived from Bacillus subtilis has been introduced No. 61014). For (R) -CHPO synthesis, co-expression plasmids (both pSG-YOL1 (this) were introduced together with either the ScGRE2, ScYGD9, or TdCR1 carbonyl reductase gene and the Bacillus subtilis-derived glucose dehydrogenase gene). Inventive Examples), pSG-YGD9 (Japanese Patent Application 2003-113402), pSG-TDR1 (Japanese Patent Application 2003-113402)).

  The reduction reaction using the enzyme of the present invention is an organic solvent that is difficult to dissolve in water or water, for example, an organic solvent such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, and methyl tertiary butyl ester. It can be carried out in a medium or in a two-phase system with an aqueous medium, or in a mixed system with an organic solvent that dissolves in water, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide and the like. The reaction of the present invention can also be performed using an immobilized enzyme, a membrane reactor, or the like.

In the reaction of the present invention, the reaction temperature is 4-60 ° C, preferably 10-40 ° C, pH 3-9, preferably pH 5-7, substrate concentration 0.01-50%, preferably 0.1-10%, more preferably Can be carried out at 0.1-5%. Since the propargyl ketone represented by Formula 1 which is the substrate of the reaction of the present invention is generally unstable in water, particularly on the alkali side, it is desirable to carry out the reaction at a low temperature in a weakly acidic region. In addition, the coenzyme NAD +, NADH, NADP ⁺ or NADPH may be added to the reaction system in an amount of 0.001 mM-100 mM, preferably 0.01-10 mM, if necessary. The substrate can be added all at once at the start of the reaction, but it is desirable to add it continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high.

  For regeneration of NADH, NADPH, for example, glucose when using glucose dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, formate when using formate dehydrogenase, amino acid dehydrogenase when using formate dehydrogenase Amino acid when using malate, malic acid when using malate dehydrogenase, glycerol when using glycerol dehydrogenase, and the like are added to the reaction system. These compounds can be added in an excess of 0.1 to 20, preferably 1 to 5 times in molar ratio to the substrate ketone. Meanwhile, NADH and NADPH regeneration enzymes such as glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, amino acid dehydrogenase, malate dehydrogenase, and glycerol dehydrogenase are NADH and NADPH of the present invention. The enzyme activity can be added in an amount of 0.1 to 100 times, preferably 0.5 to 20 times that of the dependent enzyme.

  The substrate, propargyl ketone, can be synthesized, for example, by the method described in the literature (J. Org. Chem., 52, 2860 (1987)).

  The optically active propargyl alcohol produced by the reduction of the propargyl ketone of the present invention can be purified by appropriately combining microbial cells and proteins, separation by membrane treatment, solvent extraction, distillation and the like.

For example, in (S) -CHPO synthesis, an organic solvent such as ethyl acetate, butyl acetate, toluene, hexane, benzene, methyl isobutyl ketone, methyl tertiary butyl ether, butanol, hexane, heptane is directly added to the reaction solution containing microbial cells. After adding and extracting, etc., it can extract | collect as optically active propargyl alcohol by concentrating under reduced pressure. In order to further increase the purity of the reaction product, it can be further purified by performing silica gel column chromatography, distillation, crystallization, or the like.
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

[Example 1] Simultaneous expression of (R) -2-octanol dehydrogenase derived from Pichia finlandica and glucose dehydrogenase derived from Bacillus subtilis Pichia obtained by the method shown in the patent document (WO01 / 61014) -E. coli HB101 strain (HB101 (pSG-PFO1)) carrying plasmid pSG-PFO1 coexpressing (R) -octanol dehydrogenase derived from Finrandica and glucose dehydrogenase derived from Bacillus subtilis in 2 × YT medium The cells were cultured in 50 mL (Difco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2), and gene expression was induced with 0.1 mM IPTG. The obtained microbial cells were collected, crushed with a sealed ultrasonic crusher UCD-200TM (Cosmo Bio), and the centrifuged supernatant was used as a cell-free extract.

[Example 2] Enzymatic activity of (R) -2-octanol dehydrogenase derived from Pichia finlandica and glucose dehydrogenase derived from Bacillus subtilis Using the cell-free extract obtained in Example 1, enzyme activity was measured did. The (R) -2-octanol dehydrogenase activity was measured at 30 ° C. in a reaction solution containing 50 mM Tris-HCl buffer (pH 8.5), 2.5 mM NAD +, 5 mM (R) -2-octanol and the enzyme. The increase in absorbance at 340 nm resulting from the increase in NADH was measured. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute. The cell-free extract obtained from recombinant Escherichia coli showed (R) -2-octanol oxidation activity in a NAD ⁺ dependent manner, and the specific activity was 35.9 U / mg-protein. In addition, CHPN reduction activity was measured by reacting at 30 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADH, 1% dimethyl sulfoxide, 2 mM CHPN, and enzyme. The decrease in absorbance at 340 nm derived from was measured. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute. The cell-free extract obtained from recombinant Escherichia coli exhibited CHPN reduction activity in a NADH-dependent manner, and the specific activity was 8.01 U / mg protein.

Glucose dehydrogenase activity was measured by reacting at 30 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 2.5 mM NAD ⁺ , 100 mM D-glucose and enzyme, and derived from an increase in NADH. The increase in absorbance at 340 nm was measured. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute. In the cell-free extract obtained from recombinant E. coli, the specific activity was 1.18 U / mg protein.

[Example 3] Synthesis of (S) -CHPO by (R) -2-octanol dehydrogenase derived from Pichia finlandica and glucose dehydrogenase derived from Bacillus subtilis 2 transformed E. coli obtained in Example 1 × Cultured in 50 mL of YT medium (Difco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2), induced gene expression with 0.1 mM IPTG, and collected . Add half of the resulting bacterial mass to a 50 mL reaction solution containing 400 mM potassium phosphate buffer (pH 6.5), 1% CHPN (73 mM), 1% dimethyl sulfoxide, 1.6% glucose (88 mM), The reaction was started at 20 ° C. with shaking. One hour after the start of the reaction, half of the collected cells were further added, and the reaction was continued overnight.

[Example 4] Isolation of (S) -CHPO from reaction solution The reaction solution prepared in Example 3 was extracted three times with 150 mL of hexane. A portion of this hexane extract was taken and the conversion rate was measured by gas chromatography. For the analysis, FFAP 20% Chromosorb W (AW), 80-100 mesh (diameter 0.32 × 210 cm, Shinwa Kako) was used. The column chamber temperature and inlet temperature were 200 ° C., the detector temperature was 230 ° C., nitrogen was used as the carrier gas, and a hydrogen flame ionization detector was used for detection. The retention time for each compound was 5.0 minutes for CHPN and 8.5 minutes for CHPO. As a result of quantification, the conversion rate was 100%. The organic layer was filtered to remove insoluble components, and then the solvent was distilled off under reduced pressure. Crystals were precipitated by cooling the obtained oil. The precipitated crystals were collected by filtration to obtain 380 mg of (S) -CHPO crystals as the target product. The yield was 75%.

[Example 5] Optical purity of synthesized (S) -CHPO Using the hexane extract described in Example 4, optical purity was measured using high performance liquid chromatography using an optical resolution column. For the analysis, CHIRALCEL OD (φ0.46 × 25 cm, Daicel Chemical Industries) was used. The column chamber temperature was 40 ° C., elution was hexane / 2-propanol (97/3, flow rate 1 mL / min), and detection wavelength was 220 nm. The retention time of each enantiomer was 13.0 minutes for (S) and 14.9 minutes for (R). As a result, the optical purity of the product was 100% ee (S).

[Example 6] Simultaneous expression of (R) -2-octanol dehydrogenase derived from Pichia finlandica and formate dehydrogenase derived from Mycobacterium baccae Obtained by the method shown in the patent document (WO01 / 61014) E. coli strain HB101 (HB101 (pSF-PFO3)) carrying plasmid pSF-PFO3 that co-expresses (R) -octanol dehydrogenase derived from Pichia finlandica and formate dehydrogenase derived from Mycobacterium baccae The cells were cultured in 50 mL of 2 × YT medium (Difco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2), and gene expression was induced with 0.1 mM IPTG. The obtained microbial cells were collected, crushed with a sealed ultrasonic crusher UCD-200TM (Cosmo Bio), and the centrifuged supernatant was used as a cell-free extract.

[Example 7] Enzymatic activity of (R) -2-octanol dehydrogenase derived from Pichia finlandica and formate dehydrogenase derived from Mycobacterium baccae Using the cell-free extract obtained in Example 6, enzyme activity Was measured. The cell-free extract obtained from recombinant Escherichia coli exhibited (R) -2-octanol oxidation activity in a NAD ⁺ dependent manner, and the specific activity was 18.2 U / mg-protein.

Formate dehydrogenase activity was measured at 30 ° C. in a reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 2.5 mM NAD ⁺ , 100 mM sodium formate and enzyme. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute. In the cell-free extract obtained from recombinant E. coli, the specific activity was 1.11 U / mg protein.

[Example 8] Synthesis of (S) -CHPO by (R) -2-octanol dehydrogenase derived from Pichia finlandica and formate dehydrogenase derived from Mycobacterium baccae. Transformed Escherichia coli obtained in Example 6 Is cultured in 50 mL of 2 × YT medium (Difco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2), and the expression of the gene is induced with 0.1 mM IPTG. Fungus. Half of the resulting cells were added to 25 mL of 400 mM potassium phosphate buffer (pH 6.5), 1% CHPN (73 mM), 1% dimethyl sulfoxide, 1.6% glucose (88 mM), 1 mM NAD ⁺ In addition to the reaction solution, the reaction was continued overnight at 20 ° C. with shaking. The reaction solution was extracted twice with 75 mL of ethyl acetate and quantified by gas chromatography. As a result, the conversion rate was 84%.

[Example 9] Purification of chromosomal DNA from Saccharomyces cerevisiae Saccharomyces cerevisiae X2180-1B (Yeast Genetic Stock Center) was cultured in a YM medium to prepare bacterial cells. Purification of chromosomal DNA from the bacterial cells was performed by the method described in Meth. Cell Biol. 29, 39-44 (1975).

[Example 10] Cloning of carbonyl reductase ScGRE2
PCR primer YOL1-ATG1 (SEQ ID NO: 9), YOL- based on DNA sequence (DDBJ Accession No. Z74893) corresponding to predicted protein YOL151w (SWISS-PROT Accession No. Z74893-1) registered in DDBJ XbaIR (SEQ ID NO: 10), YOL-XbaIF (SEQ ID NO: 11), and YOL1-TAA1 (SEQ ID NO: 12) were synthesized.
SEQ ID NO: 9
YOL-ATG1: GACACATGTCAGTTTTTGTTTCAGGTGCTAAC
SEQ ID NO: 10
YOL-XbaIR: TCCTCTAGAAATTCCCAAGCTG
SEQ ID NO: 11
YOL-XbaIF: CTGTCTAGAGGAGAATCGTGACTCTGTAAAATTC
SEQ ID NO: 12
YOL-TAA1: GTCGAATTCCTCTATTAAATACGGCCCTCAAATTTTAAAATTTGGGAG

  Denature using 50 μL of the primer YOL-ATG1 and YOL-XbaIR each containing 25 pmol, dNTP 10 nmol, Saccharomyces cerevisiae-derived chromosomal DNA 50 ng, Pfu DNA polymerase buffer (STRATAGENE), Pfu DNA polymerase 2U (STRATAGENE) (95 ° C, 45 seconds), annealing (55 ° C, 30 seconds), extension (72 ° C, 1 minute 20 seconds) 30 cycles, PCR was performed using GeneAmp PCR System 2400 (Perkin Elmer). Amplification product was obtained.

  The amplified product was treated with phenol, then double digested with restriction enzymes BspLU11I and XbaI, and the resulting fragment was purified.

  Next, 50 μL of the reaction solution containing primers YOL-Xba1F and YOL-TAA1 each containing 25 pmol, dNTP 10 nmol, Saccharomyces cerevisiae-derived chromosomal DNA 50 ng, Pfu DNA polymerase buffer (STRATAGENE), and Pfu DNA polymerase 2U (STRATAGENE) PCR was performed using GeneAmp PCR System 2400 (manufactured by PerkinElmer) using 30 cycles of denaturation (95 ° C., 45 seconds), annealing (55 ° C., 30 seconds), extension (72 ° C., 1 minute 20 seconds). As a result, a specific amplification product was obtained.

  The obtained amplification product was treated with phenol, then double digested with restriction enzymes XbaI and EcoRI, and the resulting fragment was purified.

  The obtained two kinds of PCR-amplified DNA fragments digested with restriction enzymes were ligated with a vector pSE420D (Japanese Patent Laid-Open No. 2000-189170) double-digested with restriction enzymes NcoI and EcoRI and TAKARA Ligation Kit. Escherichia coli JM109 strain was transformed with the ligated DNA, grown on LB medium containing ampicillin (50 mg / L), and the plasmid was purified by FlexiPrep from the obtained transformant. As a result of analyzing the base sequence of the inserted DNA portion of the obtained plasmid, the base sequence registered in DDBJ was completely identical except for the primer portion. The obtained plasmid was designated as pSE-YOL1. The process of plasmid construction is shown in FIG.

[Example 11] Construction of plasmid pSG-SCR1 co-expressing carbonyl reductase ScGRE2 and a glucose dehydrogenase gene derived from Bacillus subtilis
pSE-YOL1 was double-digested with NheI and Csp45I, and a DNA fragment consisting of 719 bp was purified. Further, this plasmid was double-digested with Csp45I and EcoRI, and a DNA fragment consisting of 469 bp was purified. Plasmid pSE-BSG1 (Japanese Patent Application No. 2000-374593) containing the glucose dehydrogenase gene derived from Bacillus subtilis was double digested with two restriction enzymes NheI and EcoRI, and the above 2 DNA fragment excised from pSE-YOL1 and Takara Ligation Ligation was performed using Kit. Escherichia coli JM109 is transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the resulting transformant can be purified with FlexiPrep to simultaneously express glucose dehydrogenase and ScGRE2. PSG-SCR1, a novel plasmid, was obtained. The process of plasmid construction is shown in FIG.

[Example 12] Simultaneous expression of carbonyl reductase ScGRE2 and glucose dehydrogenase derived from Bacillus subtilis Escherichia coli HB101 transformed with pSG-SCR1 obtained in Example 11 (E. coli HB101 (pSG-SCR1)) was treated with ampicillin. After overnight culture in LB medium containing 50 mg / L, 0.1 mM IPTG was added to induce gene expression, followed by further incubation for 4 hours. The obtained cells were collected and then crushed with a closed ultrasonic crusher UCD-200TM (manufactured by Cosmo Bio), and the centrifuged supernatant was used as a cell-free extract.

[Example 13] Enzyme activity of carbonyl reductase ScGRE2 Using the cell-free extract obtained in Example 12, the enzyme activity was measured. The measurement of CHPN reduction activity was performed at 25 ° C. in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADPH, 2 mM CHPN, 1% dimethyl sulfoxide and an enzyme. 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol NADH per minute. The cell-free extract obtained from recombinant Escherichia coli showed CHPN reduction activity in a NADPH-dependent manner, and the specific activity was 10.8 U / mg-extract.

[Example 14] Synthesis of (R) -CHPO using carbonyl reductase ScGRE2
Contains 200 mM potassium phosphate buffer (pH 6.5), 1 mM NADP ⁺ , CHPN 50 mM, D-glucose 250 mM, BSA 0.1%, glucose dehydrogenase (manufactured by Amano) 2 U, and ScGRE2 enzyme solution 1 U The reaction was allowed to react overnight at 25 ° C. with shaking.

[Example 15] Optical purity of (R) -CHPO The reaction solution prepared in Example 14 was extracted with 2 volumes of ethyl acetate. The solvent was distilled off under reduced pressure, and the residue was dissolved in 1 volume of hexane / 2-propanol (97/3). The optical purity of this sample was measured using high performance liquid chromatography using an optical resolution column. The analysis was performed by the method described in Example 5. As a result, the optical purity of the product was 99.8% ee (R).

[Example 16] Conversion rate of (R) -CHPO Using the sample prepared in Example 14, the conversion rate was measured by gas chromatography according to the method described in Example 4. The conversion rate calculated from the peak area was 28.4%.

[Example 17] Cloning of carbonyl reductase ScYGD9 and construction of plasmid pSG-YGD9 co-expressing carbonyl reductase ScYGD9 and a glucose dehydrogenase gene derived from Bacillus subtilis according to the method described in Japanese Patent Application No. 2003-113402, I went there.
(1) Cloning of carbonyl reductase homolog YGL039w
Primers for PCR YGL2-ATG2 (SEQ ID NO: 13), YGL2-TAA2 (sequence) based on the DNA sequence (DDBJ Accession No. Z72561) corresponding to the predicted protein YGL039w (SWISS-PROT Accession No. P53183) registered in DDBJ No. 14) was synthesized. Using a 50 μL reaction solution containing 25 pmol of each primer, 10 nmol of dNTP, 50 ng of chromosomal DNA derived from Saccharomyces cerevisiae, buffer solution for Pfu DNA polymerase (manufactured by STRATAGENE), Pfu DNApolymerase 2U (manufactured by STRATAGENE), denaturation (95 ° C., 45 seconds), PCR was performed using GeneAmp PCR System 2400 (manufactured by PerkinElmer) for 30 cycles of annealing (50 ° C., 30 seconds) and extension (72 ° C., 1 minute 15 seconds). As a result, a specific amplification product was obtained. .
The amplified product was treated with phenol, double-digested with restriction enzymes BspHI and NheI, and ligated with vectors pSE420D and TAKARA Ligation Kit double-digested with restriction enzymes NcoI and XbaI. Escherichia coli JM109 strain was transformed with the ligated DNA, grown on LB medium containing ampicillin (50 mg / L), and the plasmid was purified by FlexiPrep from the obtained transformant. The nucleotide sequence of the inserted DNA portion of the plasmid was analyzed, and the result is shown in SEQ ID NO: 15. The obtained base sequence completely matched the base sequence registered in DDBJ. The obtained plasmid was designated as pSE-YGD9.

(2) Construction of plasmid pSG-YGD9 co-expressing carbonyl reductase homolog YGL039w and glucose dehydrogenase gene from Bacillus subtilis
Glucose dehydrogenation gene obtained by double digestion of pSE-YGD9 with two restriction enzymes EcoRI and HindIII and excised from plasmid pSE-BSG1 (JP 2000-189170) containing the glucose dehydrogenase gene derived from Bacillus subtilis Ligation was performed using a DNA fragment containing and Takara Ligation Kit. Escherichia coli JM109 is transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the resulting transformant is purified by FlexiPrep to allow simultaneous expression of glucose dehydrogenase and YGL039w PSG-YGD9, a novel plasmid, was obtained.

[Example 18] Expression of carbonyl reductase ScYGD9 E. coli strain HB101 transformed with pSG-YGD9 obtained in Example 17 (E. coli HB101 (pSG-YGD9)) was cultured in LB medium containing 50 mg / L of ampicillin. After overnight culture, 0.1 mM IPTG was added to induce gene expression, and the culture was further continued for 4 hours. The obtained cells were collected and then crushed with a closed ultrasonic crusher UCD-200TM (manufactured by Cosmo Bio), and the centrifuged supernatant was used as a cell-free extract.

[Example 19] Enzyme activity of carbonyl reductase ScYGD9 Using the cell-free extract obtained in Example 18, the enzyme activity was measured according to the method of Example 13. The cell-free extract obtained from recombinant Escherichia coli exhibited CHPN reduction activity in a NADPH-dependent manner, and the specific activity was 22.7 U / mg-extract.

[Example 20] Synthesis of (R) -CHPO using carbonyl reductase ScYGD9
Reaction solution containing 200 mM potassium phosphate buffer (pH 6.5), 1 mM NADP ⁺ , CHPN 50 mM, D-glucose 250 mM, BSA 0.1%, glucose dehydrogenase (Amano) 2U, and ScYGD9 enzyme solution 1U Was reacted overnight at 25 ° C. with shaking.

[Example 21] Optical purity of (R) -CHPO The optical purity of the reaction solution prepared in Example 20 was measured in the same manner as in Example 5. As a result, the optical purity of the product was 99.3% ee (R).

[Example 22] (R) -CHPO conversion rate Using the sample prepared in Example 20, the conversion rate was measured in the same manner as in Example 4. As a result, the conversion rate calculated from the peak area was 11.6%.

[Example 23] Cloning of carbonyl reductase TdCR1 and construction of plasmid pSG-TDR1 that co-expresses carbonyl reductase TdCR1 and a glucose dehydrogenase gene derived from Bacillus subtilis according to the method described in the literature (Japanese Patent Application 2003-113402). I went there.

(1) Construction of plasmid pUC-TDX containing a part of the carbonyl reductase gene TdCR1 In order to clone from the XbaI site to the 3 ′ end of the ORF of the carbonyl reductase gene, primers Td-XbaF (SEQ ID NO: 16), Td -TAA1 (SEQ ID NO: 17) was synthesized.
50 μL each containing primers Td-XbaF and Td-TAA1, 50 pmol each, dNTP10 nmol, 50 ng chromosomal DNA derived from Torlas pora delbrecki, Pfu Turbo DNA polymerase buffer (STRATAGENE), Pfu Turbo DNA polymerase 3.75 U (STRATAGENE) 30 cycles of denaturation (95 ° C, 2 minutes 30 seconds), annealing (55 ° C, 1 minute), extension (72 ° C, 1 minute), using GeneAmp PCR System 2400 (Perkin Elmer) went. The obtained PCR product was designated as Td-PCR2.

The obtained PCR product was recovered as an ethanol precipitate after phenol-chloroform extraction. Td-PCR2 was digested with XbaI, and then subjected to agarose gel electrophoresis. The target band was excised and purified by Sephaglas BandPrep Kit (Pharmacia).
The restriction enzyme-digested Td-PCR2 was ligated using pUC18 (Takara Shuzo) digested with XbaI and TakaraLigation Kit to transform Escherichia coli JM109 strain. The transformed strain was grown on LB medium containing ampicillin, and the nucleotide sequence of the inserted fragment was analyzed. The plasmid obtained here was designated as pUC-TDX.

(2) Construction of plasmid pSG-TDR1 that co-expresses carbonyl reductase gene TdCR1 and glucose dehydrogenase gene derived from Bacillus subtilis Plasmid pUC-TDX is digested with restriction enzyme XbaI, ethanol precipitated, and then subjected to agarose electrophoresis. A band of about 0.8 bp containing a part of the TdCR1 gene was excised, purified and collected by SephaglasBandPrep (Amersham Pharmacia Biotech). The obtained DNA fragment was digested with the same restriction enzyme, and after alkaline phosphatase treatment, the plasmid pSG-TDX obtained by phenol extraction, phenol-chloroform extraction, chloroform extraction and ethanol precipitation was ligated using TaKaRa Ligation Kit. Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by FlexiPrep. As a result, pSG-TDR1, which is a plasmid capable of simultaneously expressing glucose dehydrogenase and TdCR1, was obtained.

[Example 24] Simultaneous expression of carbonyl reductase TdCR1 and glucose dehydrogenase derived from Bacillus subtilis Escherichia coli HB101 transformed with pSG-TDR1 obtained in Example 23 (E.coli HB101 (pSG-TDR1)) After overnight culture in LB medium containing 50 mg / L of ampicillin, 0.1 mM of IPTG was added to induce gene expression, and further cultured for 4 hours. The obtained cells were collected and then crushed with a closed ultrasonic crusher UCD-200TM (manufactured by Cosmo Bio), and the centrifuged supernatant was used as a cell-free extract.

[Example 25] Enzyme activity of carbonyl reductase TdCR1 Using the cell-free extract obtained in Example 24, the enzyme activity was measured according to the method of Example 13. The cell-free extract obtained from recombinant Escherichia coli exhibited CHPN reduction activity in a NADPH-dependent manner, and the specific activity was 5.7 U / mg protein.

[Example 26] Synthesis of (R) -CHPO using carbonyl reductase TdCR1
Reaction solution containing 200 mM potassium phosphate buffer (pH 6.5), 1 mM NADP +, CHPN 50 mM, D-glucose 250 mM, BSA 0.1%, glucose dehydrogenase (manufactured by Amano) 1U, and TdCR1 enzyme solution 0.5U Was reacted overnight at 25 ° C. with shaking.

[Example 27] Optical purity of (R) -CHPO The optical purity of the reaction solution prepared in Example 26 was measured in the same manner as in Example 5. As a result, the optical purity of the product was 99.7% ee (R).

[Example 28] (R) -CHPO conversion rate Using the sample prepared in Example 26, the conversion rate was measured in the same manner as in Example 4. As a result, the conversion rate calculated from the peak area was 9.0%.

FIG. 1 shows the construction of the ScGRE2 expression plasmid pSE-YOL1. FIG. 2 shows the construction of a co-expression plasmid pSG-SCR1 with ScGRE2 and glucose dehydrogenase derived from Bacillus subtilis.

Claims (8)

The following (a) to (e);
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
(C) a protein composed of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having an activity to produce an optically active propargyl alcohol derivative represented by Formula 2:
(D) a polynucleotide that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 1, wherein the propargyl ketone derivative represented by Formula 1 is reduced, and the optical activity represented by Formula 2 A polynucleotide encoding a protein having an activity of producing a propargyl alcohol derivative; or (e) a propargyl ketone represented by formula 1 having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2 A polynucleotide encoding a protein having an activity of reducing the derivative to produce an optically active propargyl alcohol derivative represented by formula 2:
A protein encoded by the polynucleotide according to any one of the above, a microorganism or a transformant functionally expressing the protein, or a processed product thereof, is represented by the formula 1:

Wherein R represents a C1-C10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group or alkynyl group, or a C6-C20 aryl group.
Is reacted with a propargyl ketone derivative represented by formula (2), and the ketone is reduced to give a compound of formula 2:

(Wherein R has the same meaning as in formula 1)
A process for producing an optically active propargyl alcohol derivative, comprising producing an optically active propargyl alcohol derivative represented by the formula: The propargyl ketone derivative represented by Formula 1 is represented by Formula 3:

Wherein the optically active propargyl alcohol derivative represented by formula 2 is represented by formula 4:

The manufacturing method of the optically active propargyl alcohol derivative of Claim 1 which is (S) -1-cyclohexyl-2-propyn-1-ol shown by these. A protein encoded by the polynucleotide according to any one of (a) to (e), a corresponding coenzyme, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) Using a transformant that expresses a dehydrogenase having the activity of regenerating the compound, or a processed product of the transformant, the propargyl ketone represented by formula 1 is reduced, and the optically active propargyl alcohol represented by formula 2 is reduced. The method according to claim 1 or 2, wherein the method is produced. The method according to claim 3, wherein the dehydrogenase having NADH or NADPH regeneration activity is glucose dehydrogenase or formate dehydrogenase. The following (a) to (e);
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3, 5 or 7;
(B) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4, 6 or 8;
(C) a protein comprising amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 4, 6 or 8, and a propargyl ketone derivative represented by formula 1 A polynucleotide that encodes a protein having an activity to produce an optically active propargyl alcohol derivative represented by formula 5;
(D) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, 5 or 7, wherein the propargyl ketone derivative represented by formula 1 is reduced; A polynucleotide encoding a protein having activity to produce the optically active propargyl alcohol derivative shown; or (e) having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 4, 6 or 8 A polynucleotide encoding a protein having an activity of reducing the propargyl ketone derivative represented by formula 1 to produce an optically active propargyl alcohol derivative represented by formula 5:
A protein encoded by the polynucleotide according to any one of the above, a microorganism or a transformant functionally expressing the protein, or a processed product thereof, is represented by the formula 1:

Wherein R represents a C1-C10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group or alkynyl group, or a C6-C20 aryl group.
And reacting with a propargyl ketone derivative represented by

(Wherein R has the same meaning as in formula 1)
A process for producing an optically active propargyl alcohol derivative, comprising producing an optically active propargyl alcohol derivative represented by the formula: The propargyl ketone derivative represented by Formula 1 is represented by Formula 3:

1-cyclohexyl-2-propyn-1-one represented by formula (5), and the optically active propargyl alcohol derivative represented by formula (5)

The manufacturing method of the optically active propargyl alcohol derivative of Claim 5 which is (R) -1-cyclohexyl-2-propyn-1-ol shown by these. A protein encoded by the polynucleotide according to any one of (a) to (e), a corresponding coenzyme, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) The propargyl ketone represented by formula 1 is reduced using a transformant that also expresses a dehydrogenase having an activity to regenerate the compound, or a processed product of the transformant, and the optically active propargyl alcohol represented by formula 5 is reduced. The method according to claim 5 or 6, wherein the method is produced. The method according to claim 7, wherein the dehydrogenase having NADH or NADPH regeneration activity is glucose dehydrogenase or formate dehydrogenase.

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