JP2006238813A - Method for producing optically active alcohol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for profitably producing a 2-halo-1-(3,4-methylenedioxyphenyl)ethanol. <P>SOLUTION: This method for producing the 2-halo-1-(3,4-methylenedioxyphenyl)ethanol is characterized by comprising a process for treating a 2-halo-1-(3,4-methylenedioxyphenyl)ethanone with a transformant or its died cell to which an ability for asymmetrically reducing the 2-halo-1-(3,4-methylenedioxyphenyl)ethanone to produce the 2-halo-1-(3,4-methylenedioxyphenyl)ethanol and an ability to reproduce a coenzyme depended by an enzyme having the ability are artificially imparted, and a process for collecting the produced 2-halo-1-(3,4-methylenedioxyphenyl)ethanol. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing an optically active alcohol.

  2-Halo-1- (3,4-methylenedioxyphenyl) ethanol is a high blood pressure, pulmonary hypertension, Raynaud's disease, acute renal failure, myocardial infarction, narrow, which is described in an endothelin antagonist (see, for example, Patent Document 1). Heart disease, cerebral infarction, cerebral vasospasm, arteriosclerosis, asthma, gastric ulcer, diabetes, recurrent stenosis, prostatic hypertrophy, endotoxin shock, endotoxin-induced multiple organ failure or disseminated intravascular coagulation, and / or It is a compound useful as an intermediate of a compound which becomes an active ingredient of a drug for the treatment of renal failure or hypertension induced by cyclosporine.

U.S. Pat.No. 5,565,485

  Development of a method for industrially advantageously producing 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is desired.

  As a result of intensive studies on a method for producing 2-halo-1- (3,4-methylenedioxyphenyl) ethanol, the present inventors have determined that 2-halo-1- (3,4-methylenedioxyphenyl) ethanone 2-Halo-1- (3,4-methylenedioxyphenyl) ethanol is produced by the action of a certain biological catalyst, which is collected to produce 2-halo-1- (3,4- The present inventors have found that methylenedioxyphenyl) ethanol can be produced and completed the present invention.

That is, the present invention
1. A process for producing 2-halo-1- (3,4-methylenedioxyphenyl) ethanol, wherein 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is asymmetrically reduced. Transformants that are artificially imparted with the ability to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol and the ability to regenerate the coenzyme on which the enzyme having the ability depends (hereinafter, This may be referred to as this transformant.) Or a process in which 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is allowed to act on the dead cells, and the produced 2-halo-1- (3,4-methylenedioxyphenyl) a production method comprising a step of collecting ethanol (hereinafter sometimes referred to as the production method of the present invention);
2. The transformant is
One plasmid containing DNA consisting of a base sequence encoding the amino acid sequence of an enzyme having the following two artificially conferred abilities simultaneously:
DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and base sequence encoding the amino acid sequence of the enzyme having the ability to be artificially imparted (ii) below One plasmid having the DNA consisting of
A plasmid containing a DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and the amino acid sequence of the enzyme having the ability to artificially grant (ii) below Two plasmids comprising a plasmid having a DNA comprising a coding base sequence,
The production method according to item 1 above, wherein the ability is artificially imparted, characterized in that at least a transformant is introduced.
(I) Ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol ( ii) the ability to regenerate the coenzyme on which the enzyme having the ability of (i) depends;
3. The production method according to item 1 or 2, wherein the transformant is Escherichia coli;
4). 4. The production method according to the above 1 to 3, wherein the coenzyme is NADH / NAD ⁺ (nicotinamide adenine dinucleotide) or NADPH / NADP ⁺ (nicotinamide adenine dinucleotide phosphate);
5. [2] The production method according to [1] to [4] above, wherein 2-oxocycloalkanecarboxylic acid ester is allowed to act on a transformant or a killed cell thereof in the presence of glucose;
6. The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol, The production method according to the above item 1 to 5, which is an ability of an enzyme having an amino acid sequence selected from the following amino acid sequence group;
<Amino acid sequence group>
(A) an amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence in which one or a plurality of amino acid sequences are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and 2-halo- Amino acid sequence of an enzyme having the ability to asymmetrically reduce 1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol (c ) Having the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 2 (d) having the amino acid sequence encoded by the base sequence of DNA that hybridizes under stringent conditions with the DNA comprising the base sequence represented by SEQ ID NO: 2 And 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is asymmetrically reduced to give 2-halo-1- (3,4-methylenedioxy). Amino acid sequence of an enzyme having the ability to produce (phenyl) ethanol (e) 2-halo-1- (3,4-methylenedioxyphenyl) ethanone derived from a microorganism belonging to the genus Penicillium asymmetrically reduced to 2 The amino acid sequence of an enzyme capable of producing halo-1- (3,4-methylenedioxyphenyl) ethanol;
7. The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol, The production method according to the above 1 to 5, wherein the enzyme having the amino acid sequence represented by SEQ ID NO: 1 has the ability;
8). The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is SEQ ID NO: [6] The production method according to the above [1] to [5], wherein the enzyme having the amino acid sequence encoded by the base sequence represented by 2 has the ability
9. Catalyst for asymmetric reduction of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol as Use of a transformant or a killed cell thereof artificially imparted with an ability to regenerate a coenzyme on which the enzyme having the ability depends;
Etc. are provided.

  According to the present invention, a 2-halo-1- (3,4-methylenedioxyphenyl) ethanol compound useful as an intermediate for producing a physiologically active substance can be produced.

Hereinafter, the present invention will be described in detail.
2-Halo-1- (3,4-methylenedioxyphenyl) ethanone is, for example, the general formula (1)

（式中、XはF、Cl、Br、I）で示される２−ハロ−１−（３、４−メチレンジオキシフェニル）エタノン等をあげることができる。
２−ハロ−１−（３、４−メチレンジオキシフェニル）エタノールとは、前記の２−ハロ−１−（３、４−メチレンジオキシフェニル）エタノンのケトン部分を還元して得られるヒドロキシ体又はその塩である。具体的には、本発明製造方法において原料化合物として一般式（１）で示される２−ハロ−１−（３、４−メチレンジオキシフェニル）エタノンを用いる場合には、これに対応するヒドロキシ体である
一般式（２）

(Wherein, X is 2-halo-1- (3,4-methylenedioxyphenyl) ethanone represented by F, Cl, Br, I).
2-Halo-1- (3,4-methylenedioxyphenyl) ethanol is a hydroxy compound obtained by reducing the ketone moiety of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone. Or a salt thereof. Specifically, when 2-halo-1- (3,4-methylenedioxyphenyl) ethanone represented by the general formula (1) is used as a raw material compound in the production method of the present invention, the corresponding hydroxy compound The general formula (2)

（式中、XはF、Cl、Br、I）で示される２−ハロ−１−（３、４−メチレンジオキシフェニル）エタノールが製造される。

2-halo-1- (3,4-methylenedioxyphenyl) ethanol represented by the formula (wherein X is F, Cl, Br, I) is produced.

The transformant used in the production method of the present invention is an asymmetric reduction of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedi). Oxyphenyl) is a transformant artificially imparted with the ability to produce ethanol and the ability to regenerate the coenzyme on which the enzyme having the ability depends. As such a transformant, for example, one plasmid containing DNA consisting of a base sequence encoding an amino acid sequence of an enzyme having the following two artificially conferred abilities,
DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and base sequence encoding the amino acid sequence of the enzyme having the ability to be artificially imparted (ii) below One plasmid having the DNA consisting of
A plasmid containing a DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and the amino acid sequence of the enzyme having the ability to artificially grant (ii) below Two plasmids comprising a plasmid having a DNA comprising a coding base sequence,
And a transformant in which at least is introduced.
<Artificially granted ability>
(I) Ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol ( ii) Ability to regenerate the coenzyme on which the enzyme having the ability of (i) depends.

Such a transformant is usually (1) 2-halo-1- (3,4-methylene) by asymmetric reduction of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone. (2) 2-halo-1- (2) an enzyme having two artificially conferred abilities, namely, the ability to produce dioxyphenyl) ethanol and the ability to regenerate the coenzyme upon which the enzyme having the ability depends. Enzyme capable of asymmetrically reducing 3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol and coenzyme on which the enzyme depends 2 types of enzymes having the ability to regenerate (hereinafter, the enzymes (1) and (2) may be collectively referred to as the present enzyme).

In addition, “ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol” As a specific example, for example, the ability of an enzyme having an amino acid sequence selected from the following amino acid sequence group can be given.
<Amino acid sequence group>
(A) an amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence in which one or a plurality of amino acid sequences are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and 2-halo- Amino acid sequence of an enzyme having the ability to asymmetrically reduce 1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol (c ) Having the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 2 (d) having the amino acid sequence encoded by the base sequence of DNA that hybridizes under stringent conditions with the DNA comprising the base sequence represented by SEQ ID NO: 2 And 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is asymmetrically reduced to give 2-halo-1- (3,4-methylenedioxy). Amino acid sequence of an enzyme having the ability to produce (phenyl) ethanol (e) 2-halo-1- (3,4-methylenedioxyphenyl) ethanone derived from a microorganism belonging to the genus Penicillium asymmetrically reduced to 2 -Amino acid sequence of an enzyme having the ability to produce halo-1- (3,4-methylenedioxyphenyl) ethanol

  The transformant may be prepared using a genetic engineering technique. In addition, 2-halo-1- (3,4-methylenedioxyphenyl) ethanone, which is used in preparing such a transformant, is asymmetrically reduced to give 2-halo-1- (3, 4-Methylenedioxyphenyl) A gene having a base sequence encoding the amino acid sequence of an enzyme (hereinafter also referred to as the present reductase) having at least the ability to produce ethanol (hereinafter referred to as the present reductase gene). (1) may have been cloned from naturally occurring genes, or (2) may be naturally occurring genes in the base sequence of the cloned gene. A gene in which deletion, substitution or addition of a part of its bases has been artificially introduced (that is, a naturally occurring gene was subjected to mutation treatment (partial mutation introduction method, mutation treatment, etc.) It may be I, may be one which is (3) artificially synthesized.

Here, the “amino acid sequence in which an amino acid is deleted, substituted or added” in (b) or the “amino acid sequence encoded by the base sequence of DNA that hybridizes under stringent conditions” in (d) above. Include, for example, the processing that the enzyme having the amino acid sequence represented by SEQ ID NO: 1 undergoes in the cell, the species that the enzyme is derived from, species differences, individual differences, differences between tissues, and the like, Amino acid mutations and the like are included.
As a technique for artificially performing the “(amino acid) deletion, substitution, or addition (done)” in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, a sequence is used. Examples include a method in which a conventional site-directed mutagenesis is performed on a DNA having a base sequence encoding the amino acid sequence represented by No. 1 or 3, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method utilizing amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc.
The number of amino acids modified as described above is at least one residue, specifically one or several, or more. A number of such modifications can find the ability to reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to 2-halo-1- (3,4-methylenedioxyphenyl) ethanol. Any range is acceptable.
Among the deletions, substitutions or additions, modifications relating to amino acid substitution are particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine; (4) serine, threonine; (5) lysine, arginine. And (6) substitution within the group of phenylalanine and tyrosine.

In the present invention, “(amino acid) deletion, substitution, or addition (done)” includes, for example, high sequence identity (specifically, 80% or more sequence identity) between two proteins. Preferably 90% or more, more preferably 95% or more sequence identity) must be present. In addition, “hybridizes under stringent conditions” means sequence identity with respect to the base sequence between two DNAs (specifically, sequence identity of 80% or more, preferably 90% or more, more preferably 95%). The above sequence identity) must exist.
As used herein, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENETYX-MAC, or a public database, for example, the homepage address http It is generally available at: //www.ddbj.nig.ac.jp.
Regarding “hybridize under stringent conditions” in (d) above, the hybridization used here is, for example, by Sambrook J., Frisch EF, Maniatis T., Molecular Cloning Second Edition (Molecular Cloning 2nd edition), described in Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press) and “Cloning and Sequence” (supervised by Watanabe, edited by Masahiro Sugiura, 1989, published by Rural Bunkasha) It can be performed according to a conventional method such as Southern hybridization. “Stringent conditions” means, for example, 6 × SSC (a solution containing 900 mM NaCl, 90 mM trisodium citrate. Here, a solution containing 175.3 g NaCl and 88.2 g trisodium citrate is added with 800 ml of water. After dissolving and adjusting the pH with 10N NaCl, a solution with a total volume of 1000 ml is made 20 × SSC.) Is hybridized at 65 ° C., and then washed with 2 × SSC at 50 ° C. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6). The salt concentration in the washing step can be selected from, for example, conditions of 2 × SSC at 50 ° C. (low stringency conditions) to 0.1 × SSC at 65 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.

The present reductase gene may be prepared, for example, according to the following preparation method.
Ordinary genetic engineering techniques from microorganisms belonging to the genus Penicillium such as Penicillium citrinum (for example, “New Cell Engineering Experimental Protocol” (The University of Tokyo Institute of Medical Science, Division of Cancer Research, Shujunsha, 1993) A cDNA library was prepared according to the method described in (1)), and the amino acid sequence represented by SEQ ID NO: 1 was obtained by performing PCR using the prepared cDNA library as a template and using appropriate primers. DNA consisting of a base sequence encoding, DNA consisting of a base sequence encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, base sequence shown in SEQ ID NO: 2 The present reductase gene is prepared by amplifying DNA or the like having
When PCR is performed using a cDNA library derived from Penicillium citrinum as a template and an oligonucleotide having the base sequence shown in SEQ ID NO: 3 and an oligonucleotide having the base sequence shown in SEQ ID NO: 4 as primers In this method, the reductase gene is prepared by amplifying a DNA comprising the base sequence shown in SEQ ID NO: 2.
The PCR conditions include, for example, 20 μM each of 4 types of dNTP, 15 pmol of each of 2 types of oligonucleotide primers, 1.3 U of Taq polymerase, and a reaction solution mixed with a cDNA library as a template at 97 ° C. (2 minutes) ), Followed by 10 cycles of 97 ° C. (0.25 minutes) −50 ° C. (0.5 minutes) −72 ° C. (1.5 minutes), then 97 ° C. (0.25 minutes) −55 ° C. (0.5 minutes) -72 ° C. (2.5 minutes) is repeated 20 times, and the conditions are further maintained at 72 ° C. for 7 minutes.
A restriction enzyme recognition sequence or the like may be added to the 5 ′ end side of the primer used in the PCR.
The amplified DNA as described above, Sambrook J., Frisch EF, Maniatis T. et al., ^{"Molecular Cloning: A Laboratory Manual 2 nd} edition " (1989), Cold Spring Harbor Laboratory Press, "Current Protocols in Molecular Biology" (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X and the like, and can be cloned into a vector to obtain a recombinant vector. Specific examples of vectors used include pUC119 (Takara Shuzo), pTV118N (Takara Shuzo), pBluescriptII (Toyobo), pCR2.1-TOPO (Invitrogen), pTrc99A (Pharmacia) ), PKK223-3 (manufactured by Pharmacia) and the like. If the reductase gene is prepared in such a manner that it is incorporated into a vector, it is convenient for use in later genetic engineering techniques.

An enzyme having the ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol An enzyme having the ability to regenerate dependent coenzyme (hereinafter also referred to as the present coenzyme regenerating enzyme gene) is, for example, when the coenzyme regenerating enzyme gene is an enzyme different from the reductase. It may be prepared according to the following preparation method.
Chromosomal DNA is prepared from microorganisms belonging to the genus Bacillus such as Bacillus megaterium according to the usual genetic engineering technique, and PCR is performed using the prepared chromosomal DNA as a template and using appropriate primers. A DNA comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 8, and a base sequence encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 8 The coenzyme regenerating enzyme gene is prepared by amplifying DNA comprising the above, DNA having the base sequence represented by SEQ ID NO: 7, and the like.
Here, when PCR is performed using Bacillus megaterium-derived chromosomal DNA as a template and an oligonucleotide having the base sequence shown in SEQ ID NO: 5 and an oligonucleotide having the base sequence shown in SEQ ID NO: 6 as primers Will amplify DNA consisting of the base sequence shown in SEQ ID NO: 7 to prepare this coenzyme regenerating enzyme gene.
The PCR conditions include, for example, 20 μM each of 4 types of dNTP, 15 pmol of each of 2 types of oligonucleotide primers, 1.3 U of Taq polymerase, and a reaction solution mixed with a cDNA library as a template at 97 ° C. (2 minutes) ), Followed by 10 cycles of 97 ° C. (0.25 minutes) −50 ° C. (0.5 minutes) −72 ° C. (1.5 minutes), then 97 ° C. (0.25 minutes) −55 ° C. (0.5 minutes) -72 ° C. (2.5 minutes) is repeated 20 times, and the conditions are further maintained at 72 ° C. for 7 minutes.
A restriction enzyme recognition sequence or the like may be added to the 5 ′ end side of the primer used in the PCR.
The amplified DNA as described above, Sambrook J., Frisch EF, Maniatis T. et al., ^{"Molecular Cloning: A Laboratory Manual 2 nd} edition " (1989), Cold Spring Harbor Laboratory Press, "Current Protocols in Molecular Biology" (1987), John Wiley & Sons, Inc. ISBNO-471-50338-X and the like, and can be cloned into a vector to obtain a recombinant vector. Specific examples of vectors used include pUC119 (Takara Shuzo), pTV118N (Takara Shuzo), pBluescriptII (Toyobo), pCR2.1-TOPO (Invitrogen), pTrc99A (Pharmacia) ), PKK223-3 (manufactured by Pharmacia) and the like. If the reductase gene is prepared in such a manner that it is incorporated into a vector, it is convenient for use in later genetic engineering techniques.

Methods for preparing the transformant include, for example, (1) DNA in which both the reductase gene and the coenzyme regenerating enzyme gene and a promoter capable of functioning in the host cell are connected in a functional form. A method of preparing a single recombinant plasmid such that the present gene can be expressed in the host cell and introducing it into the host cell, (2) regeneration of the present reductase gene and the present coenzyme One gene of this gene can be expressed in the host cell, such as DNA in which one of the genes with the enzyme gene and a promoter capable of functioning in the host cell are operably connected. Such a recombinant plasmid is prepared separately for each of the above genes and introduced into a host cell. Furthermore, a method of introducing one or both genes into the chromosome of the host cell can also be used.
As a method for preparing the single recombinant plasmid by introducing it into a host cell, for example, a recombinant plasmid is constructed by linking regions involved in expression control, such as a promoter and terminator, to both genes. Or a method for constructing a recombinant plasmid that can be expressed as an operon containing a plurality of cistrons such as lactose operon.
Here, the recombinant plasmid includes, for example, genetic information that can be replicated in the host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, Preferable examples include those in which a gene encoding the present enzyme is introduced into a functional form in an expression vector having a promoter that can function in the above and a detectable marker. Various types of expression vectors are commercially available.
Here, “in a functional form” means that when the host cell is transformed by introducing the above-described recombinant plasmid into the host cell, this gene (or one of the genes) is , Means in a state of being linked to a promoter so as to be expressed under the control of the promoter. Examples of the promoter include an E. coli lactose operon promoter, an Escherichia coli tryptophan operon promoter, a synthetic promoter capable of functioning in E. coli, such as a tac promoter or a trc promoter, and the like. In addition, a promoter controlling the expression of this gene in Penicillium citrinum or Bacillus megaterium may be used.
In addition, when a vector containing a selection marker gene (for example, a gene for imparting antibiotic resistance such as a kanamycin resistance gene or a neomycin resistance gene) is used as an expression vector, the transformant into which the vector has been introduced is used as the selection marker gene. It is possible to easily select a phenotype or the like as an index.
When it is necessary to induce higher expression, a ribosome binding region may be linked upstream of a gene having a base sequence encoding the amino acid sequence of the present reductase and / or the present coenzyme regenerating enzyme. Examples of the ribosome binding region used include those described in reports by Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).
Host cells include microbial cells, insect cells or mammals that are prokaryotes (eg, Escherichia, Bacillus, Corynebacterium, Staphylococcus, Streptomyces) or eukaryotes (eg, Saccharomyces, Kluyveromyces, Aspergillus). An animal cell etc. can be mentioned. For example, Escherichia coli and the like can be preferably mentioned from the viewpoint that mass preparation of the present transformant becomes easy.
As a method for introducing a plasmid into which the present reductase and / or the present coenzyme regenerating enzyme can be expressed in a host cell, any method commonly used depending on the host cell to be used may be used. ^{"Molecular Cloning: a Laboratory Manual 2 nd} edition " (1989), Cold Spring Harbor Laboratory Press, "Current Protocols in Molecular Biology" (1987), John Wiley & Sons , is described in Inc. ISBNO-471-50338-X, etc. And the electroporation method described in “Methods in Electroporation: Gene Pulser / E. Coli Pulser System” Bio-Rad Laboratories, (1993) and the like.
In order to select a transformant introduced with a plasmid in which the present reductase gene and / or the present coenzyme regenerating enzyme gene can be expressed in the host cell, as described above, for example, the selection included in the vector Selection may be performed using the phenotype of the marker gene as an index.
Host cells plasmids were introduced (i.e., transformants) that owns the reductase gene and the coenzyme regenerating enzyme gene, for example, ^{"Molecular Cloning: A Laboratory Manual 2 nd} edition " (1989) In accordance with the usual methods described in Cold Spring Harbor Laboratory Press, etc., confirmation can be performed by confirming restriction enzyme sites, analyzing base sequences, Southern hybridization, Western hybridization, and the like.

The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing a suitable carbon source, nitrogen source and trace nutrients such as vitamins. The culture method may be any of solid culture, test tube shake culture, reciprocating shake culture, jar fermenter culture, liquid culture such as tank culture, and preferably aeration and agitation culture method. A liquid culture can be mentioned.
The culture temperature can be appropriately changed within a range in which the present transformant can grow, but is usually about 10 to 50 ° C, preferably about 20 to 40 ° C. The pH of the medium is preferably in the range of about 6-8. The culture time varies depending on the culture conditions, but is usually preferably about 1 day to about 5 days.
As a medium for culturing the transformant, for example, various media appropriately containing a carbon source and a nitrogen source, organic salts, inorganic salts and the like that are usually used for culturing host cells such as microorganisms can be used. .
Examples of the carbon source include sugars such as glucose, dextrin and sucrose, sugar alcohols such as glycerol, organic acids such as fumaric acid, citric acid and pyruvic acid, animal oils, vegetable oils and molasses. The amount of these carbon sources added to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.
Nitrogen sources include, for example, natural organic nitrogen sources such as meat extract, peptone, yeast extract, malt extract, soy flour, Corn Steep Liquor, cottonseed flour, dry yeast, casamino acid, and amino acids And ammonium salts of inorganic acids such as sodium nitrate, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, and urea. Of these, ammonium salts of organic acids, natural organic nitrogen sources, amino acids and the like can be used as carbon sources in many cases. The amount of these nitrogen sources added to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.
Examples of the organic salt and inorganic salt include chlorides such as potassium, sodium, magnesium, iron, manganese, cobalt, and zinc, sulfates, acetates, carbonates, and phosphates. Specifically, for example, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, monopotassium hydrogen phosphate and dipotassium hydrogen phosphate Is mentioned. The amount of these organic salts and / or inorganic salts added to the medium is usually about 0.0001 to 5% (w / v) with respect to the culture solution.
In addition, DNA is introduced in which allolactose-inducible promoters such as tac promoter, trc promoter, and lac promoter are functionally connected to a gene having a base sequence encoding the amino acid sequence of this enzyme. In the case of the thus obtained transformant, for example, isopropyl thio-β-D-galactoside (IPTG) can be added to the medium in a small amount as an inducer for inducing production of the enzyme.

  The transformant can be obtained, for example, by collecting the transformant as a precipitate by centrifuging the culture obtained by the above culture. As needed, you may wash | clean the said transformant before collection | recovery using buffer solutions, such as a 100 mM phosphate 1 potassium-phosphate 2 potassium buffer (pH 6.5) etc., for example.

Furthermore, the dead cells can be prepared from the transformant by the following method.
Examples of killing treatment methods include physical sterilization methods (heating, drying, freezing, light, ultrasonic waves, filtration, energization), and sterilization methods using chemicals (alkali, acid, halogen, oxidizing agent, sulfur, Boron, arsenic, metal, alcohol, phenol, amine, sulfide, ether, aldehyde, ketone, cyan and antibiotics). Of these sterilization methods, a treatment method which does not deactivate the enzyme activity of the present enzyme as much as possible and has little influence on the reaction system, such as residue and contamination, may be appropriately selected according to various reaction conditions.

  The transformant prepared as described above or the killed cell thereof can be used, for example, in the form of freeze-dried cells, organic solvent-treated cells, dried cells, etc., or in a fixed form (immobilized product). Also good.

  Examples of a method for obtaining an immobilized product include a carrier binding method (a method of adsorbing the present transformant or its dead cells on an inorganic carrier such as silica gel or ceramic, cellulose, an ion exchange resin, etc.) and a comprehensive method (polyacrylamide). And a method of confining the present transformant or dead cells thereof in a polymer network such as a sulfur-containing polysaccharide gel (for example, carrageenan gel), alginate gel, or agar gel.

Next, the catalytic reaction in the production method of the present invention will be described.
In the production method of the present invention, the reaction for converting 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is 2-halo- This is achieved by allowing 1- (3,4-methylenedioxyphenyl) ethanone to act on the transformant or its killed cells.
The reaction is usually performed in the presence of water. Water may be in the form of a buffer solution. Examples of the buffer used in this case include alkali metal phosphates such as sodium phosphate and potassium phosphate, and alkali metals of acetic acid such as sodium acetate and potassium acetate. Salt.
In addition, when using a buffer solution as a solvent, the amount is usually 1 to 300 times by weight, preferably 5 to 100 times with respect to 1 part by weight of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone. Weight times.
In the reaction, 2-halo-1- (3,4-methylenedioxyphenyl) ethanone may be continuously or sequentially added to the reaction system.

The reaction temperature can be about 0 to 70 ° C., preferably about 10 to 40 ° C. from the viewpoint of the stability and reaction rate of the enzyme contained in the transformant or its killed cells. It is done.
The reaction pH can be appropriately changed within the range in which the reaction proceeds, and examples thereof include 5 to 8.

The reaction can be carried out in the presence of an organic solvent in addition to water. Examples of the organic solvent in this case include ethers such as tetrahydrofuran, t-butyl methyl ether and isopropyl ether, hydrocarbons such as toluene, hexane, cyclohexane, heptane, isooctane and decane, t-butanol, methanol, ethanol, Examples thereof include alcohols such as isopropanol and n-butanol, sulfoxides such as dimethyl sulfoxide, ketones such as acetone, nitriles such as acetonitrile, and mixtures thereof.
The amount of the organic solvent used for the reaction is usually 100 times or less, preferably 70 times or less, relative to 2-halo-1- (3,4-methylenedioxyphenyl) ethanone.

The reaction is usually preferably performed by adding a coenzyme such as NADH or NADPH.
The amount of coenzyme used in the reaction is usually 0.5 times or less, preferably 0.1 times or less, relative to 2-halo-1- (3,4-methylenedioxyphenyl) ethanone. .

In the catalytic reaction in the production method of the present invention, a stoichiometric amount of reduced coenzyme (electron donor) is consumed in the asymmetric reduction reaction of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone. The ability to convert the resulting oxidized coenzyme (electron acceptor) back into reduced coenzyme (electron donor), ie 2-halo-1- (3,4-methylenedioxyphenyl) ethanone An enzyme having the ability to regenerate the coenzyme on which the enzyme having the ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is regenerated. Use of enzyme regenerating enzyme is essential. In this case, the present coenzyme regenerating enzyme may be an enzyme different from the present reductase that performs the asymmetric reduction reaction, and the present reductase also has a function as a coenzyme regenerating enzyme. It may be a thing. Of course, a combination of both may be used.
Here, “the reductase also has a function as a coenzyme regenerating enzyme” means that, for example, the isolated reductase is used in the presence of an oxidized coenzyme (electron acceptor). It may be confirmed by examining whether or not a reduced coenzyme (electron donor) is produced by carrying out a reaction that oxidizes the regenerative raw material compound that is a substrate of the coenzyme regenerating enzyme.
Examples of the coenzyme regenerating enzyme include glucose dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, amino acid dehydrogenase, and organic dehydrogenase (malate dehydrogenase, etc.). Among these, a coenzyme regenerating enzyme that regenerates coenzyme by oxidizing glucose is preferable. The amount of glucose used in this case is 100 mol times or less, preferably 10 mol times or less, relative to 2-halo-1- (3,4-methylenedioxyphenyl) ethanone.

  In the reaction, for example, water, 2-halo-1- (3,4-methylenedioxyphenyl) ethanone, the present transformant or its killed cell, and a coenzyme, an organic solvent, etc. are mixed as necessary. , Stirring and shaking.

  The end point of the reaction can be determined, for example, by following the abundance of the raw material compound in the reaction solution by liquid chromatography, gas chromatography, or the like. The range of the reaction time is usually 5 minutes to 10 days, preferably 30 minutes to 4 days.

  After completion of the reaction, the target product may be collected by a compound recovery method usually used in a method for producing a compound using an enzyme as a catalyst. For example, first, the reaction solution is extracted with an organic solvent such as hexane, heptane, tert-butyl methyl ether, ethyl acetate, toluene and the like. The extraction operation may be performed after filtering the reaction solution as necessary or removing insolubles by a treatment such as centrifugation. Next, after the extracted organic layer is dried, the target product can be recovered as a concentrate. The desired product can be further purified by column chromatography or the like, if necessary.

  Hereinafter, the present invention will be described in more detail with reference to production examples and the like, but the present invention is not limited to these examples.

Example 1 (Preparation of the present reductase gene)
(1-1) Preparation of cDNA library 100 ml of a medium (potato dextrose broth (Becton Dickinson) dissolved in water at a rate of 24 g / L) was placed in a 500 ml flask and sterilized at 121 ° C. for 15 minutes. did. To this, 0.5 ml of a culture solution of Penicillium citrinum IFO4631 strain cultured in a medium having the same composition (30 ° C., 48 hours, shaking culture) was added, followed by shaking culture at 30 ° C. for 72 hours. Thereafter, the obtained culture broth was centrifuged (8000 × g, 10 minutes), and the resulting precipitate was collected. This precipitate was washed 3 times with 50 ml of 20 mM potassium phosphate buffer (pH 7.0) to obtain about 1.0 g of washed cells.
Total RNA was prepared by the guanidine thiocyanate phenol chloroform method using washed cells of Penicillium citrinum IFO4631 strain. From the prepared total RNA, RNA having poly (A) was obtained using Oligotex (dT) 30-Super (Takara Shuzo).
A cDNA library was prepared based on the Gubler and Hoffman method. First, RNA having poly (A) obtained as described above, Oligo (dT) 18-linker primer ((XhoI site) manufactured by Takara Shuzo Co., Ltd.), RAV-2 Rtase and SuperScriptII Rtase are used to make a single strand. cDNA was prepared. By adding E. coli DNA polymerase, E. coli Rnase / E. Coli DNA Ligase Mixture and T4 DNA Polymerase to the prepared single-stranded cDNA (including the above reaction solution), double-stranded cDNA synthesis and The blunt end treatment of the double-stranded cDNA was performed.
Ligation of the double-stranded cDNA thus obtained and an EcoRI-NotI-BamHI adapter (Takara Shuzo Co., Ltd.) was performed. The DNA obtained after ligation was ligated with λZapII (EcoRI-XhoI cleavage) in the following order: phosphorylation treatment, cleavage treatment with XhoI, removal of low molecular weight DNA using a spin column (Takara Shuzo) Thereafter, a cDNA library (hereinafter sometimes referred to as cDNA library (A)) was prepared by packaging using an in vitro packaging kit (manufactured by STRATAGENE).

(1-2) Preparation of plasmid containing this reductase gene (construction of plasmid pTrcRPc)
Using the oligonucleotide having the base sequence shown in SEQ ID NO: 3 and the oligonucleotide shown in SEQ ID NO: 4 as primers, using the cDNA library prepared in (1-1) as a template, the following reaction solution composition, PCR was performed under the reaction conditions. (Using Expand High Fidelity PCR System from Roche Diagnostics)

[Reaction solution composition]
1 μl of cDNA library stock solution
dNTP (each 2.5 mM-mix) 0.4 μl
Primer (20 pmol/μl) 0.75μl each
10xbuffer (with MgCl ₂ ) 5μl
enz.expandHiFi (3.5 × 10 ³ U / ml) 0.375μl
Ultrapure water 41.725μl

[Reaction conditions]
A container containing the reaction solution having the above composition was set in the PERKIN ELMER-GeneAmp PCR System 2400, heated to 97 ° C. (2 minutes), then 97 ° C. (0.25 minutes) -55 ° C. (0.5 minutes) -72. Cycle of 10 ° C. (1.5 minutes), followed by 20 cycles of 97 ° C. (0.25 minutes) -55 ° C. (0.5 minutes) -72 ° C. (2.5 minutes). For 7 minutes.

Two kinds of restriction enzymes (NcoI and BamHI) were added to the PCR-amplified DNA fragment obtained by purifying the PCR reaction solution, whereby the DNA fragment was double-digested. The resulting DNA fragment was then purified.
On the other hand, the vector pTrc99A (manufactured by Pharmacia) was subjected to double digestion by adding two types of restriction enzymes (NcoI and BamHI). The digested DNA fragment was then purified.
Two kinds of DNA fragments obtained by purification in this way were mixed and ligated with T4 DNA ligase. E. coli DH5α was transformed with the obtained ligation solution. A plasmid containing the present reductase gene (hereinafter sometimes referred to as plasmid pTrcRPc) was taken out from the obtained transformant using QIAprep Spin Miniprep Kit (manufactured by Qiagen).

Example 2 (Preparation of the present coenzyme regenerating enzyme gene)
(2-1) Preparation for preparing a gene having a base sequence encoding an amino acid sequence of an enzyme having the ability to convert oxidized β-nicotinamide adenine dinucleotide or the like to a reduced form Into a flask, LB medium (1% tryptone , 0.5% yeast extract, 1% sodium chloride) was added and sterilized. The medium thus prepared was inoculated with 0.3 ml of a culture solution in which Bacillus megaterium IFO12108 strain was previously cultured in a liquid medium having the above composition, and this was cultured with shaking at 30 ° C. for 10 hours.
After culturing, the obtained culture was centrifuged (15000 × g, 15 minutes, 4 ° C.) to recover the cells. The collected cells are suspended in 30 ml of 50 mM monopotassium phosphate-dipotassium phosphate buffer (pH 7.0), and this suspension is centrifuged (15000 × g, 15 minutes, 4 ° C.). Washed cells were obtained.
Chromosomal DNA was purified from the washed cells thus obtained using Qiagen Genomic Tip (Qiagen) according to the method described in the manual attached thereto.

(2-2) Preparation of a gene having a base sequence encoding an amino acid sequence of an enzyme having the ability to convert oxidized β-nicotinamide adenine dinucleotide or the like into a reduced form (Part 1: Construction of plasmid pSDGDH12)
Based on the amino acid sequence of glucose dehydrogenase derived from the known Bacillus megaterium IWG3 described in The Journal of Biological Chemistry Vol. 264, No. 11, 6381-6385 (1989), it has the base sequence represented by SEQ ID NO: 5. An oligonucleotide and an oligonucleotide having the base sequence represented by SEQ ID NO: 6 are synthesized.
Example using the chromosomal DNA purified in (2-1) as a template, using the oligonucleotide having the base sequence shown by SEQ ID NO: 5 and the oligonucleotide having the base sequence shown by SEQ ID NO: 6 as primers PCR is performed with the reaction solution composition and reaction conditions described in 1 (1-2). (Using Expand High Fidelity PCR System from Roche Diagnostics)
The PCR amplified DNA fragment obtained by purifying the PCR reaction solution is ligated to the existing “PCR Product insertion site” of the pCR2.1-TOPO vector using TOPO ^™ TA cloning kit manufactured by Invitrogen. The resulting ligation solution is E. coli. Transform E. coli DH5α.
A plasmid containing glucose dehydrogenase (hereinafter sometimes referred to as plasmid pSDGDH12) is taken out from the resulting transformant using QIAprep Spin Miniprep Kit (manufactured by Qiagen).
Next, after the sequencing reaction using Dye Terminator Cycle sequencing FS ready Reaction Kit (manufactured by PerkinElmer) using the extracted plasmid pSDGDH12 as a template, the base sequence of the resulting DNA was converted with DNA sequencer 373A (manufactured by PerkinElmer). To analyze. The result is shown in SEQ ID NO: 7.

Example 3 (Preparation of plasmid containing the present reductase gene and the present coenzyme regenerating enzyme gene: construction of plasmid pTrcRSbG12)
By adding two types of restriction enzymes (BamHI and XbaI) to the plasmid pSDGDH12 prepared in Example 2 (2-2), the plasmid was double-digested. The digested DNA fragment was then purified.
On the other hand, by adding two types of restriction enzymes (BamHI and XbaI) to the plasmid pTrcRPc prepared in Example 1, the plasmid was double-digested. The digested DNA fragment was then purified.
Two kinds of DNA fragments obtained by purification in this way were mixed and ligated with T4 DNA ligase. The resulting ligation solution was used for E. coli. E. coli DH5α was transformed. A plasmid containing the present reductase gene and the present coenzyme regenerating enzyme gene (hereinafter sometimes referred to as plasmid pTrcRSbG12) was taken out from the obtained transformant using QIAprep Spin Miniprep Kit (manufactured by Qiagen).

Example 4 (Preparation of transformant containing the present reductase gene and the present coenzyme regenerating enzyme gene)
Using the pTrcRSbG12 plasmid prepared in Example 3, E. coli HB101 was transformed. The obtained transformant was inoculated into a sterile LB medium (100 ml × 3) containing 0.1 mM IPTG and 50 μg / ml ampicillin, and then cultured with shaking (30 ° C., 18 hours). After culturing, the culture broth was centrifuged and washed to recover 1.2 g of washed cells.

Example 5 (Method for producing 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol)
To 2 ml of 100 mM potassium phosphate-potassium phosphate buffer (pH 6.5), 0.5 g of washed cells prepared in Example 4, NAD ⁺ 2.4 mg, NADP ⁺ 2.4 mg, glucose 0.1 g and Glucose dehydrogenase (Amano Enzyme) 36U was added. To this mixture was added an additional 20 mg of 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanone. The mixture (reaction solution) thus obtained was reacted by stirring at 30 ° C. for 15 hours. After completion of the reaction, 5 ml of t-butyl methyl ether was added to the reaction solution, stirred, and then centrifuged to collect the organic layer and the aqueous layer separately. The organic layer was subjected to content analysis by liquid chromatography and optical purity analysis under the following conditions. 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol is 4.8% based on the amount of 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanone used in the reaction. It was generated. Moreover, as a result of measuring the optical purity of 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol in the organic layer under the following conditions, the (S) form was 99% e.e. e. Met. The steric configuration of the obtained 1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol was determined by liquid chromatography (LC) analysis under the following analysis conditions in the 1- and S- and R-forms. Determined by retention time comparison with (3,4-methylenedioxyphenyl) -2-chloro-ethanol. For the absolute configuration, reference was made to data of a prior document (Eur. J. Org. Chem. (2000), 3313-3321). Further, the organic layer is concentrated to obtain crude (S) -1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol.
(Content analysis conditions)
Column: SUMPAX ODS A-212 (5 μm, 6 mmΦ × 15 cm)
Mobile phase: Liquid A Water, Liquid B Acetonitrile time (min) Liquid A (%): Liquid B (%)
0 80:20
30 30:70
40 30:70
40.1 80:20
Flow rate: 0.5 ml / min Column temperature: 30 ° C
Detection: 254 nm

(Optical isomer analysis conditions)
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane / 2-propanol / trifluoroacetic acid = 98/2 / 0.1
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 254 nm
(S) -1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol: 54.5 minutes (R) -1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol: 76.7 minutes

Reference Example 1 Method for Acquiring Microorganisms that Generate 2-Halo-1- (3,4-Methylenedioxyphenyl) ethanol (1-1) Preparation of Washed Bacteria Sterilized microorganisms isolated from commercially available microorganisms or soil After inoculating LB medium (10 ml), this is cultured with shaking (30 ° C., 18 hours). After culturing, the washed cells are collected by centrifuging and washing the culture solution.
(1-2) Screening 1 ml of washed cells prepared in the above (1-1), NADP ⁺ 12 mg, NAD ⁺ 12 mg, glucose 2 in 20 ml of 100 mM monopotassium phosphate-dipotassium phosphate buffer (pH 6.5) Add 5 g and 150 U of glucose dehydrogenase. After adding an additional 240 mg of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to the mixture, the pH of the mixture is adjusted to 6.5 with 15% aqueous sodium carbonate. The reaction is carried out by stirring the mixture (reaction solution) thus obtained at 30 ° C. for 4 hours. After completion of the reaction, 25 ml of t-butyl methyl ether is poured into the reaction solution and stirred, and then the organic layer and the aqueous layer are collected separately by centrifugation. The same operation is performed by adding 25 ml of t-butyl methyl ether to the recovered aqueous layer. The organic layers thus obtained are concentrated together, and then dissolved in 30 ml of chloroform and dried using anhydrous Na ₂ SO ₄ . After drying, the residue is obtained by distilling off chloroform. Qualitative and / or quantitative analysis (optical purity analysis is also possible) by liquid chromatography or gas chromatography that the resulting residue contains 2-halo-1- (3,4-methylenedioxyphenyl) ethanol Confirm by. Incidentally, the absolute configuration of 2-halo-1- (3,4-methylenedioxyphenyl) ethanol was referred to the data of a prior document (Eur. J. Org. Chem. (2000), 3313-3321). Since it can be determined with reference to the data of the prior literature (Eur. J. Org. Chem. (2000), 3313-3321), optically active 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is produced. Microorganisms to be selected can also be selected. The analysis conditions in liquid chromatography are described below.

<Chemical purity analysis conditions>
Column: CHIRALPAK OD-H (manufactured by Daicel Chemical Industries)
Mobile phase: hexane / 2-propanol / trifluoroacetic acid = 98/2 / 0.1
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 254 nm
(S) -1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol: 54.5 minutes (R) -1- (3,4-methylenedioxyphenyl) -2-chloro-ethanol: 76.7 minutes

  According to the present invention, a 2-halo-1- (3,4-methylenedioxyphenyl) ethanol compound useful as an intermediate for producing a physiologically active substance can be produced.

SEQ ID NO: 3
Oligonucleotide SEQ ID NO: 4 which is a primer designed for PCR
Oligonucleotide SEQ ID NO: 5 which is a primer designed for PCR
Oligonucleotide SEQ ID NO: 6 which is a primer designed for PCR
Oligonucleotides are primers designed for PCR

Claims (9)

A process for producing 2-halo-1- (3,4-methylenedioxyphenyl) ethanol, wherein 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is asymmetrically reduced to produce 2- Transformant artificially provided with the ability to produce halo-1- (3,4-methylenedioxyphenyl) ethanol and the ability to regenerate the coenzyme on which the enzyme having the ability depends, or killing the transformant A step of allowing 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to act on cells, and a step of collecting the produced 2-halo-1- (3,4-methylenedioxyphenyl) ethanol The manufacturing method characterized by having. The transformant is
One plasmid containing DNA consisting of a base sequence encoding the amino acid sequence of an enzyme having the following two artificially conferred abilities simultaneously:
DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and base sequence encoding the amino acid sequence of the enzyme having the ability to be artificially imparted (ii) below One plasmid having the DNA consisting of
A plasmid containing a DNA comprising a base sequence encoding the amino acid sequence of an enzyme having the ability to be artificially imparted (i) below and the amino acid sequence of the enzyme having the ability to artificially grant (ii) below Two plasmids comprising a plasmid having a DNA comprising a coding base sequence,
The production method according to claim 1, wherein at least a transformant is introduced.
<Artificially granted ability>
(I) Ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol ( ii) Ability to regenerate the coenzyme on which the enzyme having the ability of (i) depends. The method according to claim 1 or 2, wherein the transformant is Escherichia coli. The production method according to claim 1, wherein the coenzyme is NADH / NAD ⁺ (nicotinamide adenine dinucleotide) or NADPH / NADP ⁺ (nicotinamide adenine dinucleotide phosphate). The production method according to claim 1, wherein 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is allowed to act on a transformant or a killed cell thereof in the presence of glucose. The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is 6. The production method according to claim 1, wherein the enzyme has an amino acid sequence selected from the amino acid sequence group.
<Amino acid sequence group>
(A) an amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence in which one or a plurality of amino acid sequences are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and 2-halo- Amino acid sequence of an enzyme having the ability to asymmetrically reduce 1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol (c ) Having the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 2 (d) having the amino acid sequence encoded by the base sequence of DNA that hybridizes under stringent conditions with the DNA comprising the base sequence represented by SEQ ID NO: 2 And 2-halo-1- (3,4-methylenedioxyphenyl) ethanone is asymmetrically reduced to give 2-halo-1- (3,4-methylenedioxy). Amino acid sequence of an enzyme having the ability to produce (phenyl) ethanol (e) 2-halo-1- (3,4-methylenedioxyphenyl) ethanone derived from a microorganism belonging to the genus Penicillium asymmetrically reduced to 2 -Amino acid sequence of an enzyme having the ability to produce halo-1- (3,4-methylenedioxyphenyl) ethanol The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is SEQ ID NO: The production method according to claim 1, wherein the enzyme has the amino acid sequence represented by 1. The ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol is SEQ ID NO: The production method according to claim 1, wherein the enzyme has an amino acid sequence encoded by the base sequence represented by 2. As a catalyst for the asymmetric reduction of 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol Ability to asymmetrically reduce 2-halo-1- (3,4-methylenedioxyphenyl) ethanone to produce 2-halo-1- (3,4-methylenedioxyphenyl) ethanol and the ability Use of a transformant artificially imparted with an ability to regenerate a coenzyme on which an enzyme having a phenotype depends, or a dead cell thereof.