JPWO2005005648A1 - Process for producing novel optically active carboxylic acids - Google Patents

Abstract

A 4-halocrotonic acid derivative represented by the following general formula (I) is allowed to act on enoate reductase or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, and the following general An optically active 4-halobutyric acid derivative represented by the formula (II) is produced (in the formulas (I) and (II), R represents a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, a substituted group. An optionally substituted aryl group or an optionally substituted alkoxy group, X represents a halogen atom, and A1 and A2 represent a hydrogen atom or a halogen atom.

Description

  The present invention stereoselectively bonds the carbon-carbon double bond of a 4-halocrotonic acid derivative using enoate reductase or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell. The present invention relates to a method for producing an optically active 4-halobutyric acid derivative, which is a compound useful as an intermediate material for pharmaceuticals, agricultural chemicals and the like by reduction. The present invention also provides (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o) utilizing the method for producing the 4-halobutyric acid derivative. -Tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5-carboxamide.

  Optically active 4-halobutyric acid derivatives are industrially useful compounds as intermediate materials for pharmaceuticals, agricultural chemicals and the like. For example, (R) -4,4,4-trifluoro-2-methylbutyric acid (2R-methyl-4,4,4-trifluorobutanoic acid) is a useful (R) -4,4 as a leukotriene antagonist. , 4-trifluoro-2-methylbutylamine is known to be a synthetic intermediate (European Patent Application No. 489548). As a method for obtaining an optically active 4-halobutyric acid derivative, there has been known a method of reducing a 4-halocrotonic acid derivative using a complex catalyst comprising an optically active phosphine compound and a ruthenium compound (European Patent Application No. 673911). book). However, this reaction needs to be performed under high pressure, and the catalyst used is expensive.

  Enoate reductase is an enzyme that catalyzes a reaction that reduces the carbon-carbon double bond of enoate, but its presence has been reported in microorganisms such as Clostridia (Journal of Biological Chemistry). , Vol. 276, No. 8, p5779-5787, 2001). Asymmetric reduction of various enoates using microorganisms having enoate reductase activity has been reported (Studies in Natural Products Chemistry, vol. 20, 817, 1998), but the 4-position carbon of enoate is halogenated. Regarding 4-halocrotonic acid derivatives, there was no report of asymmetric reduction using enoate reductase.

  An object of the present invention is to provide a novel method for producing an optically active 4-halobutyric acid derivative with higher optical purity at low cost and in a simple manner. The present invention also provides (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole- It is an object to provide a novel method for producing 5-carboxamide.

  In order to solve the above-mentioned problems, the present inventors diligently studied a method for producing an optically active 4-halobutyric acid derivative. It has been found that it catalyzes an asymmetric reduction reaction of a 4-halocrotonic acid derivative. Furthermore, a transformed cell into which a gene encoding the protein is introduced is prepared, and the culture solution obtained by culturing the cell, the preparation of the cell, or the cell is converted into a 4-halocrotonic acid derivative as a raw material. It was found that the optically active 4-halobutyric acid derivative, which is the target product, can be obtained with high optical purity and high concentration by acting. Furthermore, by using (R) -4,4,4-trifluoro-2-methylbutyric acid obtained by this method, (R) -N- (4,4,4-trifluoro-2-methylbutyl)- It was found that 3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5-carboxamide can be efficiently produced. Thus, the present invention has been completed.

で表される４−ハロクロトン酸誘導体に、エノエートレダクターゼまたはそれを含む細胞、同細胞の調製物もしくは同細胞を培養して得られた培養液を作用させて、下記一般式（ＩＩ）

で表される光学活性４−ハロ酪酸誘導体を生成させることを特徴とする、一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体の製造方法（式（Ｉ）、（ＩＩ）中、Ｒはハロゲン原子、ニトロ基、ヒドロキシル基、置換されていてもよいアルキル基、置換されていてもよいアリール基または置換されていてもよいアルコキシ基を、Ｘはハロゲン原子を、Ａ_１およびＡ_２は水素原子またはハロゲン原子を示す）。
（２）エノエートレダクターゼが以下の（Ａ）、（Ｂ）または（Ｃ）に示すタンパク質である、（１）の製造方法：
（Ａ）配列番号２または配列番号４に記載のアミノ酸配列を有するタンパク質、または
（Ｂ）配列番号２または配列番号４に記載のアミノ酸配列と５０％以上の相同性を有するアミノ酸配列からなり、かつ、一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質。
（Ｃ）配列番号２または配列番号４に記載のアミノ酸配列において、１または数個のアミノ酸が置換、欠失もしくは付加されたアミノ酸配列からなり、かつ、一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質。
（３）エノエートレダクターゼを含む細胞が、エノエートレダクターゼをコードするＤＮＡで形質転換された細胞である、（１）の製造方法。
（４）エノエートレダクターゼをコードするＤＮＡで形質転換された細胞が、前記ＤＮＡを染色体上に組み込むことによって形質転換された細胞である、（３）の製造方法。
（５）エノエートレダクターゼをコードするＤＮＡで形質転換された細胞が、前記ＤＮＡを含むベクターで形質転換された細胞である、（３）の製造方法。
（６）エノエートレダクターゼをコードするＤＮＡが以下の（Ａ）、（Ｂ）または（Ｃ）に示すタンパク質をコードするＤＮＡである、（３）〜（５）のいずれかの製造方法：
（Ａ）配列番号２または配列番号４に記載のアミノ酸配列を有するタンパク質、または
（Ｂ）配列番号２または配列番号４に記載のアミノ酸配列と５０％以上の相同性を有するアミノ酸配列からなり、かつ、一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質。
（Ｃ）配列番号２または配列番号４に記載のアミノ酸配列において、１または数個のアミノ酸が置換、欠失もしくは付加されたアミノ酸配列からなり、かつ、一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質。
（７）エノエートレダクターゼをコードするＤＮＡが以下の（Ｄ）、（Ｅ）または（Ｆ）に示すＤＮＡである、（３）〜（５）のいずれかの製造方法：
（Ｄ）配列番号１または配列番号３に記載の塩基配列を有するＤＮＡ、または
（Ｅ）配列番号１または配列番号３に記載の塩基配列またはその相補配列とストリンジェントな条件でハイブリダイズする塩基配列を有し、一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質をコードするＤＮＡ。
（Ｆ）配列番号１または配列番号３に記載の塩基配列において１または数個の塩基が置換、欠失もしくは付加された塩基配列及びその相補鎖からなり、かつ一般式（Ｉ）で表される４−ハロクロトン酸誘導体を一般式（ＩＩ）で表される光学活性４−ハロ酪酸誘導体に変換する酵素活性を有するタンパク質をコードするＤＮＡ。
（８）一般式（Ｉ）および（ＩＩ）の化合物において、Ｒがメチル基であり、Ｘ、Ａ_１およびＡ_２がそれぞれフッ素原子であることを特徴とする、（１）〜（７）のいずれかの製造方法。
（９）（８）の方法により一般式（ＩＩＩ）で表される４，４，４−トリフルオロチグリン酸を一般式（ＩＩ）で表される（Ｒ）−４，４，４−トリフルオロ−２−メチル酪酸に変換する工程、前記（Ｒ）−４，４，４−トリフルオロ−２−メチル酪酸を、一般式（Ｖ）で表される（Ｒ）−４，４，４−トリフルオロ−２−メチルブチルアミンに変換する工程、及び前記（Ｒ）−４，４，４−トリフルオロ−２−メチルブチルアミンに一般式（ＩＩ）で表される化合物を反応させて、一般式（ＶＩＩ）で表される（Ｒ）−Ｎ−（４，４，４−トリフルオロ−２−メチルブチル）−３−［２−メトキシ−４−（ｏ−トリルスルホニルカルバモイル）ベンジル］−１−メチルインドール−５−カルボキサミドを生成させる工程を含む、（Ｒ）−Ｎ−（４，４，４−トリフルオロ−２−メチルブチル）−３−［２−メトキシ−４−（ｏ−トリルスルホニルカルバモイル）ベンジル］−１−メチルインドール−５−カルボキサミドの製造方法．

That is, the present invention is as follows.
(1) The following general formula (I)

A haloate reductase or a cell containing the same, a preparation of the same cell, or a culture solution obtained by culturing the same cell is allowed to act on the 4-halocrotonic acid derivative represented by the following general formula (II):

A method for producing an optically active 4-halobutyric acid derivative represented by the general formula (II) (formula (I), (II), R represents a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkoxy group, X represents a halogen atom, A ₁ and A ₂ Represents a hydrogen atom or a halogen atom).
(2) The production method of (1), wherein the enoate reductase is a protein shown in the following (A), (B) or (C):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or (B) an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and A protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(C) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added, and represented by the general formula (I) 4- A protein having an enzyme activity for converting a halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(3) The production method of (1), wherein the cell containing enoate reductase is a cell transformed with DNA encoding enoate reductase.
(4) The production method of (3), wherein the cell transformed with DNA encoding enoate reductase is a cell transformed by integrating the DNA onto a chromosome.
(5) The production method of (3), wherein the cell transformed with DNA encoding enoate reductase is a cell transformed with a vector containing the DNA.
(6) The production method of any one of (3) to (5), wherein the DNA encoding enoate reductase is a DNA encoding a protein shown in the following (A), (B), or (C):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or (B) an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and A protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(C) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added, and represented by the general formula (I) 4- A protein having an enzyme activity for converting a halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(7) The production method of any of (3) to (5), wherein the DNA encoding enoate reductase is the DNA shown in the following (D), (E) or (F):
(D) DNA having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or (E) a base sequence that hybridizes with the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary sequence thereof under stringent conditions And a DNA encoding a protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(F) The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 consists of a nucleotide sequence in which one or several bases are substituted, deleted or added and its complementary strand, and is represented by the general formula (I) A DNA encoding a protein having an enzyme activity for converting a 4-halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(8) In the compounds of the general formulas (I) and (II), R is a methyl group, X, A ₁ and A ₂ are each a fluorine atom, (1) to (7) Either manufacturing method.
(9) The 4,4,4-trifluorotiglinic acid represented by the general formula (III) is converted to the (R) -4,4,4-trifluoro represented by the general formula (II) by the method of (8). The step of converting to 2-methylbutyric acid, the (R) -4,4,4-trifluoro-2-methylbutyric acid is represented by the general formula (V) (R) -4,4,4-tri A step of converting to fluoro-2-methylbutylamine, and reacting the compound represented by the general formula (II) with the (R) -4,4,4-trifluoro-2-methylbutylamine to give a general formula (VII (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole- (R) -N comprising the step of producing 5-carboxamide (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4-(o-tolyl sulfonylcarbamoyl) benzyl] -1-production method of methyl indole-5-carboxamide.

で表される４−ハロクロトン酸誘導体に作用させることにより、該化合物の炭素・炭素二重結合を不斉還元させ、下記一般式（ＩＩ）

で表される光学活性４−ハロ酪酸誘導体を製造する。The present invention is described in detail below.
<1> Method for producing optically active 4-halobutyric acid derivative In the present invention, enoate reductase or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell is a reaction substrate. The following general formula (I)

The carbon-carbon double bond of the compound is asymmetrically reduced by acting on a 4-halocrotonic acid derivative represented by the following general formula (II):

An optically active 4-halobutyric acid derivative represented by:

In the above general formulas (I) and (II), R represents a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted alkoxy group. , X represents a halogen atom, and A ₁ and A ₂ represent a hydrogen atom or a halogen atom.

  Here, examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tertiary butyl, n-pentyl, isopentyl, and neopentyl. A straight chain, branched chain or cyclic alkyl group having 1 to 8 carbon atoms such as a group, tertiary pentyl group, isoamyl group or n-hexyl group. In these, a C1-C4 alkyl group is preferable and a methyl group or an ethyl group is especially preferable. Examples of the aryl group include a phenyl group, a mesityl group, and a naphthyl group. Examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, and a tertiary butoxy group. Among these, an alkoxy group having 1 to 4 carbon atoms is preferable.

  The alkyl group, aryl group and alkoxy group may be substituted. The substituent is not particularly limited as long as it does not adversely affect the asymmetric reduction reaction. Specifically, an alkyl group, an aryl group, an alkoxy group, a halogen group, a cyano group, an amino group, a nitro group, and A hydroxyl group etc. are mentioned. Accordingly, in the general formulas (I) and (II), as the substituted alkyl group, specifically, benzyl group, phenethyl group, trifluoromethyl group, cyanomethyl group, aminomethyl group, hydroxymethyl group, nitromethyl group, methoxy A methyl group etc. are mentioned. Specific examples of the substituted aryl group include a chlorophenyl group, an aminophenyl group, a hydroxyphenyl group, a nitrophenyl group, and a methoxyphenyl group. Specific examples of the substituted alkoxy group include a benzyloxy group, a phenoxy group, and a trifluoromethoxy group.

X represents a halogen atom, and A ₁ and A ₂ represent a hydrogen atom or a halogen atom, and these halogen atoms are preferably a fluorine atom, a chlorine atom or a bromine atom, and more preferably a fluorine atom. It is particularly preferable that all of X, A ₁ and A ₂ are fluorine atoms.

  R in the general formulas (I) and (II) is preferably an alkyl group having 1 to 4 carbon atoms, a benzyl group or a phenyl group, more preferably an alkyl group having 1 to 4 carbon atoms, A methyl group is preferred.

The compound represented by the general formula (I) has a molecular weight of 1000 or less, preferably 500 or less, more preferably 300 or less. Specifically, for example, 4 represented by the general formula (III) 4,4-trifluorotiglic acid and the like. The compound represented by the above general formula (I) is, for example, an aldehyde represented by the following general formula (VIII) (X, A ₁ and A ₂ represent the same substituents as described above) and the fourth edition experimental chemistry It can be easily synthesized by conducting a Wittig reaction with a desired stabilized ylide by the method described in the lecture (19, p62, 1992) and hydrolyzing the resulting unsaturated ester.

Moreover, as a compound represented by the said general formula (II), molecular weight is 1000 or less, Preferably it is 500 or less, More preferably, it is a 300 or less thing, Specifically, (R)-of general formula (IV)- Examples include 4,4,4-trifluoro-2-methylbutyric acid.

  Enoate reductase generally refers to an enzyme that catalyzes the reduction reaction of a carbon-carbon double bond of enoate (Studies in Natural Products Chemistry, vol. 20, p817, 1998). In the present invention, 4- A protein capable of asymmetric reduction of the carbon-carbon double bond of a halocrotonic acid derivative to produce an optically active 4-halobutyric acid derivative. Here, the optical purity of the produced 4-halobutyric acid derivative is 60% e.e. e. Or more, preferably 90% e.e. e. More preferably, it is 98% e.e. e. More preferably, it is 99.5% e.e. e. It is particularly preferred that Such an activity can be measured by measuring the initial decrease rate of NADH in a reaction system containing a 4-halocrotonic acid derivative as a substrate and further containing NADH as a coenzyme.

  The enoate reductase that can be used in the production method of the present invention is not particularly limited as long as it has the above activity, and examples thereof include those having the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4. These are enoate reductases derived from Moorella thermoautotropica and Clostridium acetobutylicum, respectively.

  In the present invention, those homologs having the above enzyme activity may be used. Examples of homologs include those having an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 within a range that does not impair the activity. be able to. Here, the term “several” specifically means 20 or less, preferably 10 or less, more preferably 5 or less.

  The homolog may be a protein having a homology of 35% or more, preferably 50% or more, more preferably 80%, particularly preferably 95% or more with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Good. Incidentally, the homology search of the above protein can be performed using programs such as FASTA and BLAST, for example, for GenBank and DNA Database of JAPAN (DDBJ). Using the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and performing a homology search with BLAST program for GenBank, the sequence of SEQ ID NO: 2 is derived from Clostridium tyrolticum-derived 2-enoate reduce (Accession No. CAA71086) It showed 59% homology, and the sequence of SEQ ID NO: 4 showed 49% homology with Clostridium tyrobutyricum-derived 2-enoate reductase (Accession No. CAA71086). Further, the sequences of SEQ ID NO: 2 and SEQ ID NO: 4 showed 50% homology with each other. In this specification, Accession No. Indicates that of GenBank.

  The enoate reductase used in the production method of the present invention is an enoate from any microorganism having enoate reductase activity using a probe prepared based on the base sequence of a gene encoding part or all of the enoate reductase. After the DNA encoding reductase is isolated, it can be obtained by expressing the DNA in a host such as E. coli. Moreover, it can also obtain by refine | purifying from the microorganisms which have enoate reductase activity, for example, the microbial cell body of Clostridium (Clostridium) genus bacteria and Moorella (Moorella) genus bacteria. As a method for obtaining enoate reductase from bacterial cells, for example, Eur. J. et al. Biochem. Vol. 97, p103 (1979).

  As a Clostridium bacterium, for example, Clostridium acetobutylicum ATCC 824 has enoate reductase that can be suitably used in the present invention. As a bacterium belonging to the genus Moorella, for example, Moellera thermoautotropica DSM1974 Has an enoate reductase that can be suitably used in the present invention. The former is described in the ATCC (American Type Culture Collection) online catalog (http://www.atcc.org/) and can be obtained from the ATCC. The latter is also available from the online catalog (http: //www.DSM) of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (available from German Collection of Microorganisms and Cell Cultures). is there.

  In the production method of the present invention, a 4-halocrotonic acid derivative may be reacted with a purified enzyme of enoate reductase, but it is obtained by culturing a cell containing enoate reductase, a preparation of the cell, or the cell. The culture medium may be reacted with a 4-halocrotonic acid derivative to produce an optically active 4-halobutyric acid derivative. The cell containing enoate reductase is preferably a cell transformed with DNA encoding enoate reductase.

  In this case, DNA encoding enoate reductase includes DNA encoding enoate reductase having the amino acid sequence of SEQ ID NO: 2 or 4. A 4-halocrotonic acid derivative having an amino acid sequence having 50% or more homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and represented by the general formula (I) is represented by the general formula (II): It may be a DNA encoding a protein having an enzymatic activity to be converted into the optically active 4-halobutyric acid derivative represented. Specific examples include DNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Such DNA can be obtained by PCR using primers designed based on the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In the production method of the present invention, a DNA homologue having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having enoate reductase activity may be used.

  Here, the homolog is a base in which one or several bases are deleted, substituted or added to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 as long as it encodes a protein having enoate reductase activity. Includes DNA consisting of sequence and prime complementary strand. Here, the term “several” means specifically 60 or less, preferably 30 or less, and more preferably 10 or less.

  In addition, the homolog may be DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof under stringent conditions and encodes a protein having enoate reductase activity. Here, “DNA that hybridizes under stringent conditions” means that a probe DNA is used to perform colony hybridization, plaque hybridization, Southern blot hybridization, or the like under stringent conditions. For example, in the colony hybridization method and the plaque hybridization method, a colony or plaque-derived DNA or a filter on which the DNA fragment is immobilized is used as the “stringent conditions”. After hybridization at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride, 0.1 to 2 × SSC solution (1 × SSC composition is 150 mM sodium chloride, 15 mM sodium citrate) The conditions for washing the filter at 65 ° C. can be mentioned.

  DNA encoding enoate reductase can be isolated by, for example, the following method. First, enoate reductase is purified from microbial cells by the above method and the like, and then the N-terminal amino acid sequence is analyzed. N-terminal amino acid sequence analysis is performed by cleaving a purified protein with an enzyme such as lysyl endopeptidase or V8 protease, purifying a peptide fragment by reverse phase liquid chromatography, etc., and then analyzing the amino acid sequence with a protein sequencer. It is done by deciding. Using a primer designed based on the determined amino acid sequence and performing PCR using a chromosomal DNA or cDNA library of an enoate reductase-producing microbial strain as a template, a part of the DNA encoding enoate reductase (DNA fragment) is obtained. Obtainable. Furthermore, the above-mentioned DNA fragment is used as a probe from a library or cDNA library obtained by introducing a restriction enzyme digest of chromosomal DNA of an enoate reductase-producing microorganism strain into a phage or plasmid, and transforming E. coli. By performing colony hybridization, plaque hybridization and the like, DNA encoding enoate reductase can be obtained.

  In addition, the base sequence of the DNA fragment obtained by the above PCR is analyzed, and from the obtained sequence, a PCR primer for extending outside the sequence-determined region is designed, and the enoate reductase-producing microorganism strain Enzyme reductase is encoded by digesting chromosomal DNA with an appropriate restriction enzyme and performing inves PCR (Genetics vol. 120, p621-623 (1988)) using the DNA cyclized by self-cyclization as a template. It is also possible to obtain DNA.

  The DNA encoding enoate reductase can also be obtained by chemically synthesizing DNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

  Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA of SEQ ID NO: 1 or 3 (Nucleic Acid Res. Vol. 10, p6487 (1982), Methods in Enzymol. Vol. 100, p448 (1983)). , Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989), PCR: A Practical Approach, IRL Press, p200 (1991)), etc., as appropriate, to introduce substitution, deletion, insertion and / or addition mutations Thus, DNA encoding enoate reductase that can be used in the production method of the present invention can be obtained.

  Further, a homology search is performed on a database such as GenBank or DDBJ based on all or part of the amino acid sequence of SEQ ID NO: 2 or 4 and all or part of the base sequence of SEQ ID NO: 1 or 3. It is also possible to obtain the base sequence information of a DNA homolog encoding enoate reductase. A person skilled in the art can obtain DNA encoding enoate reductase by PCR or the like from the deposited strain (available from ATCC, DSMZ, etc.) based on this nucleotide sequence information.

  Furthermore, the DNA encoding enoate reductase is a colony of DNA prepared from an arbitrary microorganism having enoate reductase activity using DNA having all or part of the nucleotide sequence of SEQ ID NO: 1 or 3 as a probe. It can also be obtained by performing hybridization under stringent conditions by a hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like to obtain a hybridizing DNA. Here, “part” refers to DNA having a length sufficient to be used as a probe, specifically 15 bp or more, preferably 50 bp or more, more preferably 100 bp or more.

  Each hybridization was performed using Molecular Cloning 2nd Edt. , Cold Spring Harbor Laboratory Press (1989) and the like.

  An enoate reductase expression vector is provided by inserting the DNA encoding enoate reductase isolated as described above into a known expression vector so as to allow expression. By culturing cells transformed with the expression vector, enoate reductase can be obtained from the cells. A transformed cell can also be obtained by incorporating an enoate reductase-encoding DNA into a chromosomal DNA of a known host cell so that it can be expressed.

  As a method for producing transformed cells, specifically, DNA encoding enoate reductase is incorporated into a plasmid vector or phage vector stably present in the microorganism, and the constructed expression vector is introduced into the microorganism. Alternatively, it is necessary to introduce DNA encoding enoate reductase directly into the host genome and to transcribe and translate the genetic information.

  At this time, if the DNA encoding enoate reductase does not contain a promoter that can be expressed in the host microorganism, it is necessary to incorporate an appropriate promoter upstream of the DNA strand encoding enoate reductase. Furthermore, it is preferable to incorporate a terminator downstream of the 3 'side. The promoter and terminator are not particularly limited as long as they are known to function in microorganisms used as hosts, and vectors, promoters and terminators that can be used in these various microorganisms are, for example, “ Microbiology Basic Course 8, Genetic Engineering, Kyoritsu Shuppan ", especially regarding yeast, Adv. Biochem. Eng. vol. 43, p75-102 (1990), Yeast vol. 8, p423-488 (1992).

  The host microorganism to be transformed to express the enoate reductase of the present invention is not particularly limited as long as the host itself does not adversely affect this reaction. Specifically, for example, The microorganisms shown can be mentioned.

  Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacter, Streptococcus b Bacteria with established host vector systems belonging to the genus.

  Actinomycetes with established host vector systems belonging to the genus Rhodococcus, the genus Streptomyces, and the like.

  Saccharomyces (genus), Kluyveromyces (genus), Schizosaccharomyces (genus), Zygosaccharomyd (genus) Yeast with established host vector systems belonging to the genus Hansenula, Pichia, Candida and the like.

  Molds with established host vector systems belonging to the genera Neurospora, Aspergillus, Cephalosporia, Trichoderma, and the like.

  Among the microorganisms, the host is preferably the genus Escherichia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, particularly preferably the genus Escherichia, It belongs to the genus Corynebacterium.

  The procedure for preparing transformed cells, the construction of a recombinant vector suitable for the host, and the method for culturing the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering ( See, for example, “Molecular Cloning 2nd Edition”, Cold Spring Harbor Laboratory Press (1989)).

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

  In the genus Escherichia, particularly Escherichia coli, plasmid vectors include pBR and pUC series plasmids, and promoters include lac (β-galactosidase), trp (tryptophan operon), tac, trc ( lac, trp fusion), promoters derived from λ phage PL, PR, and the like. Examples of the terminator include trpA-derived, phage-derived, and rrnB ribosomal RNA-derived terminators.

  In the genus Bacillus, examples of the vector include a pUB110-type plasmid and a pC194-type plasmid, and can also be integrated into a chromosome. As the promoter and terminator, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and α-amylase can be used.

  In the genus Pseudomonas, vectors include general host vector systems established in Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in degradation of toluene compounds, and TOL plasmids. A basic broad host range vector (including genes necessary for autonomous replication derived from RSF1010 and the like) pKT240 (Gene, vol. 26, p273-82 (1983)) can be mentioned.

  In the genus Brevibacterium, in particular, Brevibacterium lactofermentum, examples of the vector include plasmid vectors such as pAJ43 (Gene vol. 39, p281 (1985)). As the promoter and terminator, various promoters and terminators used in E. coli can be used.

  In the genus Corynebacterium, in particular, Corynebacterium glutamicum, as vectors, pCS11 (Japanese Patent Laid-Open No. 57-183799), pCB101 (Mol. Gen. Genet. Vol. 196, p175 (1984) ) And the like.

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

  In the genus Schizosaccharomyces, the vector is Mol. Cell. Biol. vol. 6, p80 (1986), and a plasmid vector derived from Schizosaccharomyces pombe. In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

  In the genus Aspergillus, Aspergillus niger and Aspergillus oryzae are the most studied among molds, and integration into plasmids and chromosomes is available. Promoters derived from external proteases or amylases can be used (Trends in Biotechnology vol. 7, p283-287 (1989)).

  In addition to the above, host vector systems corresponding to various microorganisms have been established, and these can be used as appropriate. In addition to microorganisms, various host / vector systems have been established in plants and animals, especially in animals such as insects using moths (Nature vol. 315, p592-594 (1985)), rapeseed, corn, A system for expressing a heterogeneous protein in a large amount in a plant such as potato and a system using a cell-free protein synthesis system such as an E. coli cell-free extract or wheat germ have been established and can be suitably used.

  In the production method of the present invention, a cell transformed with DNA encoding enoate reductase may be allowed to act on a 4-halocrotonic acid derivative which is a reaction substrate. The transformed cell may be allowed to act as it is in the reaction solution, but the transformed cell preparation, for example, the transformed cell is treated with an organic solvent such as acetone, dimethyl sulfoxide (DMSO), toluene or a surfactant. Treated cells, freeze-dried cells, microbial cell preparations such as those physically or enzymatically disrupted, those obtained by removing the enzyme fraction of the present invention in the transformed cells as a crude product or a purified product, Further, those immobilized on a carrier represented by polyacrylamide gel, carrageenan gel or the like may be allowed to act. In the production method of the present invention, a culture solution obtained by culturing the cells, that is, a culture solution containing the cells may be directly used.

In the production method of the present invention, it is preferable to add coenzyme NAD ⁺ or NADH to the reaction solution. The addition concentration is 0.001 mM to 100 mM, preferably 0.01 to 10 mM. When these coenzymes are added, it is preferable to regenerate NAD ⁺ produced from NADH into NADH for improving production efficiency.
(I) a method utilizing the NAD ⁺ reducing ability of the host microorganism itself,
(Ii) Microorganisms having the ability to produce NADH from NAD ⁺ and preparations thereof, or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase) A method of adding an enzyme (regenerative enzyme) that can be used for regeneration of NADH, such as an elementary enzyme, or the like, or (iii) an enzyme that can be used for regeneration of NADH to produce a transformed cell And a method for introducing a gene for a regenerating enzyme into a host simultaneously with DNA encoding enoate reductase.

  Among these, in the method (i), it is preferable to add glucose, ethanol, 2-propanol, formic acid or the like to the reaction system.

  In the above method (ii), a microorganism preparation containing a regenerative enzyme, a microorganism prepared by treating the microorganism with acetone, freeze-dried, physically or enzymatically disrupted, etc. In addition, a product obtained by removing the enzyme fraction as a crude product or a purified product, a product obtained by immobilizing the enzyme fraction on a carrier represented by polyacrylamide gel, carrageenan gel, or the like may be used. May be used. In this case, the amount of the regenerative enzyme used is specifically about 0.01 to 100 times, preferably about 0.5 to 20 times the enzyme activity as compared with the enoate reductase of the present invention. Added. Addition of compounds that serve as substrates for the regenerative enzyme, such as glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. Although it is also necessary, the addition amount is 0.1 to 20 times molar equivalent, preferably 1 to 5 times molar equivalent, relative to the 4-halocrotonic acid derivative which is the reaction raw material.

  In the method (iii), a method for incorporating DNA encoding enoate reductase and a DNA for the regenerative enzyme into a chromosome, a method for transforming a host by introducing both DNAs into a single vector, Alternatively, the method of transforming the host after separately introducing both DNAs into the vector can be used, but in the case of the method of transforming the host after separately introducing both DNAs into the vector, there is no problem between the two vectors. It is necessary to select a vector in consideration of compatibility. When introducing multiple genes into a single vector, methods such as linking promoter-terminator-related regions such as promoters and terminators to each gene, or expressing them as an operon containing multiple cistrons such as the lactose operon. Is possible.

  The production method of the present invention includes, for example, an aqueous medium containing a reaction substrate, the transformed cell of the present invention and / or the transformed cell preparation, and various coenzymes added as necessary and a regeneration system thereof. In or in a mixture of the aqueous medium and an organic solvent.

  The compound represented by the general formula (I) which is a reaction substrate in the production method of the present invention usually has a substrate concentration of 0.01 to 90% w / v, preferably 0.1 to 30% w / v. Can be used. The reaction substrate may be added all at once at the start of the reaction. However, from the viewpoint of reducing the effects when there is enzyme substrate inhibition and improving the accumulated concentration of the product, it is continuous or intermittent. It is desirable to add to.

  Examples of the aqueous medium include water or a buffer, and examples of the organic solvent include those having high solubility of the reaction substrate such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, dimethyl sulfoxide, and the like. be able to.

  The method of the present invention can be carried out, for example, at a reaction temperature of 4 to 60 ° C., preferably 10 to 45 ° C., and pH 3 to 11, preferably pH 5 to 8. It is also possible to use a membrane reactor or the like. In addition, in order to prevent inactivation of enoate reductase by oxygen, it is also effective to remove oxygen by adding sodium sulfite to the reaction solution or sealing the reaction solution with nitrogen or argon gas. .

  The optically active 4-halobutyric acid derivative produced by the method of the present invention is extracted with an organic solvent such as ethyl acetate and toluene after the reaction is completed, after the cells and proteins in the reaction solution are separated by centrifugation, membrane treatment, etc. , Distillation, column chromatography, crystallization, etc. can be used for appropriate purification.

<2> (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5 Method for producing carboxamide (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methyl of the present invention A method for producing indole-5-carboxamide comprises enoate reductase or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell. 4,4 represented by the general formula (III) (R) -4,4,4-trifluoro-2-methylbutyric acid represented by the general formula (IV) by reaction with 1,4-trifluorotiglic acid (a), (R) -4 A step (b) of converting 4,4-trifluoro-2-methylbutyric acid into (R) -4,4,4-trifluoro-2-methylbutylamine represented by the general formula (V); (R) -4,4,4-trifluoro-2-methylbutylamine is reacted with a compound represented by general formula (VI) to represent (R) -N- (4,4) represented by general formula (VII) , 4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5-carboxamide.

Step (a) can be performed in the same manner as described above. Step (b) can use conventional methods for converting carboxylic acids to amines. For example, after reacting with ammonia and amidating and then reducing (reaction formula (IX) below), benzylamine or the like or ammonia equivalent (for example, HN (SiMe ₃ ) ₂ etc.), A method of reduction and further deprotection by catalytic hydrogenolysis or the like (the following reaction formula (X)) can be used.

  In step (c), (R) -4,4,4-trifluoro-2-methylbutylamine of formula (V) obtained in step (b) is reacted with a compound of formula (VI) to give a compound of formula (V) VII) (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5 Carboxamide is obtained. This step can be performed in the same manner as the reaction of a normal amine and a carboxylic acid. Specifically, for example, the method is carried out in the same manner as disclosed in European Patent Application Publication No. 489547 (particularly Example 5) and European Patent Application Publication No. 499548 (particularly Example 2). Can do. Further, the compound of the general formula (VI) used in this step may be obtained by any production method. For example, as disclosed in the above-mentioned patent publication, 4- (5-methoxycarbonyl-1-methyl) is used. Using a compound obtained by reacting indol-3-ylmethyl) -3-methoxybenzoic acid (general formula XI) with 2-methylbenzenesulfonamide (general formula XII) and hydrolyzing the resulting compound. it can.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to this.

(1) Preparation of transformed cells transformed with enoate reductase gene derived from Moorella thermoacetica (Moorella thermoacetica) 2-enoate reductase (Accession No. CA2 Accession No. CA2 Accession No. 60) The primers of SEQ ID NOS: 5 and 6 were synthesized based on the DNA sequence (Accession No. Y16136, REGION: 47.2050, SEQ ID NO: 1). 15 pmol of each primer, 10 nmol each of dNTP, 25 ng of genomic DNA of Moorella thermoacetica DSM 1974, 10 × buffer solution for KOD-plus- (manufactured by Toyobo Co., Ltd.), KOD-plus-1 unit (Toyobo Co., Ltd.) PTC-200 (MJ Research) using 50 μL of the reaction solution containing Spinning Co., Ltd., 30 cycles of denaturation (94 ° C., 15 seconds), annealing (57 ° C., 30 seconds), and extension (68 ° C., 2 minutes). PCR reaction was carried out using As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, a band considered to be specific could be detected.

  The reaction solution was purified with MinElute PCR Purification kit (Qiagen). The purified DNA fragment was digested with restriction enzymes EcoRI and XbaI, and subjected to agarose gel electrophoresis. The target band portion was excised and collected after purification with Qiagen Gel Extraction kit (Qiagen). The obtained DNA fragment was ligated to pUC118 digested with EcoRI and XbaI using Ligation high (manufactured by Toyobo Co., Ltd.), and Escherichia coli JM109 strain was transformed by the heat shock method.

  The transformed cells were grown on an LB agar medium containing ampicillin (50 μg / mL), and colony direct PCR was performed on the obtained colonies to confirm the size of the inserted fragment. Transformed cells that are considered to have the target DNA fragment inserted are cultured in LB medium containing 50 μg / mL ampicillin, and the plasmid is purified using QIAPrepSpin Mini Prep kit (manufactured by Qiagen) to obtain pUCMTER1 . When the base sequence of the DNA inserted into the plasmid was analyzed by the dye terminator method, the inserted DNA fragment was identical to the base sequence of SEQ ID NO: 1.

(2) Preparation of transformed cells transformed with enoate reductase gene derived from Clostridium acetobutylicum (Clostridium acetobutylicum). Based on the DNA sequence to be used (Accession No. AE007834, REGION: 859..2853, SEQ ID NO: 3), primers described in SEQ ID NOs: 7 and 8 were synthesized. 15 pmol each of these primers, 10 nmol each of dNTP, 25 ng genomic DNA of Clostridium acetobutylicum ATCC824, 10 × buffer for KOD-plus- (manufactured by Toyobo), KOD-plus-1 unit (manufactured by Toyobo Co., Ltd.) PTC-200 (manufactured by MJ Research) in 30 cycles of denaturation (94 ° C., 15 seconds), annealing (57 ° C., 30 seconds), extension (68 ° C., 2 minutes) A PCR reaction was performed using As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, a band considered to be specific could be detected.

  The reaction solution was purified with MinElute PCR Purification kit (Qiagen). The purified DNA fragment was digested with restriction enzymes EcoRI and XbaI, and subjected to agarose gel electrophoresis. The target band portion was excised, purified and collected by Qiagen Gel Extraction kit (manufactured by Qiagen). The obtained DNA fragment was ligated to pUC118 digested with EcoRI and XbaI using Ligation high (manufactured by Toyobo Co., Ltd.) to transform Escherichia coli JM109 strain. The transformed cells were grown on an LB agar medium containing ampicillin (50 μg / mL), and colony direct PCR was performed on the obtained colonies to confirm the size of the inserted fragment. Transformed cells that are considered to have the target DNA fragment inserted are cultured in LB medium containing 50 μg / mL ampicillin, and the plasmid was purified using QIAPrepSpin Mini Prep kit (manufactured by Qiagen) to obtain pUCCaER1. . When the base sequence of the DNA inserted into the plasmid was analyzed by the dye terminator method, the inserted DNA fragment was identical to the base sequence of SEQ ID NO: 3.

(3) Synthesis of (R) -2-methyl-4,4,4-trifluorobutyric acid using E. coli transformed with DNA encoding enoate reductase obtained in (1) and (2) above The transformed cells were cultured at 37 ° C. under anaerobic conditions with Aneropac Kenki (manufactured by Mitsubishi Gas Chemical Company) in an LB medium containing ampicillin (50 μg / mL) and 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). Grow for 30 hours. The resulting bacterial cell broth (5 mL) was collected by centrifugation to obtain bacterial cells, and then the enoate reductase activity of the bacterial cells with 4,4,4-trifluorotiglic acid as a substrate was obtained by the following method. confirmed.

  The substrate 4,4,4-trifluorotiglic acid was synthesized as follows. That is, 50% aqueous methanol (20 mL) and 8 mol / L NaOH (3 mL) were added to 4,4,4-trifluorotiglic acid ethyl ester (3.64 g, 20 mmol), and the resulting mixture was added for about 2 hours. Stir. The reaction mixture was evaporated under reduced pressure, concentrated hydrochloric acid was added to adjust the pH to 2 or less, methylene chloride extraction was performed, and the obtained organic layer was concentrated under reduced pressure to give 4,4,4- Trifluorotiglic acid (2.9 g, 94% yield) was obtained.

A 200 μL reaction solution (0.6 g / L NAD ⁺ (manufactured by Oriental Yeast), 50 mM potassium phosphate buffer (pH 7.0), 100 mM glucose, 0.2 g / L glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) 76 unit / mg), 25 mM 4,4,4-trifluorotiglic acid) was added, followed by reaction at 37 ° C. for 20 hours under anaerobic conditions by Aneropac Kenki (manufactured by Mitsubishi Gas Chemical Company). The reaction was terminated by adding 10 μL of 6N HCl, extracted with ethyl acetate, and 2-methyl-4,4,4-trifluorobutyric acid was quantified. Quantification was performed by measuring the ethyl acetate solution using gas chromatography (GC).

The GC conditions are as follows.
Column: β-DEX225 (manufactured by SUPELCO, 30 m × 0.25 mm ID, 0.25 μm film)
Carrier: He 1.5 ml / min, split 1/50
Column temperature: 110 ° C
Injection temperature: 250 ° C
Detection: FID 250 ° C
GC: Shimadzu GC-14A (Shimadzu Corporation)

  As a result, when E. coli transformed with pUCMTER1 was used, the yield of (R) -2-methyl-4,4,4-trifluorobutyric acid was 625 μg and the optical purity was 67.2% e.e. e. Met. In addition, when using E. coli transformed with pUCCaER1, the yield of (R) -2-methyl-4,4,4-trifluorobutyric acid was 757 μg and the optical purity was 99.8% e.e. e. That was all.

  The present invention makes it possible to obtain an optically active 4-halobutyric acid derivative, which is an industrially useful compound as an intermediate raw material for pharmaceuticals, agricultural chemicals and the like, with high optical purity. Also useful as a leukotriene antagonist (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1- Methylindole-5-carboxamide can be efficiently produced.

Claims (9)

The following general formula (I)

A haloate reductase or a cell containing the same, a preparation of the same cell, or a culture solution obtained by culturing the same cell is allowed to act on the 4-halocrotonic acid derivative represented by the following general formula (II):

A method for producing an optically active 4-halobutyric acid derivative represented by the general formula (II) (general formulas (I) and (II)) , R represents a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkoxy group, X represents a halogen atom, A ₁ and A ₂ represents a hydrogen atom or a halogen atom). The production method according to claim 1, wherein the enoate reductase is a protein represented by the following (A), (B) or (C):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or (B) an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and A protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(C) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added, and represented by the general formula (I) 4- A protein having an enzyme activity for converting a halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II). The production method according to claim 1, wherein the cell containing enoate reductase is a cell transformed with DNA encoding enoate reductase. The production method according to claim 3, wherein the cell transformed with DNA encoding enoate reductase is a cell transformed by integrating the DNA on a chromosome. The production method according to claim 3, wherein the cell transformed with DNA encoding enoate reductase is a cell transformed with a vector containing the DNA. The production method according to any one of claims 3 to 5, wherein the DNA encoding enoate reductase is a DNA encoding a protein shown in the following (A), (B) or (C):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or (B) an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and A protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(C) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added, and represented by the general formula (I) 4- A protein having an enzyme activity for converting a halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II). The production method according to any one of claims 3 to 5, wherein the DNA encoding enoate reductase is the following DNA (D), (E) or (F):
(D) DNA having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or (E) hybridizing under stringent conditions with DNA consisting of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary sequence thereof And a DNA encoding a protein having an enzyme activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (II).
(F) The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 consists of a nucleotide sequence in which one or several bases are substituted, deleted or added and its complementary strand, and is represented by the general formula (I) A DNA encoding a protein having an enzyme activity for converting a 4-halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II). In the compounds of the general formulas (I) and (II), R is a methyl group, X, A ₁ and A ₂ are each a fluorine atom, The manufacturing method as described. The method according to claim 8, wherein 4,4,4-trifluorotiglic acid represented by general formula (III) is converted to (R) -4,4,4-trifluoro represented by general formula (IV). The step of converting to 2-methylbutyric acid, the (R) -4,4,4-trifluoro-2-methylbutyric acid is represented by the general formula (V) (R) -4,4,4-tri A step of converting to fluoro-2-methylbutylamine, and reacting the compound represented by the general formula (VI) with the (R) -4,4,4-trifluoro-2-methylbutylamine, to give a general formula (VII) (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4- (o-tolylsulfonylcarbamoyl) benzyl] -1-methylindole-5 represented by -Comprising the step of producing carboxamide (R)- - (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4-(o-tolyl sulfonylcarbamoyl) benzyl] -1-production method of methyl indole-5-carboxamide.