JPH08103269A - Base sequence of carbonyl reductase gene and method for utilizing the same - Google Patents

Abstract

PURPOSE: To obtain the subject enzyme, having a specific amino acid sequence, producible according to the manner of the genetic engineering, capable of acting on a γ-substituted acetoacetic ester and providing an (R)-γ-substituted-β- hydroxybutyric acid ester, etc., useful as a synthetic raw material, etc., as medicines and agrochemicals. CONSTITUTION: This new carbonyl reductase has an amino acid sequence containing an amino acid sequence represented by the formula and is capable of acting on a γ-substituted acetoacetic ester and producing an (R)-γ- substituted-β-hydroxybutyric acid ester useful as a synthetic raw material for medicines, agrochemicals, etc., and used for synthesis, etc., of L-carnitine useful as the medicines. This enzyme is obtained by extracting an mRNA from Sporobolomyces salmonicolor IFO1038, etc., belonging to the genus Sporobolomyces, synthesizing a cDNA using the mRNA as a template, cloning the resultant cDNA with a primer comprising a part of a gene capable of coding the carbonyl reductase according to the polymerase chain reaction(PCR) method, sorting out a gene, joining the sorted gene to a vector and expressing the gene in a host cell.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a DNA having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing and encoding a polypeptide having a carbonyl reductase (hereinafter abbreviated as ALD) activity, its DNA and a vector. Recombinant DNA, transformant transformed with the recombinant DNA, method for producing ALD using the transformant, and method for producing (R) -γ-substituted-β-hydroxybutyric acid ester Regarding In particular, (R) -γ-substituted-β-hydroxybutyric acid ester is a compound useful as a raw material for the synthesis of medicines, agricultural chemicals and the like, and for example, it is known that it can be used for the synthesis of L-carnitine used as a medicine. Has been.

[0002]

2. Description of the Related Art Conventionally, yeast-derived enzymes have been used as an enzyme of microbial origin that catalyzes the synthesis of (R) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester. L-β-hydroxyacyl CoA dehydrogenase (EC1.1.1.35)
(JP-A-59-118093) and an alcohol dehydrogenase derived from Thermoanaerobium brockii (EC 1.1.1.
2) (JACS, 107 , 4028 (1985)) is known.

However, the former has significant disadvantages such as reactivity and temperature stability, and the latter requires anaerobic culture, and (R) -γ-substituted-β-hydroxy is used. It was difficult to say that the production method of butyric acid ester was practical. As a solution to such a point, the present inventors extensively searched natural and conserved strains, and as a result, reduced nicotinamide adenine dinucleotide phosphate (NAD) derived from the genus Sporobolomyces.
(PH) -dependent carbonyl reductase (ALD) was found (JP-A-63-304979), and by using the enzyme, (R) -γ-substituted-β-hydroxybutyric acid was selectively selected from γ-substituted acetoacetic acid esters. It has been found that an ester can be produced (JP-A-63-304).
No. 991).

Using such an ALD, (R) -γ-
In order to efficiently produce a substituted -β-hydroxybutyric acid ester, it was necessary to obtain a large amount of ALD at low cost. In the past, in order to efficiently obtain this enzyme in large quantities, attempts have been made extensively to obtain high-producing strains from soil, etc., or breeding improvement has been made by artificial mutation of existing production strains, but it is satisfactory. No results were obtained.

[0005]

[Means for Solving the Problems] Under these circumstances, the present inventors have conducted diligent research, and as a result, obtained a gene involved in ALD production from a microorganism by a gene recombination technique, The base sequence of the gene was determined. As a result of further research by the present inventors, the DNA encoding ALD was
In addition to having the advantage that it can be combined with a gene expression control sequence relatively easily, that the control of its expression can be facilitated, and that it can be used for breeding high ALD-producing transformants, The present invention has been completed by discovering that substitution, deletion or addition of a nucleotide sequence can be easily performed by a known method.

That is, the present invention provides (1) a vector having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and encoding a polypeptide having ALD activity, (2) the DNA of (1) above. Recombinant D incorporated into
NA, (3) a transformant transformed with the recombinant DNA of (2) above, (4) culturing the transformant of (3) above, and then collecting the obtained ALD. Characteristic ALD production method, and (5) The transformant of (3) or a treated product thereof is reacted with γ-substituted acetoacetic acid ester to produce (R) -γ-substituted-β-hydroxybutyric acid. (R) -γ- characterized by collecting the ester
It relates to a method for producing a substituted-β-hydroxybutyric acid ester.
The present invention also provides a highly-expressing recombinant DNA using the DNA of (1) above, a method for producing the same, and a breeding method of an ALD high-producing transformant using the DNA of (1) above. To do.

The present invention will be described in more detail below. (1) Isolation of DNA Carrying Genetic Information Involved in ALD Synthesis DN having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing of the present invention and encoding a polypeptide having ALD activity
A (hereinafter, also simply referred to as ALD gene) can be produced from DNA carrying genetic information involved in ALD synthesis.

[0008] Examples of the DNA carrying the genetic information involved in the synthesis of ALD include, for example, Sporobomyces.
lomyces) yeasts. This Sporoboromyces yeast is not particularly limited as long as it is an ALD-producing Sporoboromyces yeast, but particularly preferred is Sporoboromyces salmonicolor.
(Sporobolomyces salmonicolor) IFO1038 can be mentioned. The DNA fragment having the ALD gene can be obtained from the gene donor by a usual method. For example, D synthesized from a chromosomal DNA library or cDNA library of a gene donor prepared by a conventional method based on the partial amino acid sequence of purified ARD.
A target fragment can be obtained by a hybridization method using an NA probe.

Further, this DNA, once isolated and obtained, utilizes most or part of the nucleotide sequence in the DNA according to a conventional method and uses it as a probe to detect other organisms. Searching for genes that carry the genetic information involved in ALD synthesis among the genes possessed by the organism, then cutting out the genes thus identified by applying the method of gene recombination technology. Then, it is easily understood by those skilled in the art to obtain a large amount of it and use it in the same manner as that derived from this yeast of the genus Sporoboromyces.

Therefore, as the DNA source for carrying the genetic information involved in the synthesis of ALD of the present invention, not only yeast of the genus Sporoboromyces but also microorganisms to which the above-mentioned method can be applied and which is a gene encoding ALD There is a thing which has. In addition to this, as long as it has an ALD gene, it can be used without distinction between animals, plants, lower organisms, and higher organisms.

(2) Determination of Nucleotide Sequence of DNA Fragment Containing ALD Gene Genes The Maxam-Gilbert method, dideoxy sequence method, dideoxy chain termination method can be used to determine the DNA nucleotide sequence of a DNA fragment having an ALD gene. Etc. Some of these methods include
A method in which an appropriate restriction enzyme is allowed to act to create a restriction enzyme map, and then a necessary fragment is subcloned, a shotgun cloning method, a method for amplifying a gene by PCR, and a method for deleting with a nucleolytic enzyme. Of course, various methods such as are included.

DNA structurally analyzed as described above
In the case where a region other than the gene involved in ALD synthesis is carried, a region unnecessary for ALD synthesis can be deleted by various methods. Examples of such methods include BAL31 nuclease and exonuclease III.
And a recombination method using a restriction enzyme cleavage site. At this time, changing the promoter specific to the gene to another one or changing the strength of the promoter by introducing a site-specific mutation can be easily carried out by the current gene manipulation technique, and therefore, , Even if a partially modified DNA fragment is ALD
DN containing gene responsible for production of active polypeptide
It is clear that all A fragments are included in the present invention.

Once the base sequence of the gene has been clarified, the base substitution can be easily carried out by applying a method known in the art so long as the function is not impaired. For example, the substitution of the genetic code that encodes the corresponding amino acid, the substitution of the base for the conversion that considers the utilization rate of the genetic code of the organism, or the conversion of the amino acid sequence that does not adversely affect the function of ALD. , Addition or deletion treatment and the like. Furthermore, such modifications also include altering the sequence and length of the DNA so that only the active centers of ALD are preserved and other moieties are significantly altered.

Therefore, those skilled in the art can easily understand that the DNA carrying the genetic information involved in the synthesis of ALD of the present invention includes all those modified as described above. Is. In view of the above circumstances, the DNA involved in the synthesis of ALD of the present invention can be obtained by substantially utilizing the idea of the present invention, and any DNA having substantially the same function as the DNA of the present invention can be used. It is contained.

(3) Recombinant DNA having ALD gene
Preparation of a host cell by reintegrating a DNA containing a DNA carrying the genetic information involved in the synthesis of ALD into a suitable vector DNA by applying appropriate means to delete a region unnecessary for DNA synthesis. Can be reintroduced into. The vector DNA used for introducing the DNA carrying the genetic information involved in the synthesis of ALD of the present invention into a host cell and expressing it in the introduced host cell is , ALD gene can be used without particular limitation.

[0016] Such a vector DNA is one which can incorporate a DNA which carries the genetic information involved in the synthesis of ALD, which can transform a host cell with the recombinant vector DNA, and which is obtained. The transformant is not particularly limited as long as it can express the DNA that carries the genetic information involved in the synthesis of ALD introduced into the cells of the transformant, and any one can be used.
Examples of such vector DNA include those that are capable of autonomously replicating in a host cell and are further provided with an appropriate selectable marker and the like capable of selecting only recombinant host cells. Furthermore, such vector DNA may be one that can be easily produced by those skilled in the art from known vector DNA and the like.

Examples of such vector DNA include those selected from plasmid vectors, phage vectors and cosmid vectors. Further, it may be a shuttle vector capable of gene exchange with other host strains, and is specially devised to improve the expression efficiency of gene products such as runaway vector and sleeper vector. May be. In addition, such vector DNA has the lacUV5 promoter, tr
p promoter, tac promoter, lpp promoter, tufB promoter, recA promoter,
It may be one to which a regulatory factor such as PL promoter is appropriately added.

In order to incorporate the DNA carrying the genetic information involved in the synthesis of ALD into the above vector DNA, first, an appropriate restriction enzyme is allowed to act on the above vector DNA,
The obtained vector DNA fragment is mixed with the DNA fragment which carries the genetic information involved in the synthesis of ALD, and D
This can be done by acting NA ligase. At this time, if necessary, treatments such as linker addition, blunt end formation and the like known in the art can be added. The recombinant DNA thus obtained is then introduced into a suitable host cell.

(4) Introduction of Recombinant DNA into Host Cell Host cells for introducing the recombinant DNA prepared as described above are transformed with the recombinant vector obtained above. As long as it can express the ALD gene, it can be used without particular limitation. As such a host cell, as long as the expression of the ALD gene can be achieved for the purpose of the present invention, there is no distinction between Gram-negative bacteria and Gram-positive bacteria, and further,
It is possible to use animal-derived cells or plant-derived cells regardless of lower cells or higher cells.

The host cells that can be used here include those that can achieve the expression of DNA that carries the genetic information involved in the synthesis of ALD of the present invention. Examples of such host cells include E. coli, Xanthomonas bacteria,
Acetobacter, Pseudomonas, Gluconobacter, Azotobacter, Rhizobium, Alcaligenes, Klebsiella,
Those selected from Salmonella and Serratia.

A preferred host cell to be used here is, for example, Escherichia coli. The above recombinant DN
In transforming the host cell with A, a method widely known in the DNA recombination technique can be applied. As such a method, a host cell is made into a competent state and mixed with a recombinant DNA in a transformation buffer, or
Examples include a method of transferring by conjugal transfer using a helper plasmid.

(5) Production of ALD by Recombinant The ALD-producing transformant obtained by the present invention is the ALD-producing transformant obtained by culturing it in a nutrient medium as it is or as a single ALD from the culture. Away, (R) -γ-substitution-β
It can be used for the synthesis of hydroxybutyric acid ester or its derivative.

The ALD-producing transformant can be obtained in a large amount by culturing in an appropriate nutrient medium. The medium used for culturing is an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of microorganisms. In addition, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results. The culture may be carried out under aerobic conditions at a pH of 4 to 10 and at a temperature of 20 to 60 ° C in an arbitrary range for 1 to 10 days. When using a transformant, depending on the strain used, ampicillin,
An antibiotic such as chloramphenicol may be added to the culture solution, or isopropyl β-D (-)-thiogalactopyranoside [Isopropyl β-D (-)-Thiogalactopyranoside (IP
It is also possible to use an inducer such as TG)].

For the isolation and purification of ALD from cells of the ALD-producing transformant obtained by culturing in a large amount or the culture solution thereof, a method similar to the method described in JP-B-63-304991 can be used, Since the production amount is very high, a simpler method can be used. In order to carry out such a method, first, the organism is disrupted by a method such as a mechanical method, an enzyme treatment method, an autolysis method or an ultrasonic treatment method to obtain a crude extract, which is then subjected to ammonium sulfate or phosphoric acid. Protein precipitation method with salting-out agent such as sodium or solvent such as acetone or ethanol, electrophoresis method, gel filtration method or molecular sieve chromatography method, ultrafiltration method, reverse phase chromatography method, high performance liquid chromatography method, ion The purification can be carried out by using a method such as an exchange chromatography method, an affinity chromatography method, an adsorption chromatography method, etc., alone or in appropriate combination.

(6) Production of (R) -γ-substituted-β-hydroxybutyric acid ester The recombinant ALD thus obtained is disclosed in JP-A-63-3049.
No. 91 or JP-A No. 63-309195, it has been conventionally found in the natural world when producing (R) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester as described in JP-A-63-309195. Although it can be used in the same manner as the ALD of the non-recombinant microorganism, the present invention can produce the ALD in a large amount and with high purity.
The objective (R) -γ-substituted-β-hydroxybutyric acid ester can be produced better.

ALD which has been conventionally found in the natural world
In the producing microorganism, since the activity of synthesizing (S) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester (hereinafter, abbreviated as S-activity) is also present, γ-substituted From acetoacetic ester (R) -γ
When trying to produce a -substituted-β-hydroxybutyric acid ester, the S-activity is separated or selectively inactivated in order to avoid the by-product of (S) -γ-substituted-β-hydroxybutyric acid ester. Was necessary. Since the recombinant microorganism of the present invention can produce only ALD in a large amount, it is said that the S-activity produced by the host is removed when producing (R) -γ-substituted-β-hydroxybutyric acid ester. It is possible to omit complicated operations.

In the γ-substituted acetoacetic acid ester used in this method, the γ-position substituent is preferably a chloro, bromo, fluoro, or azido group, and the ester group is an alkyl group having 1 to 10 carbon atoms. Is preferred. The enzyme used in this method is not limited to an isolated one as long as it has a form capable of exerting an enzymatic activity. The enzyme may be a semi-refined product, a crude extract,
Further, it may be a culture, organism, freeze-dried organism, acetone-dried organism, or a ground product of these organisms. Further, the enzyme itself or the organism itself may be immobilized and used by a known means.

The reaction temperature is 10 to 70 ° C, preferably 20 to 50 ° C. The pH during the reaction is maintained at 4 to 10, preferably 6 to 8. This reaction may be carried out batchwise or continuously. JP-A-63-309
As described in No. 195, by carrying out this reaction in a two-phase system consisting of an organic solvent capable of forming a two-phase with water such as ethyl acetate and an aqueous medium, a higher yield can be obtained as compared with a system containing an aqueous solvent alone. A high-purity and high-concentration (R) -γ-substituted-β-hydroxybutyric acid ester can be obtained.

Further, since this enzyme requires NADPH as a coenzyme, it needs to be added to the reaction solution, but the use of a commonly used NADPH regeneration system can significantly reduce the amount of expensive coenzyme used. Is possible. As a typical NADPH regeneration system, a method by adding glucose dehydrogenase (GDH), glucose as a substrate of GDH, and NADP ⁺ can be mentioned, but the present invention is not limited to this and can be suitably used for this reaction.

Further, this reaction can be carried out by carrying out the reaction for an arbitrary time, but preferably 1 to 50 hours,
More preferably, the reaction is performed for 5 to 30 hours. The (R) -γ-substituted-β-hydroxybutyric acid ester can be purified from the reaction solution by a conventional method. For example, after subjecting to treatment such as centrifugation and filtration to remove suspensions and the like, extraction with an organic solvent such as ether, carbon tetrachloride, benzene and ethyl acetate, dehydration with a dehydrating agent such as sodium sulfate, and organic The solvent is removed under reduced pressure, and if necessary, further purification such as distillation under reduced pressure or chromatographic separation provides high purity (R).
The -γ-substituted-β-hydroxybutyric acid ester is obtained in high yield.

The enzyme used in this method is a natural ALD.
It should be understood that it is not limited to those which match the peptide sequence of the above, and shows the ALD activity obtained by the method of gene conversion and gene recombination. Therefore, the recombinant ALD that can be used in the present method also includes a deletion of an amino acid in the peptide sequence of natural ALD, or a replacement of an amino acid in the peptide sequence with another amino acid.

As described above, according to the present invention, a large amount of A
LD can be produced, and the produced ALD can be easily isolated and purified to obtain high-purity ALD. By using the ALD thus obtained, (R) -γ
An excellent method for producing -substituted-β-hydroxybutyrate is provided. Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[0033]

【Example】

Example 1 Determination of nucleotide sequence of ALD gene (cDNA) Preparation of cDNA library mRNA was extracted from the cultured cells of Sporoboromyces salmonicolor IFO1038 by a conventional method. From the obtained mRNA, the method of Gubler et al. (Gene, 25 , 263 (1983))
Was used to synthesize double-stranded cDNA. After adding EcoRI / XhoI linker DNA to the obtained double-stranded cDNA, it was inserted into the EcoRI and XhoI sites of Uni-ZAP ^™ XR phage vector (Stratagene) by a conventional method. The resulting recombinant phage vector was packaged in vitro by a conventional method, and then Escherichia coli SURE ^™ (St
ratagene) transformed into about 20,000 c
A DNA library was created.

Preparation of Synthetic Oligonucleotide Probe Sporoboromyces salmonicolor IF was prepared by the method described in the literature (FEMS Microbiol. Lett., 70 , 45-48 (1990)).
ALD was singly purified from O1038. This purified AL
D was digested with lysyl endopeptidase, and the amino acid sequence of the obtained peptide fragment was determined. By comparing these sequences with the amino acid sequences of known aldehyde reductases, peptide fragments that are believed to be located on the N-terminal side and the C-terminal side of the enzyme were identified, and shown in FIG. 1 based on the amino acid sequences. The probe was synthesized. Probes 1 and 2 were ³² P by kaination according to the conventional method.
It was labeled and used in the following experiments.

Cloning of ALD gene from cDNA library ³² P-labeled synthetic DN was prepared from the prepared cDNA library.
The plaques of the phage containing the ALD gene were screened by plaque hybridization using the A probe. Plaque hybridization is performed by the method of Maniatis et al. (Molecular Cloning, p326 (1982).
Cold Spring Harbor Laboratory). ³²
Hybridization with probe 1 labeled with P resulted in 5 positive plaques. Next, Uni-ZAP ^™ XR vector kit (Stratagene
Co., Ltd., and the host Escherichia coli was co-infected with the recombinant phage and helper phage obtained from the above-mentioned 5 positive plaques, and the cDNA considered to be the ALD gene was subcloned into the plasmid pBluescript SK-. The obtained recombinant plasmid was analyzed by various restriction enzymes, and the shortest DN
ALD of the plasmid pSAL2 in which the A fragment was inserted
It was selected as a cDNA clone containing the gene. pSA
The manufacturing method and structure of L2 are shown in FIG.

Determination of nucleotide sequence Using this recombinant plasmid pSAL2, M13mp
Analysis was carried out using various restriction enzymes that can be introduced into 18 and 19, and a restriction enzyme cleavage map was prepared. Next, various DNA fragments obtained in this analysis were introduced into the multi-cloning site of M13mp18RF or M13mp19RF (both manufactured by Takara Shuzo Co., Ltd.) to obtain a recombinant phage. Using these phages, single-stranded recombinant phages were prepared by a conventional method, and the nucleotide sequences of the respective inserted fragments were analyzed by the Dideoxy method.
The base sequence was determined. This sequence is shown in SEQ ID NO: 1 in the sequence listing. The structural gene portion in this base sequence is shown in SEQ ID NO: 2 in the sequence listing. The result of comparing the amino acid sequence deduced from this base sequence with the partial amino acid sequence of the lysyl endopeptidase digested peptide fragment of purified ALD,
It shows in FIGS. The partial amino acid sequence of purified ALD is
It was present in the amino acid sequence deduced from the nucleotide sequence, and there was a perfect match in that portion (underlined portions in FIGS. 3 to 5).

Example 2 Preparation of Recombinant Plasmid A recombinant plasmid used for transformation was prepared in order to express ALD in E. coli. First, cD with SD sequence added upstream of the start codon of ALD structural gene
NA was obtained by the following method. Based on the nucleotide sequence determined in Example 1, an N-terminal side primer (PN) having an SD sequence added upstream of the start codon of the ALD structural gene
And a C-terminal side primer (PC) were synthesized. The base sequences of these two primers are shown in FIG. From the mRNA obtained in Example 1, the method of Maniatis et al. (Molecular Cloning, p.
14.20 (1989) Cold Spring Harbor Laboratory) was used to synthesize cDNA. PC for the synthesis of single-stranded cDNA
Double-stranded cDNA by PCR using
PN and PC were used as primers for the synthesis of. The obtained DNA fragment was digested with EcoRI and PstI and inserted into the EcoRI and PstI sites downstream of the lac promoter of the plasmid pUC118 (Takara Shuzo Co., Ltd.) to obtain a recombinant plasmid pAR. FIG. 7 shows the method and structure of pAR.

Example 3 Production of Recombinant Escherichia coli The recombinant plasmid pAR obtained in Example 3 was prepared by a conventional method.
And the recombinant plasmid pSAL2 obtained in Example 1 was used to transform Escherichia coli JM109 (manufactured by Stratagene) to obtain recombinant Escherichia coli JM109 (pAR) and JM109.
(PSAL2) was obtained. The transformants E. coli JM109 (pAR) and E. coli JM109 thus obtained
(PSAL2) is the accession number FERM P-, respectively.
14558 and FERM P-14559 have been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.

Example 4 Expression of ALD in Recombinant Escherichia coli Measurement of ALD Activity in Recombinant Escherichia coli The transformant obtained in Example 3 was cultured in LB medium and Isopro was used.
After induction with pyl β-D (-)-Thiogalactopyranoside (IPTG), the cells were collected and ultrasonically disrupted to obtain a cell-free extract. Using the ALD activity of this cell-free extract as a substrate, p-
It was measured by monitoring the reduction of NADPH with nitrobenzaldehyde (1 U represents the enzymatic activity of oxidizing 1 μmol NADPH per minute). The results are shown as specific activity in Table 1 in comparison with the transformant containing only the vector plasmid. E. coli JM109 (pAR)
Which was induced by IPTG was about 3.2 times that of IPTG-minus, which was a vector transformant JM10.
About 7.7-fold activity was obtained compared with 9 (pUC118). In addition, JM109 (pSAL2) also showed about 2.1 times the activity of the vector transformant.

[0040]

[Table 1]

Confirmation of immunological equivalence: SDS-polyacrylamide electrophoresis of the cell-free extract obtained in the above expression experiment and ALD purified from Sporoboromyces salmonicolor IFO1038 was carried out,
From the results of Western blotting using an anti-ALD antibody prepared using ALD purified from Sporoboromyces salmonicolor IFO1038 and the results of Coomassie blue staining of the electrophoretic gel, as shown in FIG. Induced protein around 35 KDa is Sporoboromyces salmonicolor IFO
It was confirmed to be immunologically equivalent to ALD derived from 1038. In addition, recombinant E. coli JM109 (pA
R) and ALD purified from Sporoboromyces salmonicolor IFO 1038, the immunological equivalence of ALD purified from Sporoboromyces salmonicolor IFO
Anti-ALD prepared using ALD purified from 1038
When examined by the Ouchterlony double diffusion method using an antibody, the immunoprecipitation lines of both enzymes were completely fused, and their equivalence was confirmed.

Determination of specific activity for various substrates ALD purified from recombinant E. coli JM109 (pAR)
And Sporoboromyces Salmonicolor IFO 1038
Using ALD purified from, the activity ratio for three substrates (activity for p-nitrobenzaldehyde was 100
The results are shown in Table 2. The activity was measured by monitoring the decrease in NADPH. It was found that both enzymes act on three types of substrates and their activity ratios are almost equal.

[0043]

[Table 2]

Example 5 Synthesis of (R) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester by recombinant Escherichia coli 3 recombinant E. coli JM109 (pAR) obtained in Example 3 was used.
Inoculate 1 liter of LB medium, incubate at 37 ° C overnight,
After inducing the enzyme with PTG, the cells were collected. This fungus body was
Wet cells obtained by washing with 9% saline were used in the following reaction. The enzyme reaction uses a 200 ml incubator, the temperature is 30 ° C., the stirring is 200 rpm, and the pH is NaHCO 3.
_It was carried out while adjusting to 6.0 by automatic addition of ₃ (2M). Table 3 shows the composition of the reaction solution at the start of the reaction.

[0045]

[Table 3] After the reaction was started, γ-chloroacetoacetic acid ethyl ester was added, and the addition timing and the addition concentration are shown in Table 4.

[0046]

[Table 4] Also, 10 hours, 25 hours, 34 hours and 5 hours after the start of the reaction.
Glucose 100 mg / ml, NADP ^{+ in} 1 hour
Was added in an amount of 0.6 mg / ml and GDH was added in an amount of 0.4 mg / ml (all based on the amount of the aqueous phase).

The time course of the reaction is shown in FIG. Γ-chloroacetoacetic acid ethyl ester as a substrate and γ as a product
The concentration of -chloro-β-hydroxybutyric acid ethyl ester in the butyl acetate phase was measured by gas chromatography (Shimadzu GC-9APF, PEG20M × 1 m, 150) after dehydration of the sampled butyl acetate phase with anhydrous sodium sulfate.
Analysis at 30 ° C / N ₂ ). At the end of reaction (72
Time), 96.3% of the added γ-chloroacetoacetic acid ethyl ester was consumed, of which 79.6% was γ
It had been converted to -chloro-β-hydroxybutyric acid ethyl ester. The butyl acetate phase separated from the reaction solution 72 hours after the start of the reaction by centrifugation was dehydrated with anhydrous sodium sulfate, and then distilled under reduced pressure, followed by gas chromatography (the above conditions) and IR.
(Shimadzu IR-435), NMR (JEOL Ltd. PMX60SI) analysis, γ-chloro-β-
It was confirmed to be hydroxybutyric acid ethyl ester.

The optical purity of the thus obtained γ-chloro-β-hydroxybutyric acid ethyl ester was determined by synthesizing an ester with (+)-α-methoxy-α-trifluoromethylphenylacetic acid into a diastereomer compound. It was calculated by separation and quantification by high performance liquid chromatography (HPLC). As a result, the optical purity of the (R) -form was ee = 99% or more. HPLC condition Column: Partisil 5 (4.6φ × 250 m
Mobile phase: Hexane / tetrahydrofuran / methanol = 600/100/1 Flow rate: 2 ml / min Detection: Absorbance at 217 nm

[0049]

EFFECTS OF THE INVENTION By cloning an ALD producing gene from a microorganism and analyzing its nucleotide sequence, a transformant having a high ALD producing ability can be obtained. Further, by using this transformant, it became possible to efficiently synthesize (R) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester.

[Sequence listing] SEQ ID NO: 1 Sequence length: 1055 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Sporobolomyces salmoni color
color) Strain name: IFO1038 Sequence features Characteristic symbol: mat peptide Location: 33..1001 Method of determining features: E sequence GCCAACAACC CCTGCACGCA TCACACATC ATG GTC GGC ACT ACT ACC CTC AAC 53 Met Val Gly Thr Thr Thr Leu Asn 15 ACT GGC GCT TCC CTC GAG CTC GTC GGC TAC GGC ACG TGG CAG GCA GCA 101 Thr Gly Ala Ser Leu Glu Leu Val Gly Tyr Gly Thr Trp Gln Ala Ala 10 15 20 CCG GGC GAG GTG GGC CAG GGC GTC AAG GTC GCC ATC GAG ACT GGA TAC 149 Pro Gly Glu Val Gly Gln Gly Val Lys Val Ala Ile Glu Thr Gly Tyr 25 30 35 CGT CAC CTC GAC CTT GCC AAG GTC TAC TCG AAC CAA CCT GAG GTT GGT 197 Arg His Leu Asp Leu Ala Lys Val Tyr Ser Asn Gln Pro Glu Val Gly 40 45 50 55 GCC GCC ATC AAG GAG GCT GGC GTC AAG CGC GAG GAC CTC TTC ATC ACC 245 Ala Ala Ile Lys Glu Ala Gly Val Lys Arg Glu Asp Leu Phe Ile Thr 60 65 70 TCG AAG CTC TGG AAC AAC TCG CAC CGC CCG GAG CAG GTC GAG CCT GCC 293 Ser Lys Leu Trp Asn Asn Ser His Arg Pro Glu Gln Val Glu Pro Ala 75 80 85 CTT GAC GAC ACC CTC AAG GAG CTC GGC CTC GAG TAC CT C GAC CTT TAC 341 Leu Asp Asp Thr Leu Lys Glu Leu Gly Leu Glu Tyr Leu Asp Leu Tyr 90 95 100 CTC ATT CAC TGG CCC GTC GCG TTC CCG CCC GAG GGC GAC ATC ACC CAG 389 Leu Ile His Trp Pro Val Ala Phe Pro Pro Glu Gly Asp Ile Thr Gln 105 110 115 AAC CTC TTC CCG AAG GCC AAC GAC AAG GAG GTC AAG CTC GAC CTG GAG 437 Asn Leu Phe Pro Lys Ala Asn Asp Lys Glu Val Lys Leu Asp Leu Glu 120 125 130 135 GTC AGC CTC GTC GAC ACG TGG AAG GCG ATG GTC AAG CTT CTC GAC ACT 485 Val Ser Leu Val Asp Thr Trp Lys Ala Met Val Lys Leu Leu Asp Thr 140 145 150 GGC AAG GTC AAG GCG ATC GGC GTT TCC AAC TTC GAC GCG AAG ATG GTC 533 Gly Lys Val Lys Ala Ile Gly Val Ser Asn Phe Asp Ala Lys Met Val 155 160 165 GAC GCC ATC ATC GAG GCT ACC GGC GTG ACC CCC TCC GTC AAC CAG ATC 581 Asp Ala Ile Ile Glu Ala Thr Gly Val Thr Pro Ser Val Asn Gln Ile 170 175 180 GAG CGT CAC CCT CTC CTT CTC CAG CCC GAG CTC ATC GCC CAC CAC AAG 629 Glu Arg His Pro Leu Leu Leu Gln Pro Glu Leu Ile Ala His His Lys 185 190 195 GCC AAG AAC ATT CAC ATT ACC GCA TAC TCT CCT CTC GGT AAC AAC ACC 677 Ala Lys Asn Ile His Ile Thr Ala Tyr Ser Pro Leu Gly Asn Asn Thr 200 205 210 215 GTC GGC GCG CCT CTT CTT GTC CAG CAC CCG GAG ATC AAG CGC ATC GCC 725 Val Gly Ala Pro Leu Leu Val Gln His Pro Glu Ile Lys Arg Ile Ala 220 225 230 GAG AAG AAC GGC TGC ACG CCC GCT CAG GTC CTC ATT GCC TGG GCC ATC 773 Glu Lys Asn Gly Cys Thr Pro Ala Gln Val Leu Ile Ala Trp Ala Ile 235 240 245 GTT GGC GGC CAC TCG GTT ATC CCC AAG TCG GTC ACC CCC TCC CGC ATT 821 Val Gly Gly His Ser Val Ile Pro Lys Ser Val Thr Pro Ser Arg Ile 250 255 260 GGC GAG AAC TTC AAG CAG GTC TCG CTC TCG CAG GAG GAC GTC GAT GCC 869 Gly Glu Asn Phe Lys Gln Val Ser Leu Ser Gln Glu Asp Val Asp Ala 265 270 275 GTC AGC AAG CTC GGC GAG GGT TCG GGC CGC AGG CGC TAC AAC ATC CCC 917 Val Ser Lys Leu Gly Glu Gly Ser Gly Arg Arg Arg Arg Tyr Asn Ile Pro 280 285 290 295 TGC ACG TAC TCG CCC AAG TGG GAC ATC AAC GTC TTT GGC GAG GAG GAC 965 Cys Thr Tyr Ser Pro Lys Trp Asp Ile Asn Val Phe Gly Glu Glu Asp 300 305 310 GAG AAG TCG TGC AAG AAC GCC GTG AAG ATC AAG TAGAAGGTCT 1008 Glu Lys Ser Cys Lys Asn Ala Val Lys Ile Lys 315 320 CGGGTCCCGG GTACTCTGTT AAAAGCAATC CCAATCAGTT CGGGGCG 1055 Sequence number: 2 Sequence length: 972 Sequence type: Nucleic acid Number of strands: Duplex topology Linear sequence type: cDNA to mRNA Origin organism name: Sporobolomyces salmoni color
color) Strain name: IFO1038 Sequence features Characteristic symbol: mat peptide Location: 4..972 Method of determining features: E sequence ATG GTC GGC ACT ACT ACC CTC AAC ACT GGC GCT TCC CTC GAG CTC GTC 48 Met Val Gly Thr Thr Thr Leu Asn Thr Gly Ala Ser Leu Glu Leu Val 1 5 10 15 GGC TAC GGC ACG TGG CAG GCA GCA CCG GGC GAG GTG GGC CAG GGC GTC 96 Gly Tyr Gly Thr Trp Gln Ala Ala Pro Gly Glu Val Gly Gln Gly Val 20 25 30 AAG GTC GCC ATC GAG ACT GGA TAC CGT CAC CTC GAC CTT GCC AAG GTC 144 Lys Val Ala Ile Glu Thr Gly Tyr Arg His Leu Asp Leu Ala Lys Val 35 40 45 TAC TCG AAC CAA CCT GAG GTT GGT GCC GCC ATC AAG GAG GCT GGC GTC 192 Tyr Ser Asn Gln Pro Glu Val Gly Ala Ala Ile Lys Glu Ala Gly Val 50 55 60 AAG CGC GAG GAC CTC TTC ATC ACC TCG AAG CTC TGG AAC AAC TCG CAC 240 Lys Arg Glu Asp Leu Phe Ile Thr Ser Lys Leu Trp Asn Asn Ser His 65 70 75 CGC CCG GAG CAG GTC GAG CCT GCC CTT GAC GAC ACC CTC AAG GAG CTC 288 Arg Pro Glu Gln Val Glu Pro Ala Leu Asp Asp Thr Leu Lys Glu Leu 80 85 90 95 GGC CTC GAG TAC CTC GAC CTT TAC CTC ATT CAC TGG CCC GTC GCG TTC 336 Gly Leu Glu Tyr Leu Asp Leu Tyr Leu Ile His Trp Pro Val Ala Phe 100 105 110 CCG CCC GAG GGC GAC ATC ACC CAG AAC CTC TTC CCG AAG GCC AAC GAC 384 Pro Pro Glu Gly Asp Ile Thr Gln Asn Leu Phe Pro Lys Ala Asn Asp 115 120 125 AAG GAG GTC AAG CTC GAC CTG GAG GTC AGC CTC GTC GAC ACG TGG AAG 432 Lys Glu Val Lys Leu Asp Leu Glu Val Ser Leu Val Asp Thr Trp Lys 130 135 140 GCG ATG GTC AAG CTT CTC GAC ACT GGC AAG GTC AAG GCG ATC GGC GTT 480 Ala Met Val Lys Leu Leu Asp Thr Gly Lys Val Lys Ala Ile Gly Val 145 150 155 TCC AAC TTC GAC GCG AAG ATG GTC GAC GCC ATC ATC GAG GCT ACC GGC 528 Ser Asn Phe Asp Ala Lys Met Val Asp Ala Ile Ile Glu Ala Thr Gly 160 165 170 175 GTG ACC CCC TCC GTC AAC CAG ATC GAG CGT CAC CCT CTC CTT CTC CAG 576 Val Thr Pro Ser Val Asn Gln Ile Glu Arg His Pro Leu Leu Leu Gln 180 185 190 CCC GAG CTC ATC GCC CAC CAC AAG GCC AAG AAC ATT CAC ATT ACC GCA 624 Pro Glu Leu Ile Ala His His Lys Ala Lys Asn Ile His Ile Thr Ala 195 200 205 TAC TCT CCT CTC GGT AAC AAC ACC GTC GGC GCG CCT CTT CTT GTC CAG 672 Tyr Ser Pro Leu Gly Asn Asn Thr Val Gly Ala Pro Leu Leu Val Gln 210 215 220 CAC CCG GAG ATC AAG CGC ATC GCC GAG AAG AAC GGC TGC ACG CCC GCT 720 His Pro Glu Ile Lys Arg Ile Ala Glu Lys Asn Gly Cys Thr Pro Ala 225 230 235 CAG GTC CTC ATT GCC TGG GCC ATC GTT GGC GGC CAC TCG GTT ATC CCC 768 Gln Val Leu Ile Ala Trp Ala Ile Val Gly Gly His Ser Val Ile Pro 240 245 250 255 AAG TCG GTC ACC CCC TCC CGC ATT GGC GAG AAC TTC AAG CAG GTC TCG 816 Lys Ser Val Thr Pro Ser Arg Ile Gly Glu Asn Phe Lys Gln Val Ser 260 265 270 CTC TCG CAG GAG GAC GTC GAT GCC GTC AGC AAG CTC GGC GAG GGT TCG 864 Leu Ser Gln Glu Asp Val Asp Ala Val Ser Lys Leu Gly Glu Gly Ser 275 280 285 GGC CGC AGG CGC TAC AAC ATC CCC TGC ACG TAC TCG CCC AAG TGG GAC 912 Gly Arg Arg Arg Tyr Asn Ile Pro Cys Thr Tyr Ser Pro Lys Trp Asp 290 295 300 ATC AAC GTC TTT GGC GAG GAG GAC GAG AAG TCG TGC AAG AAC GCC GTG 960 Ile Asn Val Phe Gly Glu Glu Asp Glu Lys Ser Cys Lys Asn Ala Val 305 310 315 AAG ATC AAG TAG 972 Lys Ile Lys 320

[Brief description of drawings]

FIG. 1 shows the nucleotide sequences of probe 1 and probe 2 used for plaque hybridization.

FIG. 2 shows the construction method and structure of the recombinant plasmid pSAL2.

FIG. 3 shows the results of comparison of the amino acid sequence deduced from the base sequence with the partial amino acid sequence of the lysyl endopeptidase digested peptide fragment of purified ALD.

FIG. 4 is a diagram showing a sequence subsequent to that of FIG. 3;

FIG. 5 is a diagram showing a continuation of FIG. 4;

FIG. 6 shows the nucleotide sequences of the N-terminal side primer (PN) and the C-terminal side primer (PC) used for cDNA synthesis.

FIG. 7 shows a construction method and structure of a recombinant plasmid pAR.

FIG. 8 shows the results of Coomassie blue staining of Western blotting and electrophoretic gel using an anti-ALD antibody prepared using ALD.

FIG. 9 shows the time course of the reaction in the synthesis of ethyl γ-chloro-β-hydroxybutyrate from ethyl γ-chloroacetoacetate by recombinant Escherichia coli.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 15/09 ZNA C12P 7/62 7432-4B 41/00 D 9452-4B // (C12N 9 / 04 C12R 1:19) (C12N 1/21 C12R 1:19) (C12P 7/62 C12R 1:19) (C12P 41/00 C12R 1:19) (72) Inventor Tadashi Morikawa 3 Asahicho, Machida-shi, Tokyo 5-5-1 Denka Kagaku Kogyo Co., Ltd. Research Institute (72) Inventor Terumi Miyoshi 3-5-1 Asahicho, Machida-shi, Tokyo Denka Kagaku Kogyo Co., Ltd.

Claims (7)

[Claims] 1. A carbonyl reductase having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. 2. A gene DNA having a base sequence shown in SEQ ID NO: 1 of the Sequence Listing and encoding a polypeptide having carbonyl reductase activity. 3. A gene DNA encoding a polypeptide having a carbonyl reductase activity, which has the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing. 4. The gene DNA according to claim 2 or 3,
Recombinant DN integrated in replicable expression vector
A. 5. A transformant transformed with the recombinant DNA according to claim 4. 6. A method for producing carbonyl reductase, which comprises culturing the transformant according to claim 5 and collecting the obtained carbonyl reductase. 7. The transformant according to claim 5 or a treated product thereof is reacted with γ-substituted acetoacetic acid ester, and the produced (R) -γ-substituted-β-hydroxybutyric acid ester is collected. (R) -γ-substituted-β-hydroxybutyric acid ester