JP4809660B2 - 3-quinuclidinone reductase and method for producing (R) -3-quinuclidinol using the same - Google Patents

Description

  The present invention relates to the production of (R) -3-quinuclidinol which is useful as an optically active physiologically active compound used in medicines and agricultural chemicals. More specifically, the present invention relates to an enzyme that asymmetrically reduces 3-quinuclidinone to produce (R) -3-quinuclidinol, and a method for producing (R) -3-quinuclidinol using the enzyme.

  At present, many compounds having physiological activity are used as a mixture of optical isomers. However, the desired activity is often present only in one optical isomer. It is also known that the other optical isomer that does not have the desired activity may be toxic to living organisms. Therefore, in order to provide an effective and safe pharmaceutical or physiologically active compound, it is strongly desired to develop a method for producing an optically active substance having a high optical purity used as a raw material or a synthetic intermediate.

  3-quinuclidinol, which is a reduction product of 3-quinuclidinone, is used as a synthetic intermediate such as a therapeutic agent for arteriosclerosis having a squalene synthase inhibitory activity, a bronchodilator having a muscarinic receptor antagonistic activity, and a gastrointestinal motility inhibitor. It is a known compound (see Patent Documents 1 to 5).

  Optical isomers exist in 3-quinuclidinol having an asymmetric carbon atom. Therefore, it is necessary to isolate optically active 3-quinuclidinol useful as a synthetic intermediate, and several attempts have been made so far.

  As a method for producing optically active 3-quinuclidinol, for example, 1-benzyl-3-hydroxyquinuclidium chloride is optically resolved with a D-tartaric acid derivative to produce (S)-(+)-3-quinuclidinol. A method (Non-Patent Document 1) and a method for synthesizing (S)-(+)-3-quinuclidinol from D-glucose have been reported (Non-Patent Document 2). A method for producing optically active 3-quinuclidinol by asymmetric hydrogenation of a Lewis acid adduct of 3-quinuclidinone using a rhodium complex compound as a catalyst (Patent Document 6) has also been reported. It is not a manufacturing method.

  As a method using a microorganism or an enzyme, for example, (R, S) -quinuclidinyl butyrate is allowed to act on horse serum butylcholinesterase to produce optically active (S)-(+)-3-quinuclidinyl butyrate. A method of leaving a rate (Non-patent Document 3), a method of producing optically active (R)-(−)-3-quinuclidinol with high optical purity by acting a protease derived from a microorganism belonging to the genus Bacillus (patent) There is literature 7). As an example using a racemic 3-quinuclidinol ester as a starting material, a method of producing (R) -3-quinuclidinol using subtilisin protease (Patent Document 8), an Aspergillus genus or Pseudomonas ( Pseudomonas) A method for producing optically active (R)-(−)-3-quinuclidinol and (S)-(+)-3-quinuclidinol by hydrolyzing an ester using an esterolytic enzyme derived from a microorganism belonging to the genus Pseudomonas) (Patent Document 9), and asymmetric hydrolysis by microorganisms and enzymes belonging to the genus Aspergillus, Rhizopus, Candida, or Pseudomonas, Process for producing active 3-quinuclidinol or optically active 3-quinuclidinol ester (patent text) 10) it is also known.

  However, these methods have problems such as low optical purity and difficulty in mass production due to the complexity of the synthesis process. In addition, any of these methods is a method of optically resolving racemic 3-quinuclidinol to obtain a target optical isomer, and therefore the other optical isomer that is not the target remains. Therefore, in these methods, for the non-target optical isomer, the configuration of the asymmetric carbon is reversed and converted to the target optical isomer, or the non-target optical isomer is racemized and reused again. There is a problem that a process for obtaining the desired optical isomer by optical resolution is required, resulting in high production costs.

  Furthermore, in order to efficiently produce an optically active form of quinuclidinol (particularly 3-quinuclidinol) using quinuclidinone (particularly 3-quinuclidinone) as a starting material, it has been reported that an asymmetric reduction reaction by a microorganism or its enzyme is used. (Patent Documents 11 to 14). Patent Document 11 describes that microorganisms that produce (R) -3-quinuclidinol from 3-quinuclidinone include the genus Nakazawaea, the genus Candida, and the genus Proteus. ing. Patent Document 12 includes the genus Alcaligenes, Arthrobacter, Filobasidium, Rhodotorula, Aureobasidium, or Yarrowia. And a microorganism having the ability to produce (R) -3-quinuclidinol from 3-quinuclidinone, and microorganisms belonging to the genus Rhodotorula include Rhodotorula aurantica. Is specifically described. Patent Document 13 includes the genus Geotrichum, the genus Tsukamurella, the genus Micrococcus, the genus Kurthia, the genus Microbacterium, the genus Kluyveromyces, the genus Acremonium. The use of a microorganism belonging to the genus (Acremonium) or the genus Mucor and having the ability to produce (R) -3-quinuclidinol from 3-quinuclidinone is described. Patent Document 14 includes the genus Rhodotorula, the genus Candida, the genus Sporidiobolus, the genus Rhodosporidium, the genus Schizosaccharomyces, and the genus Cryptococcus. Microorganisms belonging to the genus Trichosporon, Gordona, Pichia, and Nocardia are described, and microorganisms belonging to the genus Rhodotorula include Rhodotorula. rubra). It is also described that Rhodotorula rubra produces (R) isomers. In these methods, microbial cells are used for the asymmetric reduction reaction. In such a cell reaction, isolation of the product is often complicated due to impurities derived from the cell, side reactions by other enzymes, and the like.

  A method for producing (R) -3-quinuclidinol from 3-quinuclidinol by an asymmetric reduction reaction utilizing a plant-derived enzyme has been reported (Patent Document 15). In this method, tropinone reductase-I derived from Datura stramonium or Hyoscyamus niger is co-expressed in glucose dehydrogenase and E. coli and combined with a coenzyme regeneration system to perform asymmetric reduction. (R) -3-quinuclidinol is produced from 3-quinuclidinone by reaction.

  It has been reported that a novel enzyme that catalyzes an asymmetric reduction reaction of 3-quinuclidinone has been found for the production of optically active 3-quinuclidinol (Patent Document 16). This enzyme is produced from a microorganism belonging to the genus Microbacterium, and reduces 3-quinuclidinone or a salt thereof using NADH (reduced nicotinamide adenine dinucleotide) as a coenzyme, and (R) -3-quinuclidinol. And (2) the optimum pH when 3-quinuclidinone is used as a substrate is in the range of 7.0 to 9.0.

Thus, in order to efficiently produce optically active 3-quinuclidinol, attempts have been made to discover an enzyme having an action of asymmetric reduction from 3-quinuclidinone to (R) -3-quinuclidinol.
Japanese Patent Laid-Open No. 8-134067 EP-A-404737 European Patent Application No. 424021 International Patent Application Publication No. 92/04346 International Patent Application Publication No. 93/06098 JP-A-9-194480 US Pat. No. 5,215,918 German Patent Application No. 19715465 Japanese Patent Laid-Open No. 10-210997 Japanese Patent No. 3129663 Japanese Patent Laid-Open No. 10-243795 JP 2000-245495 A JP 2002-153293 A JP-A-11-196890 JP 2003-230398 A JP 2003-334069 A A. A. Kalir et al., Israel Journal of Chemistry, 1971, 9, pp.267-268 Gee. W. Jay. GWJ Fleet et al., Tetrahedron Letters, 1986, 27, pp. 3057-3058 M. Rehavi et al., Life Sciences, 1977, 21, pp.1293-1302

  The present invention relates to an enzyme that reduces 3-quinuclidinone to (R) isomer with high stereoselectivity, and a method for efficiently producing optically active (R) -3-quinuclidinol having high optical purity using the enzyme. The purpose is to provide.

The present invention provides enzymes having the following biochemical properties:
(1) asymmetric reduction of 3-quinuclidinone or a salt thereof using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme to produce (R) -3-quinuclidinol; and (2) nicotinamide adenine It does not catalyze the oxidation reaction of 3-quinuclidinol using dinucleotide phosphate (NADP ⁺ ) as a coenzyme. Hereinafter, this enzyme may be referred to as “3-quinuclidinone asymmetric reductase”.

  In one embodiment, the 3-quinuclidinone asymmetric reductase has a subunit molecular weight of 30,000 as measured by SDS-PAGE and a total molecular weight of 93,000 as measured by gel filtration chromatography.

  In another embodiment, the 3-quinuclidinone asymmetric reductase has the amino acid sequence set forth in SEQ ID NO: 41.

  In yet another embodiment, the 3-quinuclidinone asymmetric reductase is derived from Rhodotolura rubra.

  The present invention also provides a polynucleotide encoding the 3-quinuclidinone asymmetric reductase.

  In one embodiment, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase has the base sequence set forth in SEQ ID NO: 40.

  The present invention also provides a vector comprising a polynucleotide encoding the 3-quinuclidinone asymmetric reductase. In the following, for the sake of convenience, this vector is also referred to as “3-quinuclidinone asymmetric reductase expression vector”, which is intended to express only the 3-quinuclidinone asymmetric reductase gene. Do not mean.

In one embodiment, the 3-quinuclidinone asymmetric reductase expression vector further comprises a tac promoter and a lacI ^q repressor gene, and the tac promoter comprises a polynucleotide encoding the 3-quinuclidinone asymmetric reductase. Control.

In a further embodiment, the vector further comprises a polynucleotide encoding an enzyme that regenerates NADP ⁺ into NADPH (hereinafter also referred to as “NADPH regenerating enzyme”). Hereinafter, this vector is also referred to as “3-quinuclidinone asymmetric reductase and NADPH regenerating enzyme co-expression vector”.

In one embodiment, the 3-quinuclidinone asymmetric reductase and NADPH regenerating enzyme co-expression vector includes a lacI ^q repressor gene and two tac promoters, and one of the two tac promoters is the 3-quinuclidinone. It controls a polynucleotide encoding an asymmetric reductase, and the other controls a polynucleotide encoding an enzyme that regenerates the NADP ⁺ to NADPH.

In another embodiment, the enzyme that regenerates NADP ⁺ to NADPH is glucose dehydrogenase.

  In a further embodiment, the glucose dehydrogenase is derived from Bacillus megaterium.

  The present invention also provides a transformant that retains the vector in an expressible manner.

The present invention provides a transformant that retains a vector containing a polynucleotide encoding the 3-quinuclidinone asymmetric reductase and a vector containing a polynucleotide encoding an enzyme that regenerates NADP ⁺ into NADPH in an expressible manner. .

  In one embodiment, in the transformant, the host is E. coli.

  In a further embodiment, the E. coli is an E. coli that is not deficient in the lactose operon.

  The present invention also provides a method for producing (R) -3-quinuclidinol or a salt thereof, wherein the method comprises converting the 3-quinuclidinone asymmetric reductase or the transformant, a culture solution or a treated product thereof to 3-quinuclidinone. Or the process made to act on its salt is included.

In one embodiment, NADP ⁺ is further added in the working step.

  According to the present invention, there is provided an enzyme that reduces 3-quinuclidinone to (R) isomer with high stereoselectivity. Furthermore, by highly expressing the enzyme, a method for efficiently producing (R) -3-quinuclidinol having high optical purity is provided.

  In the present invention, the enzyme is not limited to a purified enzyme, and includes a crude product, an immobilized product, and the like. Enzyme purification is performed using methods well known to those skilled in the art, such as ammonium sulfate precipitation, ion exchange chromatography, and hydrophobic interaction chromatography using, for example, acetone-treated products and cell debris. Of enzymes (including enzymes purified to near homogeneity) can be obtained.

  In the present invention, a transformant refers to a recombinant microorganism into which a gene encoding the 3-quinuclidinone asymmetric reductase of the present invention has been introduced and expressed. The culture solution of the transformant means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like. Examples of treated microbial cells include acetone-treated products, cell disruptions by ultrasonic treatment, cell-free extracts obtained by disrupting cell walls by mechanical and enzymatic methods, surfactants, organic solvents, etc. , And their immobilizates. These can be prepared according to methods well known to those skilled in the art. For example, the acetone-treated product retains the above enzyme activity obtained by, for example, filtration, stirring a microbial cell obtained by culturing or a suspension thereof in cooled (for example, −20 ° C.) acetone. It is a processed powdery microbial cell product. Immobilization of microbial cells is performed, for example, by gelling a gelling agent such as carrageenan or acrylamide in the presence of microbial cells.

<3-Quinuclidinone asymmetric reductase>
The present invention provides 3-quinuclidinone asymmetric reductase. This enzyme has the ability to asymmetrically reduce 3-quinuclidinone or a salt thereof using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme to produce (R) -3-quinuclidinol. This enzyme does not catalyze the oxidation of 3-quinuclidinol using nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) as a coenzyme. These activities are referred to as “3-quinuclidinone asymmetric reductase activity” in the following description. This enzyme cannot use reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme.

  The 3-quinuclidinone asymmetric reductase of the present invention may vary slightly depending on the electrophoresis conditions. For example, a natural enzyme derived from Rhodotolura rubra has a subunit molecular weight of approximately about 0.1 by SDS-PAGE measurement. 30,000, molecular weight measured by gel filtration chromatography is about 93,000. This enzyme is a homotrimer.

  The enzyme has an optimum pH in the range of 6.0 to 8.0 when 3-quinuclidinone is used as a substrate. This enzyme is stable in a wide range from pH 6.0 to pH 8.5 when treated at 37 ° C. for 16 hours.

  The optimum temperature of the enzyme is preferably 0 to 50 ° C, more preferably around 40 ° C. This enzyme is stable up to 40 ° C., for example, when treated for 30 minutes in 100 mM potassium phosphate buffer (pH 7.0).

The activity of the 3-quinuclidinone asymmetric reductase of the present invention can be confirmed, for example, as follows. The activity was measured basically by incubating 200 μl of 100 mM potassium phosphate buffer (pH 7.0) containing 0.5 M of 3-quinuclidinone hydrochloride, 0.5 mM NADPH, and 50 μl of enzyme solution at 37 ° C. This can be done by measuring the decrease in absorbance at a wavelength of 340 nm. The enzyme amount of the 3-quinuclidinone asymmetric reductase is defined as 1 unit (U), which is the amount of enzyme that converts 1 micromole of NADPH to NADP ⁺ per minute. It can be confirmed that the 3-quinuclidinone asymmetric reductase of the present invention does not utilize NADH as a coenzyme by measuring NADPH in place of NADH. Also, basically, 200 μl of 100 mM potassium phosphate buffer (pH 7.0) containing 0.5 M (R) -3-quinuclidinol, 0.5 mM NADP ⁺ and 50 μl of enzyme solution was incubated at 37 ° C. By measuring the increase in absorbance at 340 nm, it can be confirmed that it does not catalyze the oxidation reaction of (R) -3-quinuclidinol to 3-quinuclidinone.

  The 3-quinuclidinone asymmetric reductase of the present invention preferably has the amino acid sequence set forth in SEQ ID NO: 41. As long as the enzyme has 3-quinuclidinone asymmetric reductase activity, one or more amino acids are substituted, deleted, inserted, or substituted for the amino acid sequence of the natural enzyme (for example, the amino acid sequence described in SEQ ID NO: 41). And / or an enzyme having an added amino acid sequence. A person skilled in the art, for example, site-directed mutagenesis (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; PCR A Practical Approach, IRL Press, 1991, pp. 200), etc. Alternatively, the structure of the protein can be altered by introducing additional mutations. In the present invention, the number of amino acid residues that can be substituted, deleted, inserted, and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, even more preferably. Is 0 to 3 amino acid residues. In addition, since amino acid mutations may occur in nature, not only enzymes that have artificially mutated amino acids, but also enzymes in which amino acids have been mutated in nature, as long as they have 3-quinuclidinone asymmetric reductase activity. Included in 3-quinuclidinone asymmetric reductase.

  As long as the protein having an amino acid sequence having homology to the amino acid sequence of the natural enzyme (for example, the amino acid sequence shown in SEQ ID NO: 41) also has 3-quinuclidinone asymmetric reductase activity, the 3- Included in quinuclidinone asymmetric reductase. The 3-quinuclidinone asymmetric reductase of the present invention is preferably at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% with the amino acid sequence set forth in SEQ ID NO: 41, More preferably, it may be a protein having an amino acid sequence having at least 99% homology. Protein homology search, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, or databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and estimation based on DNA sequences For example, it can be performed through the Internet using a program such as BLAST or FASTA for a database relating to an amino acid sequence. Confirmation of the activity of the protein can be performed using the procedure described above.

  Although the supply source of the 3-quinuclidinone asymmetric reductase of the present invention is not particularly limited, it can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Rhodotorula. Preferably, Rhodotolura rubra JCM3782 strain, Rhodotolura rubra JCM3785 strain, Rhodotolura rubra JCM8113 strain, Rhodotolura rubra JCM8117 strain, Rodotolura rubra JCM8117 strain, Examples include the IFO0389 strain, Rhodotolura graminis IFO0190 strain, and Rhodotolura minuta IFO0879 strain. Particularly preferred is Rhodotolura rubra JCM3782. Note that strains with IFO numbers and JCM numbers are known, and can be easily obtained from the Fermentation Research Institute and RIKEN Microbial System Storage Facility, respectively.

  The medium used for culturing the microorganism may be either a solid medium or a liquid medium that is used routinely and is known to those skilled in the art. Preferably, a liquid medium is used. As the carbon source and nitrogen source of the medium used for culturing the microorganism, any carbon source and nitrogen source that can be used by the microorganism to be cultured can be used. Such carbon sources preferably include sugars such as starch, sucrose or glucose, alcohols such as glycerol, and organic acids (eg acetic acid and citric acid) or salts thereof (eg sodium salts). Preferred nitrogen sources include natural nitrogen sources such as yeast extract, casein, corn steep liquor, peptone, meat extract, and inorganic nitrogen compounds such as ammonium sulfate, ammonium sulfate, and urea. The concentration of the carbon source is in the range of 1 to 20% (w / v), preferably 1 to 5% (w / v). The concentration of the nitrogen source is in the range of 1-20% (w / v), preferably 1-5% (w / v). The culture temperature is a temperature at which the above enzyme is stable and the cultured microorganism can sufficiently grow, and is preferably 20 to 30 ° C. The culture time is a time during which the above enzyme is sufficiently produced, and is preferably about 16 to 48 hours. Culturing can preferably be carried out under aerobic conditions, for example using aeration or shaking.

  The 3-quinuclidinone asymmetric reductase of the present invention is fractionated by protein solubility (precipitation by organic solvent, salting out by ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, Purification can be performed by appropriately combining known methods such as affinity chromatography using dyes, antibodies, and the like. For example, after preparing the cell-free extract by disrupting the cells of the above microorganisms, ammonium sulfate fractionation, hydrophobic chromatography, affinity chromatography using a red dye as a ligand, and affinity chromatography using a blue dye as a ligand are performed. Thus, it can be purified to a single band in polyacrylamide gel electrophoresis (SDS-PAGE).

<3-nucleotide encoding quinuclidinone asymmetric reductase>
A polynucleotide encoding the 3-quinuclidinone asymmetric reductase is also included in the present invention. The polynucleotide of the present invention may be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The polynucleotide of the present invention may be a DNA-RNA chimeric molecule. The polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention includes, for example, the base sequence set forth in SEQ ID NO: 40. The base sequence set forth in SEQ ID NO: 40 encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 41, and the protein comprising this amino acid sequence constitutes a preferred form of the 3-quinuclidinone asymmetric reductase according to the present invention. To do.

  As the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention, one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence set forth in SEQ ID NO: 41 as described above. And a polynucleotide encoding a protein having the amino acid sequence and having 3-quinuclidinone asymmetric reductase activity. Those skilled in the art will be able to introduce site-directed mutagenesis into the polynucleotide of SEQ ID NO: 40 (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; PCR A Practical Approach, IRL Press, 1991, pp. 200) It is possible to obtain polynucleotide homologs by introducing deletions, insertions, and / or addition mutations.

  Moreover, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention is hybridized under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide having the base sequence set forth in SEQ ID NO: 40. Also included are polynucleotides that can be produced and that encode proteins having 3-quinuclidinone asymmetric reductase activity.

  The polynucleotide of the present invention can obtain the target gene from the microorganism described above based on the base sequence information described in the present specification. PCR and hybridization screening are used for gene acquisition. It is also possible to chemically synthesize the full length of a gene by DNA synthesis. Based on the base sequence information, a polynucleotide encoding the 3-quinuclidinone asymmetric reductase derived from an organism other than the above can also be obtained. For example, 3-quinuclidinone derived from various organisms can be obtained by designing a probe using the above base sequence or a partial sequence thereof and performing hybridization under conditions stringent to DNA prepared from another organism. A polynucleotide encoding an asymmetric reductase can be isolated. Based on the above base sequence information, PCR primers can be designed from regions with high homology using sequence information registered in DNA databases such as DNA Databank of JAPAN (DDBJ), EMBL, and Gene-Bank. it can. By using such primers and performing PCR using chromosomal DNA or cDNA as a template, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase can also be isolated from various organisms.

  The polynucleotide that can hybridize under stringent conditions refers to any at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in the nucleotide sequence set forth in SEQ ID NO: 40. One or a plurality of probes are selected and designed using, for example, ECL direct nucleic acid labeling and detection system (manufactured by Amersham Biosciences) under the conditions described in the manual (for example, washing conditions: 42 ° C., primary containing 0.5 × SSC) In the wash buffer), it refers to the hybridizing polynucleotide.

  More specifically, “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions. More preferably, the conditions are 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  Furthermore, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably the amino acid sequence set forth in SEQ ID NO: 41. It includes a polynucleotide that encodes a protein having an amino acid sequence with at least 95%, even more preferably at least 99% homology, and having 3-quinuclidinone asymmetric reductase activity. The protein homology search is as described above.

  The polynucleotide encoding the 3-quinuclidinone asymmetric reductase can be expressed in the same or different host using genetic recombination techniques.

<Vector and transformant>
The vector of the present invention contains the above polynucleotide. The transformant of the present invention holds the vector of the present invention so that it can be expressed.

  A procedure for producing a transformant and construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In order to express a polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention in a microorganism, this DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and its genetic information is obtained. It is necessary to transcribe and translate. For this purpose, a promoter corresponding to a unit that controls transcription / translation may be incorporated upstream of the 5 ′ side of the DNA strand of the present invention, and more preferably a terminator may be incorporated downstream of the 3 ′ side. As the promoter and terminator, a promoter and terminator known to function in a microorganism used as a host are used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”, especially regarding yeast, Adv. Biochem. Eng., 1990, 43, pp. 75-102; Yeast, 1992, Vol. 8, pp.423.

For example, when E. coli is used as a host, pBR and pUC plasmids can be used as plasmid vectors. As the promoter, promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (fusion of lac, trp), λ phage PL, PR and the like can be used. From the viewpoint of expression efficiency, the tac promoter is preferable. Moreover, as the terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used. In order to increase the expression efficiency, a repressor gene (for example, a lactose repressor gene) can be further included. In particular, the highly expressed lacI ^q gene can be preferably used. The lacI ^q gene is a lacI gene connected to a regulatory promoter site that has been mutated to allow overexpression of the lacI repressor protein. Since the production amount of the repressor protein is increased by the expression of the lacI ^q gene arranged on the plasmid, the expression of the target gene by the promoter can be controlled more efficiently. In addition, in order to clarify that the lacI ^q gene is a repressor gene, it may be referred to as “lacI ^q repressor gene”.

In the transformant of the present invention, it is preferable that a regeneration system for coenzyme NADP ⁺ into NADPH is introduced. In the process of reducing 3-quinuclidinone to produce (R) -3-quinuclidinol, the 3-quinuclidinone asymmetric reductase of the present invention requires NADPH. Accompanying the reduction reaction by the 3-quinuclidinone asymmetric reductase, NADP ⁺ is produced from NADPH. NADP ⁺ can be regenerated back to NADPH, which is reduced, by utilizing the oxidation reaction of an appropriate substrate. Therefore, in order to increase the efficiency of the substrate reduction reaction, the transformant of the present invention preferably expresses an enzyme that regenerates NADP ⁺ into NADPH (“NADPH regenerating enzyme”). The NADPH regenerating enzyme in the present invention includes glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydrogenase. Enzymes, and glycerol dehydrogenase. Glucose dehydrogenase (GDH) is preferable. The glucose dehydrogenase is preferably a glucose dehydrogenase derived from Bacillus megaterium, and more preferably a glucose dehydrogenase derived from Bacillus megaterium IAM1030. A gene encoding glucose dehydrogenase derived from Bacillus megaterium strain IAM1030 has already been isolated, and its sequence information has been registered in a database (UniProtKB / Swiss-Prot Accession No. P39482). It can also be obtained from the microorganism by PCR or hybridization screening based on the base sequence registered in the database.

  When multiple genes are introduced into a single vector, the regions involved in expression control such as promoters and terminators should be linked to each gene, and expressed as an operon containing multiple cistrons such as lactose operon. Is possible.

  These two or more genes can be introduced into a host by a method of transforming the host with a recombinant vector in which genes are separately introduced into a plurality of vectors having different origins of replication in order to avoid incompatibility, A method of introducing both genes into one vector, a method of introducing one or both genes into a chromosome, and the like can be used.

In order to enhance the expression of the 3-quinuclidinone asymmetric reductase gene, a vector containing a polynucleotide encoding this enzyme may contain a tac promoter as a promoter. Further, it may contain a lacI ^q repressor gene that is highly expressed.

  Regarding introduction of a polynucleotide encoding 3-quinuclidinone asymmetric reductase and a polynucleotide encoding NADPH regenerating enzyme into a host, a) a method of introducing both genes into a single vector, and b) different origins of replication The method will be described separately with a method of transforming a host with a recombinant vector in which genes are separately introduced into a plurality of vectors.

In the case of a), in order to simultaneously express a large amount of 3-quinuclidinone asymmetric reductase and NADPH regenerating enzyme, two high-expression promoters are arranged so that each gene can be controlled separately. That is, a polynucleotide encoding 3-quinuclidinone asymmetric reductase is disposed downstream of one of the two highly expressed promoters, and a polynucleotide encoding NADPH regenerating enzyme is disposed downstream of the other, One expression unit is constructed. This promoter is preferably the tac promoter. These expression units may further comprise a terminator. Furthermore, it is preferable that a lactose repressor gene (preferably, a lacI ^q repressor gene) is arranged on a vector containing these two expression units. In one embodiment, the “3-quinuclidinone asymmetric reductase and NADPH regenerating enzyme co-expression vector” of the present invention is a polynucleotide encoding 3-quinuclidinone asymmetric reductase and a polynucleotide encoding NADPH regenerating enzyme. In addition, it contains a lacI ^q repressor gene and two tac promoters, one of the two tac promoters controlling the polynucleotide encoding the 3-quinuclidinone asymmetric reductase, and the other comprising the NADP ⁺ Controls a polynucleotide encoding an enzyme that regenerates to NADPH.

In the case of b), a 3-quinuclidinone asymmetric reductase expression vector and a vector for expressing NADPH regenerating enzyme are used. In order to increase the expression of the 3-quinuclidinone asymmetric reductase gene, the 3-quinuclidinone asymmetric reductase expression vector is a tac promoter upstream of the polynucleotide encoding 3-quinuclidinone asymmetric reductase as described above. An expression unit comprising A lactose repressor gene (preferably, a lacI ^q repressor gene) can be further arranged on a vector containing the expression unit. In one embodiment, the “3-quinuclidinone asymmetric reductase expression vector” of the present invention may contain a tac promoter and a lacI ^q repressor in addition to a polynucleotide encoding 3-quinuclidinone asymmetric reductase. Here, the tac promoter can control a polynucleotide encoding 3-quinuclidinone asymmetric reductase. The vector for expressing the NADPH regenerating enzyme preferably includes an expression unit including a high expression promoter upstream of the polynucleotide encoding the NADPH regenerating enzyme. This highly expressed promoter is preferably a tac promoter. These expression units may further comprise a terminator.

  A host organism to be transformed in order to express a polynucleotide encoding the 3-quinuclidinone asymmetric reductase is transformed with a recombinant vector containing the polynucleotide encoding the 3-quinuclidinone asymmetric reductase. There is no particular limitation as long as it is an organism capable of expressing 3-quinuclidinone asymmetric reductase activity. For example, Escherichia coli, Streptomyces lividans, Aspergillus oryzae and the like can be used. E. coli is preferred because of the ease of genetic recombination operations. In particular, from the viewpoint of expression efficiency, Escherichia coli having no deficiency in the lactose operon is preferable. Examples of such Escherichia coli include Escherichia coli SCS1 strain, W3110 strain, and BL21 strain.

<Production method of (R) -3-quinuclidinol>
(R) -3-quinuclidinol or a salt thereof is efficiently produced by allowing the 3-quinuclidinone asymmetric reductase or the transformant, a culture solution or a treated product thereof to act on 3-quinuclidinone or a salt thereof. “3-Quinuclidinone or a salt thereof” means that a compound formed with a salt of an organic acid, a mineral acid, or the like at its nitrogen atom can also be used in the asymmetric reduction of 3-quinuclidinone by the 3-quinuclidinone asymmetric reductase Intended. Specifically, the mineral acid salt includes hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like. Examples of the organic acid salt include aliphatic organic acid salts such as acetate, propionate, butyrate, fumaric acid, malonic acid, and oxalic acid, and aromatic organic acid salts such as benzoic acid. In the following description, only “3-quinuclidinone” is described, but a salt of 3-quinuclidinone as described above can also be used.

  In the method of the present invention, an appropriate reaction solution containing the 3-quinuclidinone asymmetric reductase or the transformant, a culture solution or a processed product thereof (hereinafter collectively referred to as “biological catalyst”). In, for example, 3-quinuclidinone is asymmetrically reduced to obtain (R) -3-quinuclidinol as a product.

  In one embodiment of the method of the present invention, (R) -3-quinuclidinol can be obtained by culturing the above transformant in the presence of 3-quinuclidinone. The amount of 3-quinuclidinone added to the culture solution is 0.1 to 30% (w / v), preferably 0.1 to 10% (w / v). The added 3-quinuclidinone may not necessarily be completely dissolved. When the added 3-quinuclidinone is not completely dissolved, for example, the solubility may be improved as a salt (for example, hydrochloride), or an organic solvent such as ethanol is not substantially inhibited from the reduction reaction. (For example, 5%) may be added.

  In another embodiment of the method of the present invention, (R) -3-quinuclidinol is cultured in a suitable reaction solution (water or buffer), or the transformed transformant cells or treated product thereof (eg, acetone). The treated product) can be obtained by contacting with 3-quinuclidinone. The amount of 3-quinuclidinone added to the reaction solution is 0.1 to 30% (w / v), preferably 0.1 to 10% (w / v). The added 3-quinuclidinone may not necessarily be completely dissolved. If the added 3-quinuclidinone is not completely dissolved, for example, the solubility may be improved as a salt (eg, hydrochloride), or a solvent such as ethanol may be used to substantially reduce the reduction reaction with a biological catalyst. You may add to the density | concentration (for example, up to 5%) which does not inhibit in the. In particular, when a treated bacterial cell is used, NADPH is preferably added as a coenzyme. The amount of coenzyme added is 0.1 to 1000 mg / l, more preferably 2 to 200 mg / l.

  The reaction can be performed under a controlled pH. In order to maintain an optimum pH for the reaction, a buffer solution such as a phosphate buffer solution is used. The pH of the reaction solution is 5 to 9, preferably 6 to 8. Alternatively, the pH of the reaction solution is maintained and adjusted to an optimum pH by adding an acid (for example, sulfuric acid) and an alkali (for example, sodium hydroxide) while monitoring the pH that changes as the reaction proceeds. obtain.

  The amount of the biological catalyst is appropriately determined depending on its shape and its activity, and is not particularly limited. About 0.1 to 10 g / mol substrate, preferably about 1 to 5 g / mol substrate relative to 3-quinuclidinone as its substrate.

  The reaction temperature is 10 ° C to 50 ° C, preferably 25 ° C to 35 ° C.

  The reaction time varies depending on the amount of biological catalyst used, reaction temperature, reaction pH, etc., but is usually about 1 to 72 hours.

In the method for producing (R) -3-quinuclidinol, it is preferable to combine the coenzyme regeneration system as described above. Playback from NADP ⁺ to NADPH can be carried out by an enzyme to regenerate plants, microorganisms, and NADPH from NADP ⁺ containing transformants. The enzyme that catalyzes the regeneration reaction of NADPH may be a single enzyme or a multistage reaction composed of a plurality of enzymes. When a target enzyme reaction is supported by a plurality of enzymes, an assembly of enzymes constituting a series of enzyme reactions is called an enzyme system. The NADP ⁺ reducing ability can be enhanced by adding sugars such as glucose and sucrose, organic acids, or alcohols such as ethanol and isopropanol to the reaction system. In addition, NADPH can be regenerated using an enzyme capable of generating NADPH from NADP ⁺ . Enzymes useful for the production of NADPH include glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydration Elemental enzymes, and glycerol dehydrogenase. As these enzymes, not only purified enzymes but also microorganisms having the enzymes, processed products thereof, or partially purified enzymes can be used. For example, in the case of glucose dehydrogenase, regeneration from NADP ⁺ to NADPH is performed with oxidation of glucose to δ-gluconolactone.

  The components constituting the reaction necessary for NADPH regeneration can be added to or immobilized on the 3-quinuclidinone asymmetric reductase reaction system constituting the present invention. Alternatively, the reaction system can be contacted through a membrane capable of exchanging NADPH.

  In the method for producing (R) -3-quinuclidinol, when a transformant transformed with a recombinant vector containing a polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention is used, for NADPH regeneration In some cases, an additional reaction system can be eliminated. That is, by using an organism having a high NADPH regeneration activity as a host, an efficient reaction can be performed in a reduction reaction using a transformant without adding an enzyme for NADPH regeneration. Alternatively, it is also possible to use a host into which the gene for an enzyme that can be used for NADPH regeneration is introduced at the same time as the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention. By using such a transformant, the expression of NADPH regenerating enzyme and the 3-quinuclidinone asymmetric reductase of the present invention and the reduction reaction of the substrate can be performed more efficiently.

When coenzyme regeneration systems are combined, an energy source such as coenzyme NADP ⁺ or / and glucose, sucrose, ethanol, methanol, etc. can be added to the culture or reaction. The amount of the coenzyme NADP ⁺ or energy source added can be appropriately determined based on the amount of the substrate 3-quinuclidinone and the biological catalyst. As described above, the reaction can be performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution. Depending on the case, 3-quinuclidinone which is a reaction substrate or / and the above coenzyme and an energy source may be appropriately added during the reaction to continue the reaction.

  In the method of the present invention, the produced (R) -3-quinuclidinol takes advantage of the difference in chemical or physical properties of the substrate 3-quinuclidinone and the produced (R) -3-quinuclidinol as required. Can be separated. In the method of the present invention, since (R) -3-quinuclidinol having an optical purity of 99.9% or more is generated, (R) -3-quinuclidinol can be obtained without separation from the (S) isomer.

  The produced (R) -3-quinuclidinol is usually used in the field such as solvent extraction, crystal precipitation, column chromatography, etc., depending on its properties (for example, solubility, hydrophobicity) after completion of the reaction. The unreacted 3-quinuclidinone is separated using the separation procedure used. For example, in the solvent extraction method, unreacted 3-quinuclidinone is extracted from the reaction solution with toluene under basic conditions, and then the produced (R) -3-quinuclidinol is recovered using a solvent such as chloroform or dichloromethane. Can do.

  The resulting product can be further purified using the above means if necessary. In this case, the second means is preferably different from the first means (for example, when solvent extraction is used as the first means, the second means includes, for example, column chromatography).

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

(Example 1: Purification of 3-quinuclidinone asymmetric reductase)
According to the following method, 3-quinuclidinone asymmetric reductase was electrophoretically purified from Rhodotolura rubra JCM3782 strain.

(culture)
Prepare 8,400 ml of YM medium (glucose 1%, peptone 0.5%, yeast extract 0.3%, malt extract 0.3%, pH 5.5), and dispense 600 ml each into a 3 L Erlenmeyer flask. Steam sterilization was carried out at 20 ° C. for 20 minutes. Three ml of the culture solution of Rhodotolura rubra JCM3782 previously cultured in the same medium was inoculated, and cultured with shaking at 28 ° C. for 3 days. The cells were collected from this culture by centrifugation (8,600 × g, 10 minutes, 4 ° C.) and washed once with demineralized water. 460 g of the wet cells thus obtained was stored frozen at −20 ° C. until just before use.

(Preparation of cell-free extract)
213 g of wet cells were thawed at room temperature and suspended in 20 mM potassium phosphate buffer (pH 7.0) in which 220 ml of 1 mM DTT and protease inhibitor mixed tablets (Roche) were dissolved. Subsequently, this microbial cell was crushed with a mini motor mill (manufactured by Eiger Japan). The cell residue was removed from the disrupted cells by centrifugation (11,000 × g, 10 minutes, 4 ° C.) to obtain 250 ml of a cell-free extract.

(Ammonium sulfate fraction)
Ammonium sulfate was added to the cell-free extract to 35% saturation and dissolved. The resulting precipitate was then removed by centrifugation (12,400 × g, 30 minutes, 4 ° C.) to give 270 ml of 35% saturated solution. To this solution, ammonium sulfate was further added so as to be 60% saturation, and the solution was left at 4 ° C. overnight. The salted-out product was then collected by centrifugation (12,400 × g, 30 minutes, 4 ° C.). The obtained salted-out product was dissolved in 300 ml of 100 mM potassium phosphate buffer (pH 7.0), the precipitate was removed by ultracentrifugation (100,000 × g, 60 minutes, 4 ° C.), and a clear crude enzyme solution was obtained. Got.

(Hydrophobic chromatography)
A 100 mM potassium phosphate buffer (pH 7.0) was added to the resulting crude enzyme solution to adjust the ammonium sulfate concentration to 30% saturation to obtain 550 ml of the crude enzyme solution. This was added to 100 ml of a Butyl-Toyopearl (Tosoh) column pre-equilibrated with 100 mM potassium phosphate buffer (pH 7.0) containing 30% saturated ammonium sulfate, washed with the same buffer, and then ammonium sulfate (35% The active fraction was eluted with a linear gradient (from saturation to 0% saturation).

(Affinity chromatography using red dye as a ligand)
The obtained active fraction (48 ml) was collected, desalted and concentrated by ultrafiltration and replaced with 20 mM potassium phosphate buffer (pH 7.0), and equilibrated with the same buffer. Red-Toyopearl (manufactured by Tosoh Corporation) ) Added to 20 ml column. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of potassium chloride (from 0 M to 0.5 M).

(Affinity chromatography with blue dye as ligand)
The obtained active fraction (81 ml) was collected, desalted and concentrated by ultrafiltration, and added to 25 ml of a Blue-Toyopearl (manufactured by Tosoh) column previously equilibrated with 20 mM potassium phosphate buffer (pH 7.0). . After washing the column with the same buffer, the active fraction was eluted with a linear gradient of potassium chloride (from 0 M to 0.5 M). As a result of analyzing each active fraction by SDS-PAGE, it was confirmed that it was a single band in the remaining 6 fractions excluding 1 fraction. These 6 fractions were collected to obtain a purified enzyme preparation.

(Example 2: Measurement of properties of 3-quinuclidinone asymmetric reductase)
The enzymatic properties of the purified enzyme preparation obtained in Example 1 were examined.

The enzyme activity was measured basically under the following standard reaction conditions: 0.5 M substrate 3-quinuclidinone hydrochloride, 0.5 mM coenzyme in 100 mM potassium phosphate buffer (pH 7.0). 200 μl of the reaction solution containing NADPH and 50 μl of the enzyme solution was incubated at 37 ° C., and the decrease in absorbance at a wavelength of 340 nm was measured. The amount of enzyme that converts 1 micromole of NADPH to NADP ⁺ per minute was defined as 1 unit (U). The enzymological properties of the purified enzyme preparation were as follows.

  Action: The quantitative and optical purity of 3-quinuclidinol produced under the standard reaction conditions was measured by gas chromatography analysis. In the gas chromatography analysis, a flame ionization detector was used as a detector and a γ-DEX225 capillary column (0.25 mm (inner diameter) × 30 m; manufactured by Spelco) was used as a column. The inlet temperature was 220 ° C., the column temperature was 150 ° C. (20 minutes), and the detector temperature was 220 ° C. Helium was used as a carrier gas at a flow rate of 70 KPa. As a result, NADPH was used as a coenzyme to act on 3-quinuclidinone to produce (R) -3-quinuclidinol with an optical purity of 99.9% ee or higher.

Coenzyme: Production of (R) -3-quinuclidinol was measured according to the standard reaction conditions using NADPH or NADH as a coenzyme. It was NADPH-dependent and NADH was not used as a coenzyme. When the production of 3-quinuclidinone from (R) -3-quinuclidinol was examined according to the standard reaction conditions using NADP ⁺ as a coenzyme and 3-quinuclidinol as a substrate, 3-quinuclidinol using NADP ⁺ as a coenzyme The oxidation reaction was not catalyzed.

  Optimal pH of action: 3-quinuclidinone reducing activity was measured by changing the pH using a 100 mM potassium phosphate buffer. The optimum pH of the reaction was 7.0, and showed an activity of 70% or more of the maximum activity in a wide range from pH 6.0 to pH 8.0.

  pH stability: The purified enzyme was treated in 100 mM potassium phosphate buffer having a different pH at 37 ° C. for 16 hours, and then the residual activity was measured under the above standard reaction conditions. Residual activity of 80% or more was exhibited in a wide range from pH 6.0 to pH 8.5.

  Optimum temperature of action: 3-quinuclidinone reducing activity was measured by changing only the temperature among the above standard reaction conditions. The optimum temperature for the reaction was around 40 ° C.

  Temperature stability: The purified enzyme was treated in 100 mM potassium phosphate buffer (pH 7.0) at various temperatures for 30 minutes, and the residual activity was measured under the standard reaction conditions described above. Up to 40 ° C., 98% or more of the activity before the treatment remained.

  Inhibitor: Various metal ions or inhibitors were added to the reaction solution under the above standard reaction conditions, and 3-quinuclidinone reduction activity was measured. Inhibited by mercuric chloride and p-chloromellicribenzoic acid.

  Molecular weight: The molecular weight of the purified enzyme subunit was determined by SDS-PAGE and found to be about 30,000. Further, the molecular weight measured by gel filtration chromatography using Superdex G200 column (manufactured by Amersham Bioscience) was about 93,000. From these results, the purified enzyme preparation was predicted to be a homotrimer.

  Km value: The reaction was carried out by changing the substrate concentration in the reaction solution under the above standard reaction conditions, and the Michaelis constant Km obtained by the line Weber-Burk plot was 145 mM. Further, the Michaelis constant Km obtained by changing the concentration of the coenzyme NADPH was 0.19 mM.

(Example 3: Analysis of partial amino acid sequence of 3-quinuclidinone asymmetric reductase)
The purified 3-quinuclidinone asymmetric reductase obtained in Example 1 was denatured in the presence of 8M urea, and then digested with Achromobacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). Separation was performed using a Smart system (Amersham Bioscience). The four peptide peaks thus separated were designated as peptide K14, peptide K20, peptide K24, and peptide K30, and the amino acid sequence was analyzed using a protein sequencer (Applied Biosystems, model 476A). The amino acid sequences of peptide K14, peptide K20, peptide K24, and peptide K30 were as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 in the sequence listing, respectively.

  By using the sequence similarity search programs BLAST and FASTA, these amino acid sequences were compared with sequences in three protein sequence databases (PTR, PRF, and SWISS-PROT). As a result, the K14 sequence is a partial amino acid sequence of a short-chain alcohol dehydrogenase (Nature, 2002, 415, pp.871-880) presumed to be involved in sorbitol metabolism in Schizosaccharomyces pombe. And the K30 sequence is Candida albicans peroxysome 2,4-dienoyl-CoA reductase (Proc. Natl. Acad. Sci. USA, 1998, 95, pp. 5150-). 5155) was similar to the partial amino acid sequence. These enzymes belong to the short chain dehydrogenase / reductase (SDR) family. However, there was no similarity between K20 and K24 with proteins on the database.

(Example 4: Cloning of 3-quinuclidinone asymmetric reductase gene)
Rhodotolura rubra JCM3782 strain was cultured in YM medium to prepare bacterial cells. Purification of chromosomal DNA from the bacterial cells was performed by the method described in Appl. Microbiool. Biotechnol., 2000, 53, pp.415-419. Based on the amino acid sequences of the internal peptide fragments K14 and K30 of 3-quinuclidinone asymmetric reductase, degenerate oligonucleotide primers for PCR QR1 (SEQ ID NO: 5), QR2 (SEQ ID NO: 6), QR3 (SEQ ID NO: 7) and QR4 (SEQ ID NO: 8) was designed. QR1 and QR2 were prepared as upstream primers, and QR3 and QR4 were prepared as downstream primers. QR2 and QR3 were designed based on the amino acid sequence inside QR1 and QR4, respectively, and designed to amplify the inside of the DNA fragment amplified with QR1 and QR4. Here, R in the sequence listing is A or G, N is arbitrary, H is other than G, and Y is C or T. The PCR reaction solution composition is as follows: template DNA, 10 × Ex Taq Buffer 10 μl, primer (QR1 or QR2, 100 μM each) 1 μl, primer (QR4 or QR3, 100 μM each) 1 μl, dNTP mixture (2.5 mM each) ) 8 μl, and EX Taq 0.5 μl, and distilled water was added to 100 μl. The PCR reaction conditions were as follows: Step 1: 94 ° C., 10 minutes; Step 2: 94 ° C., 30 seconds; Step 3: 65 ° C., 30 seconds; Step 4: 72 ° C., 1 minute; Step 5 : 94 ° C, 30 seconds; Step 6: 65 ° C, 30 seconds; Step 7: 72 ° C, 1 minute; Step 8: 4 ° C, ∞. The annealing temperature (Step 3) is decreased by 1 ° C. every cycle, and Step 2 to Step 4 are repeated 20 cycles. Step 5 to Step 7 were repeated 20 cycles.

  First, 100 ng of chromosomal DNA was used as a template, and 1st-PCR was performed using primers QR1 and QR4. Next, Nested-PCR was performed using primers QR2 and QR3 using a solution obtained by diluting the reaction solution amplified by 1st-PCR 10 times as a template, and a specific amplification product of about 500 bp was obtained. The PCR reaction solution was subjected to agarose electrophoresis, and the target band portion of about 500 bp was cut out and purified by Wizard SV Gel and PCR Clean-up (Promega). The obtained DNA fragment was ligated to the pCR4-TOPO vector using TOPO TA Cloning Kit for Sequencing (manufactured by Invitrogen) to transform E. coli TOP10 strain. The transformed strain is cultured in LB medium (peptone 1%, yeast extract 0.5%, sodium chloride 1%, pH 7.0) containing ampicillin 50 μg / ml, and DNA is used using QIAprep Spin Miniprep Kit (Qiagen). The plasmid for sequencing was extracted and purified. Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using the T7 primer and M13 reverse primer derived from the pCR4-TOPO vector. This base sequence (SEQ ID NO: 38) and deduced amino acid sequence (SEQ ID NO: 39) are shown in FIG. In FIG. 1, the underlined base sequence is presumed to be an intron region, and the enclosed amino acid sequence matches the sequence obtained by amino acid sequence analysis of the purified enzyme. From the result of the sequence analysis shown in FIG. 1, the PCR product is composed of 489 bp nucleotides, and the deduced amino acid sequence is the internal amino acid sequence of the natural 3-quinuclidinone asymmetric reductase purified in Example 1 (shown in Example 3). ). It was also found that the region encoding the K20 peptide is divided by two introns.

(Example 5: cDNA cloning of 3-quinuclidinone asymmetric reductase)
It was suggested that there are multiple introns in the genomic region encoding the 3-quinuclidinone asymmetric reductase of Rhodotolura rubra JCM3782. In order to clarify the coding region of 3-quinuclidinone asymmetric reductase, cDNA of 3-quinuclidinone asymmetric reductase was cloned by the method shown below.

(Extraction of mRNA from 3-quinuclidinone asymmetric reductase-producing bacterium Rhodotolura rubra JCM3782)
Rhodotolura rubra JCM3782 strain was cultured in YM medium, and the resulting cells were stored frozen at −80 ° C. until immediately before use, and total RNA was prepared by the following procedure. To about 0.1 g of frozen cells, 0.5 g of glass beads (particle size 0.5 mm), 1 ml of Sepasol RNA I reagent (manufactured by Nacalai Tesque) were added and crushed with a mini bead beater (manufactured by Biospec). 200 μl of chloroform was added to this solution and stirred, followed by centrifugation (16,500 × g, 15 minutes, 4 ° C.). The upper aqueous layer was separated, 500 μl of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. After centrifugation (16,500 × g, 10 minutes, 4 ° C.), the supernatant was removed, 1 ml of 75% ethanol was added, and the mixture was centrifuged again (16,500 × g, 5 minutes, 4 ° C.). After the precipitate was air-dried to completely remove ethanol, 100 μl of RNase-free water was added and incubated at 60 ° C. for 10 minutes to dissolve total RNA. MRNA was purified from the total RNA using Fast Track 2.0 kit (manufactured by Invitrogen).

(3′-RACE method for analyzing the nucleotide sequence of the 3 ′ end of mRNA encoding 3-quinuclidinone asymmetric reductase)
The base sequence encoding the C-terminal amino acid sequence of 3-quinuclidinone asymmetric reductase was analyzed by the 3′-RACE method. Single-stranded cDNA for 3′-RACE was synthesized using 3′-Full RACE Core Set (manufactured by Takara Bio Inc.) using total RNA as a template. Based on the DNA sequence of the DNA fragment encoding 3-quinuclidinone asymmetric reductase obtained in Example 4, the primer race-for1 (SEQ ID NO: 9) and primer race- for2 (SEQ ID NO: 10) was designed. PCR was performed using these primers and 3sites Adapter Primer included in 3′-Full RACE Core Set. Nested-PCR yielded a specific amplification product of about 500 bp, which was subcloned into the pCR4-TOPO vector and the nucleotide sequence was determined.

(A base sequence analysis of the 5 ′ end of 3-quinuclidinone asymmetric reductase mRNA by 5′-RACE method)
The base sequence encoding the N-terminal amino acid sequence of 3-quinuclidinone asymmetric reductase was analyzed by 5′-RACE method. Single-stranded cDNA for 5′-RACE was synthesized using mRNA as a template and Gene Racer Kit (manufactured by Invitrogen). From the nucleotide sequence of the DNA fragment obtained in Example 4, primer 5race-primer1 (SEQ ID NO: 11) and primer 5race-primer2 (SEQ ID NO: 12) were designed based on the DNA sequence of the region not containing the intron sequence. PCR was performed using these primers and GeneRacer 5'Primer and GeneRacer 5'Nested Primer included in the Gene Racer Kit. Since a specific amplification product of about 350 bp was obtained by Nested-PCR, it was subcloned into the pCR4-TOPO vector and the nucleotide sequence was determined.

(Cloning of cDNA encoding 3-quinuclidinone asymmetric reductase)
Based on the nucleotide sequences obtained with 5′-RACE and 3′-RACE, primer QR-forward-Eco (SEQ ID NO: 13) and primer QR-reverse-Pst (SEQ ID NO: 14) containing the start codon or stop codon are used. Designed. Single-stranded cDNA was synthesized using total RNA as a template using First-strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.), and PCR was performed using this single-stranded cDNA as a template. By this PCR, a specific amplification product of about 820 bp was obtained. This amplified fragment was double-digested with EcoRI and PstI and inserted into the EcoRI-PstI site located downstream of the tac promoter in plasmid pKK223-3 (manufactured by Amersham Biosciences) to obtain recombinant plasmid pKQ . The nucleotide sequence of the inserted fragment was determined using the sequence on plasmid pKK223-3 as a primer. The sequence of the cDNA encoding the obtained 3-quinuclidinone asymmetric reductase is shown in FIG. 2 (SEQ ID NO: 40), and the deduced amino acid sequence encoded by the gene is shown in FIG. 3 (SEQ ID NO: 41). This deduced amino acid sequence was compared with the results of amino acid sequence analysis obtained in Example 3. As a result, all four internal amino acid sequences of natural 3-quinuclidinone asymmetric reductase purified in Example 1 (shown in Example 3) are present in the amino acid sequence deduced from the cDNA base sequence, Matched perfectly.

(Example 6: Cloning of glucose dehydrogenase gene)
Glucose dehydrogenase derived from Bacillus megaterium IAM1030 strain (hereinafter also referred to as “GDHI”) was used for regeneration of the coenzyme NADP ⁺ . Based on the DNA sequence (DDBJ Accession No. D90043) corresponding to the GDHI enzyme (UniProtKB / Swiss-Prot Accession No. P39482) registered in the database, the primer fwd-gdh-eco (SEQ ID NO: 15) for PCR and The primer rvs-gdh-pst (SEQ ID NO: 16) was designed. Using chromosomal DNA of Bacillus megaterium IAM1030 prepared using QIAGEN Genomic-tip and Genomic DNA Buffer Set (both manufactured by Qiagen) as a template, using the two primers designed above, the GDHI enzyme gene Amplified. The obtained DNA fragment was digested with EcoRI and PstI and inserted into the EcoRI-PstI site located downstream of the tac promoter in plasmid pKK223-3 to construct a recombinant plasmid pKG. As a result of analyzing the nucleotide sequence of the inserted fragment using the sequence on the plasmid pKK223-3 as a primer, it completely matches the glucose dehydrogenase gene (GDHI) gene of Bacillus megaterium IAM1030 strain registered in the database did.

(Example 7: Production of recombinant Escherichia coli that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene using a commercially available plasmid for simultaneous expression of two genes)
Using plasmid pETDuet-1 (manufactured by Novagen) for simultaneous expression of two genes, plasmids pEQG and pEGQ for simultaneous expression of 3-quinuclidinone asymmetric reductase and glucose dehydrogenase were constructed. (DE3) strain was transformed. Plasmids pEQG and pEGQ were prepared by the following method. E. coli competent cells were prepared according to methods commonly used by those skilled in the art, and transformed with these plasmids using a heat shock method at 42 ° C. for 30 seconds to 1 minute (the same applies to the following examples).

(PEQG)
Using the plasmid pKQ constructed in Example 5 (d) as a template, the primer QR-forward-Bsa (SEQ ID NO: 17) and the above-mentioned primer QR-reverse-Pst are used to amplify the 3-quinuclidinone asymmetric reductase structural gene did. The obtained amplified fragment was double-digested with BsaI and PstI and inserted into the NcoI-PstI site located downstream of the T7 promoter 1 on the upstream side in the plasmid pETduet-1 to construct the plasmid pEQ. Subsequently, the GDHI structural gene was amplified using the plasmid pKG constructed in Example 6 as a template and the primer GDH-forward-Nde (SEQ ID NO: 18) and the primer GDH-reverse-Xho (SEQ ID NO: 19). The obtained amplified fragment was double-digested with NdeI and XhoI, and inserted into the NdeI-XhoI site located downstream of T7 promoter 2 of plasmid pEQ to construct plasmid pEQG.

(PEGQ)
Using the plasmid pKG constructed in Example 6 as a template, the GDHI structural gene was amplified using the primer GDH-forward-Bsa (SEQ ID NO: 20) and the primer GDH-reverse-Pst (SEQ ID NO: 21). The obtained amplified fragment was double-digested with BsaI and PstI and inserted into the NcoI-PstI site located downstream of T7 promoter 1 on the upstream side in plasmid pETduet-1, to construct plasmid pEG. Subsequently, using the plasmid pKQ constructed in Example 5 (d) as a template, the primer QR-forward-Nde (SEQ ID NO: 22) and the primer QR-reverse-Xho (SEQ ID NO: 23) were used to produce 3-quinuclidinone asymmetry. The reductase structural gene was amplified. The obtained amplified fragment was double-digested with NdeI and XhoI and inserted into the NdeI-XhoI site located downstream of the T7 promoter 2 on the downstream side in the plasmid pEG to construct the plasmid pEGQ.

(Example 8: Production of recombinant Escherichia coli that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene using a plasmid that controls two genes with one promoter)
In the industrial production of optically active 1,3-butanediol, a commercially available plasmid pSE420 for high expression (in which the transcription amount is enhanced by adding a g10 enhancer and a minicistron sequence to the trc promoter. Also, the trc promoter. The recombinant Escherichia coli co-expressing various reductase genes and the GDH gene has been produced using the lacI ^q gene upstream of (Invitrogen) (Asymmetric Catalysis on Industrial Scale, 2004, pp. 217-231). In addition, using the same system, another reductase gene was coexpressed with the GDH gene to produce (S) -4-chloro-3-hydroxybutyrate (Biosci. Biotechnol. Biochem., 2004, 68). Volume, pp. 638-649) or (R) -3-quinuclidinol can be produced (Patent Document 15). With reference to these cases, plasmids pTrcQG, pTrcQsdG, and pTrcGsdQ for co-expression of the 3-quinuclidinone asymmetric reductase gene and the glucose dehydrogenase gene were prepared. , And BL21 strains were transformed. For construction of the co-expression plasmid, plasmid pTrcHis (manufactured by Invitrogen) was used. This pTrcHis is completely identical to pSE420 except for the NcoI-HindIII site (the region containing the multiple cloning site for gene insertion), and contains the trc promoter, g10 enhancer, and minicistron. Construction of plasmids pTrcQG, pTrcQsdG, and pTrcGsdQ was performed as follows.

(PTrcQG)
Using the plasmid pKQ constructed in Example 5 (d) as a template, the 3-quinuclidinone asymmetric reductase structural gene was amplified using the QR-forward-Bsa primer and the QR-reverse-Pst primer. The obtained amplified fragment was double-digested with BsaI and PstI and inserted into the NcoI-PstI site located downstream of the trc promoter, g10 enhancer, and minicistron in the plasmid pTrcHis to construct pTrcQ. Subsequently, the GDHI structural gene was amplified using the plasmid pKG constructed in Example 6 as a template and the primer fwd-gdh-eco and the primer GDH-reverse-Hind (SEQ ID NO: 24). The obtained amplified fragment was double-digested with EcoRI and HindIII, and inserted into the EcoRI-HindIII site located downstream of the 3-quinuclidinone asymmetric reductase gene in plasmid pTrcQ to construct plasmid pTrcQG.

(PTrcQsdG)
Using the plasmid pKG constructed in Example 6 as a template, the GDHI structural gene was amplified using the primer GDH-forward-EcoW (SEQ ID NO: 25) and the primer GDH-reverse-Hind. The obtained amplified fragment was double-digested with EcoRI and HindIII and inserted into the EcoRI-HindIII site located downstream of the 3-quinuclidinone asymmetric reductase gene in plasmid pTrcQ to construct plasmid pTrcQsdG.

(PTrcGsdQ)
Using the plasmid pKG constructed in Example 6 as a template, the GDHI structural gene was amplified using the primer GDH-forward-Bsa and the primer GDH-reverse-Pst. The obtained amplified fragment was double-digested with BsaI and PstI and inserted into the NcoI-PstI site located downstream of the trc promoter, g10 enhancer, and minicistron in the plasmid pTrcHis to construct pTrcG. Subsequently, using the plasmid pKQ constructed in Example 5 (d) as a template, the primer QR-forward-EcoW (SEQ ID NO: 26) and the primer QR-reverse-Hind (SEQ ID NO: 27) were used to produce 3-quinuclidinone asymmetry. The reductase structural gene was amplified. The obtained amplified fragment was double-digested with EcoRI and HindIII and inserted into the EcoRI-HindIII site located downstream of the GDHI gene in plasmid pTrcG to construct plasmid pTrcGsdQ.

(Example 9: Production of recombinant E. coli that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene using two types of plasmids having different replication origins)
Production of (S) -N-benzyl-3-pyrrolidinol using the reductase BRD derived from Micrococcus lteus (International Patent Application Publication No. 02/010399) or Sporobolomyces salmonicolor In the production of (R) -4-chloro-3-hydroxybutyric acid ester using aldehyde reductase AR derived from salmonicolor (Appl. Microbiol. Biotechnol., 1999, 51, pp. 486-490) The use of two types of plasmids having different origins of replication shows higher activity than the case of using a plasmid that controls two genes with a promoter. With reference to these cases, a plasmid pWKQ for expressing the 3-quinuclidinone asymmetric reductase gene and a plasmid pAG for expressing the glucose dehydrogenase gene were constructed, and these two plasmids were used together to produce Escherichia coli. JM109 was transformed to obtain recombinant E. coli JM109 (pWKQ, pAG) that co-expressed the 3-quinuclidinone asymmetric reductase gene and the glucose dehydrogenase gene. The construction methods of plasmids pWKQ and pAG are described below.

(PWKQ)
Using the plasmid pKQ constructed in Example 5 (d) as a template, the 3-quinuclidinone asymmetric reductase structural gene was amplified using the QR-forward-EcoW primer and the QR-reverse-Pst primer. The obtained amplified fragment was double-digested with EcoRI and PstI, and inserted into the EcoRI-PstI site located downstream of the tac promoter in the plasmid pKK223-3 carrying the pBR322 origin of replication to construct plasmid pWKQ.

(PAG)
The plasmid pKG constructed in Example 6 was double-digested with BamHI and PstI, and an approximately 1 kbp fragment containing the tac promoter and the GDHI gene linked downstream thereof was separated and purified using agarose gel electrophoresis. This fragment was inserted into the BamHI-PstI site of plasmid pACYC177 (Nippon Gene) carrying the replication origin of pA15 to construct plasmid pAG.

(Example 10: 3-quinuclidinone reducing activity of recombinant Escherichia coli co-expressing 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene)
The recombinant Escherichia coli obtained in each of Examples 7 and 8 was added to 5 ml of 2 × YT medium (peptone) supplemented with 50 μg / ml ampicillin and Overnight Expression Kit (manufactured by Novagen) which is a lactose promoter expression induction reagent. 1%, yeast extract 2%, sodium chloride 1%, pH 7.2) or TB medium (peptone 1.2%, yeast extract 2.4%, glycerol 0.04%, KH ₂ PO ₄ 0.231%, K ₂ HPO ₄ 1.254%).

  The Escherichia coli obtained in Example 9 was cultured in a TB medium supplemented with 50 μg / ml ampicillin and 50 μg / ml kanamycin.

The cells were collected from the culture solution by centrifugation (16,500 × g, 10 minutes, 10 ° C.) and placed at −20 ° C. for 30 minutes or more to completely freeze. The cells were freeze-thawed by allowing them to stand at room temperature for 5 to 30 minutes immediately before the reaction. A reaction solution containing 270 mM substrate 3-quinuclidinone hydrochloride, 0.75 mM coenzyme NADP ⁺ , 0.4 mM glucose in 50 mM phosphate buffer (pH 7.0) was added to the cells, and 25 ° C. And shake reaction. After 60 minutes, a half amount of saturated potassium carbonate solution of the reaction solution was added, and 2.5 quinuclidinol and 3-quinuclidinol, which are residual substrates in the reaction solution, were added in 2.5 times the amount. -Extracted with ethylhexanol. These extracts were subjected to gas chromatography analysis using a γ-DEX225 capillary column (manufactured by Spelco) according to the conditions described in Example 2. The cell activity was expressed as a conversion rate (% / hour / ml) when cells of 1 ml of the culture solution were used under these reaction conditions. The activity of each cell is shown in Table 1 below.

  The 3-quinuclidinol produced | generated in any reaction was the (R)-body, and the optical purity was 99.9% ee or more.

(Example 11: Synthesis of (R) -3-quinuclidinol from 3-quinuclidinone by recombinant E. coli BL21 (DE3) (pEGQ) that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene)
Recombinant E. coli BL21 (DE3) (pEGQ) obtained in Example 7 was inoculated into 50 ml of TB medium (containing 50 μg / ml ampicillin and Overnight Expression Kit) sterilized in a 500 ml baffled Erlenmeyer flask. The culture was shaken at 24 ° C. for 24 hours. The cells were collected from this culture by centrifugation (8,400 × g, 20 minutes, 10 ° C.) and stored frozen at −20 ° C. In a 30 ml beaker, put 1 g of 3-quinuclidinone hydrochloride, 1.6 g of glucose, 3 mg of NADP ^{+ and} 3 ml of the cryopreserved cell, make the volume 10 ml with tap water, and react at 25 ° C. while stirring with a stirrer. I let you. The gluconic acid produced during the reaction was neutralized using a 48% potassium hydroxide solution, and the reaction was carried out while maintaining the pH of the reaction solution at 7.0. The reaction extract was analyzed in the same manner as in Example 10. FIG. 4 is a graph showing the time course of the reduction of 3-quinuclidinone by recombinant E. coli BL21 (DE3) (pEGQ). The vertical axis represents the reaction rate (%), and the horizontal axis represents the reaction time (h: time). FIG. 4 shows that after 21 hours, (R) -3-quinuclidinol having a conversion rate of 14.3% and an optical purity of 99.9% ee or more was produced.

(Example 12: Construction of plasmids pKLDuet-1 and pKLDuet-2 for simultaneous expression of two genes)
In order to simultaneously express a large amount of 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene in E. coli, plasmids pKLDuet-1 and pKLDuet-2 for simultaneous expression of two genes were constructed. These plasmids pKLDuet-1 and pKLDuet-2 consist of two tac promoters arranged so that each gene can be controlled separately, and a lacI ^q gene that expresses a repressor that makes expression control by the lac operon more strict. including. FIG. 5 is a schematic diagram showing the structures of these plasmids pKLDuet-1 and pKLDuet-2. Below, the construction method of plasmid pKLDuet-1 and pKLDuet-2 is demonstrated.

(PKLDuet-1)
A region containing two tac promoters and a multiple cloning site was designed. The arrangement of this region is as shown in FIG. 6 (SEQ ID NO: 42). In order to synthesize the NcoI-HindIII region of FIG. 6 as the first fragment, 130-mer oligonucleotides tac-MCS-tacForward (SEQ ID NO: 28) and tac-MCS-tacReverse (SEQ ID NO: 29) and primer tac- PCR was performed using MCSF-Nco (SEQ ID NO: 30) and tac-MCSR-Hind (SEQ ID NO: 31). The amplification product of about 230 bp obtained by PCR was double-digested with NcoI and PstI. In order to synthesize the BamHI-NcoI region of FIG. 6, which is the second fragment, the plasmid tKK-upF-Bam (SEQ ID NO: 32) and the primer tac-upR-Nco (SEQ ID NO: 32) were used using the plasmid pKK223-3 as a template. 33) was used for PCR. The approximately 260 bp amplified fragment obtained by PCR was double digested with BamHI and NcoI. As a third fragment, plasmid pKK223-3 was double digested with BamHI and PstI to generate a DNA fragment of about 4,300 bp. Plasmid pTacDuet-1 was synthesized by ligating the above three DNA fragments. Next, PCR was performed using the plasmid pTrcHis as a template and primers Bsa-lacI-forward (SEQ ID NO: 34) and Bam-lacI-reverse (SEQ ID NO: 35). By this PCR, a region containing the lacI ^q gene containing the regulatory promoter was amplified. The obtained amplified fragment of about 1400 bp was double-digested with BsaAI and BamHI. The fragment obtained by digestion was inserted into the BsaAI-BamHI site located upstream of the tac promoter on the upstream side in the plasmid pTacDuet-1 to obtain the plasmid pKLDuet-1 (FIG. 5).

(PKLDuet-2)
A terminator was inserted between the multicloning site upstream of pKLDuet-1 and the tac promoter on the downstream side as follows to synthesize plasmid pKLDuet-2. As shown in FIG. 5, plasmid pKLDuet-2 contains two units from a promoter to a terminator necessary for high expression.

PCR was performed using the plasmid pKK223-3 as a template and the primer tac-MCS-TTF-Not (SEQ ID NO: 36) and the primer tac-MCS-TTR-Afl (SEQ ID NO: 37). By this PCR, the region containing the terminator rrnBT ₁ T ₂ was amplified. The obtained amplified fragment of about 290 bp was double digested with NotI and AflII. The fragment obtained by digestion was inserted into the NotI-AflII site located between the two tac promoters in the plasmid pTacDuet-1 to obtain pKLDuet-2 (FIG. 5).

(Example 13: Production of recombinant E. coli that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene using one plasmid)
Using the plasmids pKLDuet-1 and pKLDuet-2 constructed in Example 12, plasmids pKL1GQ and pKL2GQ for simultaneously expressing the 3-quinuclidinone asymmetric reductase gene and the glucose dehydrogenase gene were constructed. These plasmids pKL1GQ and pKL2GQ were then used to transform E. coli SCS1, W3110, and BL21. The construction methods of plasmids pKL1GQ and pKL2GQ are described below.

(PKL1GQ and pKL2GQ)
The GDHI gene fragment amplified in the same manner as in the construction of the plasmid pEGQ in Example 7 was double-digested with BsaI and XhoI. The fragment obtained by double digestion was inserted into the NcoI-XhoI site located downstream of the upstream tac promoter in the plasmid pKLDuet-1, to construct the plasmid pKL1G. Subsequently, the 3-quinuclidinone asymmetric reductase gene fragment amplified in the same manner as in the construction of the plasmid pWKQ of Example 9 was double-digested with EcoRI and PstI. The fragment obtained by double digestion was inserted into the EcoRI-PstI site downstream of the tac promoter on the downstream side in the plasmid pKL1G to obtain the plasmid pKL1GQ.

  pKL2GQ was also prepared in the same manner as pKL1GQ. That is, the GDHI gene fragment amplified in the same manner as in the construction of the plasmid pEGQ of Example 7 was double-digested with BsaI and XhoI. The fragment obtained by double digestion was inserted into the NcoI-XhoI site located downstream of the upstream tac promoter in plasmid pKLDuet-2 to construct plasmid pKL2G. Subsequently, the 3-quinuclidinone asymmetric reductase gene fragment amplified in the same manner as in the construction of the plasmid pWKQ of Example 9 was double-digested with EcoRI and PstI. The fragment obtained by double digestion was inserted into the EcoRI-PstI site downstream of the tac promoter on the downstream side in the plasmid pKL2G to obtain the plasmid pKL2GQ.

(Example 14: Production of recombinant E. coli that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene using two types of plasmids)
By inserting the lacI ^q repressor into the recombinant p. Coli plasmid pWKQ constructed in Example 9, it was planned to increase the expression level with strict expression suppression. Plasmid pWKLQ was constructed as follows, and E. coli SCS1, W3110, and BL21 were transformed using this plasmid pWKLQ and plasmid pAG simultaneously. Below, the construction method of plasmid pWKLQ is described.

(PWKLQ)
The fragment containing the lacI ^q gene amplified in Example 12 was double digested with BsaAI and BamHI. The fragment obtained by double digestion was inserted into the BsaAI-BamHI site located upstream of the tac promoter in plasmid pWKQ to obtain plasmid pWKLQ.

Example 15: 3-Quinuclidinone Reducing Activity of Recombinant E. coli Coexpressing 3-Quinuclidinone Asymmetric Reductase Gene and Glucose Dehydrogenase Gene
Each recombinant Escherichia coli prepared in Example 13 was cultured in 5 ml of TB medium supplemented with 50 μg / ml ampicillin and Overnight Expression Kit (Novagen). Each recombinant Escherichia coli prepared in Example 14 was further cultured in the above medium supplemented with 50 μg / ml kanamycin. The obtained culture broth was reacted in the same manner as in Example 10, and 3-quinuclidinone reducing activity was measured. The results are shown in Table 2 below.

  The 3-quinuclidinol produced | generated in any reaction was the (R)-body, and the optical purity was 99.9% ee or more.

The recombinant E. coli produced in Example 13 and the recombinant E. coli produced in Example 14 are the activities of the recombinants produced by known methods (Examples 7, 8, and 9) (shown in Example 10). As compared with the above, it showed high 3-quinuclidinone reduction activity. The recombinant Escherichia coli prepared in Example 13 and the recombinant Escherichia coli prepared in Example 14 have all the following three conditions: (1) The tac promoter is arranged so as to control two genes independently. (2) a highly expressed lacI ^q repressor is placed on the plasmid; and (3) an E. coli host that is deficient in the lactose operon. Therefore, a recombinant produced to have the above conditions can have a higher 3-quinuclidinone reducing activity than a recombinant produced by a known method.

(Example 16: Synthesis of (R) -3-quinuclidinol from 3-quinuclidinone by recombinant E. coli W3110 (pWKLQ, pAG) that simultaneously expresses 3-quinuclidinone asymmetric reductase gene and glucose dehydrogenase gene)
Recombinant E. coli W3110 (pWKLQ, pAG) obtained in Example 14 was added with 50 μg / ml kanamycin to the medium used in Example 11, and cultured with shaking for 24 hours. The culture solution was collected by centrifugation (8,400 × g, 20 minutes, 10 ° C.) and stored frozen at −20 ° C. In a 30 ml beaker, put 1 g of 3-quinuclidinone hydrochloride, 1.4 g of glucose, NADP ⁺ 1.5 mg, 0.35 g of dipotassium hydrogenphosphate and 3 ml of frozen broth for 3 ml of the culture solution. The mixture was made 10 ml and reacted at 25 ° C. with stirring with a stirrer. The gluconic acid produced during the reaction was neutralized using a 48% potassium hydroxide solution, and the reaction was carried out while maintaining the pH of the reaction solution at 7.5. The reaction extract was analyzed in the same manner as in Example 10. FIG. 7 is a graph showing the time course of the reduction of 3-quinuclidinone by recombinant E. coli W3110 (pWKLQ, pAG). The vertical axis represents the reaction rate (%), and the horizontal axis represents the reaction time (h: time). FIG. 7 shows that (R) -3-quinuclidinol having a conversion rate of 98.6% and an optical purity of 99.9% ee or more was produced after 21 hours.

In the recombinant Escherichia coli having the three conditions shown in the above (1) to (3), even when the addition amount of NADP ⁺ used in Example 11 was halved, the reaction was almost theoretical in 21 hours. A yield of (R) -3-quinuclidinol was obtained.

  According to the present invention, an enzyme capable of efficiently producing (R) -3-quinuclidinol with high optical purity is provided. By using a transformant that highly expresses the enzyme of the present invention, a method for producing (R) -3-quinuclidinol having high optical purity in a higher yield is provided. The obtained (R) -3-quinuclidinol is a versatile compound as a common intermediate of drugs acting on muscarinic receptors, and it is used as a therapeutic agent for arteriosclerosis, bronchial asthma, dementia, or gastrointestinal motility suppression. Useful as a synthetic intermediate for production.

It is a figure which shows the base sequence and deduced amino acid sequence of the PCR product by primer QR2 and QR3. It is a figure which shows the base sequence of cDNA which codes 3-quinuclidinone asymmetric reductase. It is a figure which shows the deduced amino acid sequence of the enzyme which 3-quinuclidinone asymmetric reductase structural gene codes. It is a graph which shows the time-dependent change of the reduction | restoration of 3-quinuclidinone by recombinant Escherichia coli BL21 (DE3) (pEGQ). FIG. 2 is a schematic diagram showing the structure of plasmids pKLDuet-1 and pKLDuet-2 for simultaneous expression of two genes. It is a figure which shows the base sequence of the area | region containing the promoter and multiple cloning site of plasmid pKLDuet-1. It is a graph which shows the time-dependent change of the reduction | restoration of 3-quinuclidinone by recombinant Escherichia coli W3110 (pWKLQ, pAG).

Claims (18)

An enzyme having any one of the following amino acid sequences (a) to (c) and having the following biochemical properties (1) and (2) :
(A) the amino acid sequence set forth in SEQ ID NO: 41;
(B) an amino acid sequence having 1 to 30 amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence set forth in SEQ ID NO: 41; or
(C) an amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 41;
(1) asymmetric reduction of 3-quinuclidinone or a salt thereof using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme to produce (R) -3-quinuclidinol; and (2) nicotinamide adenine It does not catalyze the oxidation reaction of 3-quinuclidinol using dinucleotide phosphate (NADP ⁺ ) as a coenzyme.   The enzyme according to claim 1, wherein the molecular weight of the subunit when measured by SDS-PAGE is 30,000, and the molecular weight when measured by gel filtration chromatography is 93,000.   The enzyme according to claim 1, wherein the 1 to 30 amino acids are 1 to 20 amino acids.   The enzyme according to any one of claims 1 to 3, wherein the enzyme is derived from Rhodotolura rubra.   A polynucleotide encoding the enzyme according to any one of claims 1 to 4.   The polynucleotide of claim 5 having the base sequence set forth in SEQ ID NO: 40.   A vector comprising the polynucleotide according to claim 5 or 6. The vector according to claim 7, further comprising a tac promoter and a lacI ^q repressor gene, wherein the tac promoter controls the polynucleotide according to claim 5 or 6. The vector according to claim 7 or 8, further comprising a polynucleotide encoding an enzyme that regenerates NADP ⁺ into NADPH. 10. The vector of claim 9, comprising a lacI ^q repressor gene and two tac promoters, wherein one of the two tac promoters controls the polynucleotide of claim 5 or 6, and the other is A vector that controls a polynucleotide encoding an enzyme that regenerates the NADP ⁺ into NADPH. The vector according to claim 9 or 10, wherein the enzyme that regenerates NADP ⁺ into NADPH is glucose dehydrogenase.   The vector according to claim 11, wherein the glucose dehydrogenase is derived from Bacillus megaterium.   A transformant which retains the vector according to any one of claims 7 to 12 in an expressible manner. The transformant which hold | maintains the vector containing the vector of Claim 7 or 8, and the vector which contains the polynucleotide which codes the enzyme which reproduce | regenerates NADP ⁺ to NADPH so that expression is possible respectively.   The transformant according to claim 13 or 14, wherein the host is Escherichia coli.   The transformant according to claim 15, wherein the Escherichia coli is Escherichia coli not deficient in lactose operon.   A method for producing (R) -3-quinuclidinol or a salt thereof, comprising the enzyme according to any one of claims 1 to 4 or the transformant according to any one of claims 13 to 16, A method comprising a step of allowing a culture solution or a treated product to act on 3-quinuclidinone or a salt thereof. The method according to claim 17, wherein NADP ⁺ is further added in the action step.

Images