JP2008212044A - Method for producing labeled r-mevalonic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a labeled R-mevalonic acid at a low cost, by labeling R-mevalonic acid functioning which is an important physiologically active substance in vivo. <P>SOLUTION: The production method comprises making a labeled acetic acid or its salt react with acetyl CoA synthetase, aetoacetyl CoA synthetase, 3-hydroxy-3-methylglutaryl CoA synthetase and 3-hydroxy-3-methylglutaryl CoA reductase in a cell-free system, and labeled R-mevalonic acid can be produced at low cost, as compared with the conventional methods. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing labeled R-mevalonic acid (hereinafter sometimes referred to as labeled R-mevalonic acid).

  Mevalonic acid is an important bioactive substance group and a biosynthetic intermediate of a group of substances called terpenoids essential for life support. As mevalonic acid, natural R-mevalonic acid and non-natural S-mevalonic acid are known. In the production of mevalonic acid, in many cases, natural forms are obtained by fermentation methods (Patent Documents 1 and 2), and a racemic body that is an equal mixture of natural and non-natural forms is obtained by chemical synthesis methods. . In the anhydrous state, mevalonic acid is easily dehydrated and intramolecularly esterified (lactonized) to form mevalonolactone, but easily reacts with water in an aqueous solution or an alkaline aqueous solution to form mevalonic acid or mevalonate.

  Examples of the method for producing R-mevalonic acid include a method using biological cells such as fermentation production, a chemical synthesis method, and a method for extraction from natural products. Of these, the chemical synthesis method is considered to be the cheapest available if inexpensive raw materials are available. However, it is not used as an industrial production method of R-mevalonic acid because, for example, a racemic form is generally obtained in chemical synthesis, so that optical resolution is required. Further, it is known that R-mevalonic acid present in natural products is widely present in blood and urine of animals including humans. However, since the existing concentration is as low as several μg / L, it is not enough to extract them in large quantities as raw materials and use them industrially.

  Regarding the fermentative production method of mevalonic acid, a method using Saccharomycopsis fibrigera (Patent Document 3), a method using casein-derived peptone and corn steep liquor as a medium component (Patent Document 4), A method using Saccharomycopsis fibrigera having resistance to ML-236B (Patent Document 2) is known.

On the other hand, labeled R-mevalonic acid was only supplied as a chemically synthesized product at a very high price for reagent use. However, R-mevalon labeled in a culture by incorporating a labeled compound into a mevalonic acid-producing bacterium. A method for providing labeled R-mevalonic acid by accumulating acid is provided and used industrially (Patent Document 5). However, the method for producing labeled R-mevalonic acid by the fermentation method requires labor, such as the need to strictly control the fermentation conditions of microorganisms, and requires a large amount of labeled raw materials such as expensive ¹³ C ₆ -glucose. There were problems in terms of efficiency and cost.

  R-mevalonic acid is a physiologically active substance essential for life support of many organisms, and is also a biosynthetic intermediate for various terpenoids such as carotenoids, plant hormones, and ubiquinones. It is known that all biosynthesis of R-mevalonic acid to terpenoids is biosynthesized via isopentenyl diphosphate. The biosynthesis of isopentenyl diphosphate has long been believed to be biosynthesized only from the mevalonate pathway, but Seto and Katsurayama et al. Reported that animal cells, plant cell cytoplasm, fungi such as mold and yeast, In addition to the pathway via mevalonic acid existing in bacteria (mevalonic acid pathway), it is clear that biosynthesis is performed via the methylerythritol phosphate (MEP) pathway present in many eubacteria and plant plastids. (Non-Patent Document 1).

  On the other hand, terpenoids are a general term for a group of organic compounds having a 5 carbon isoprene unit as a basic skeleton, and are biosynthesized by polymerization of isopentenyl pyrophosphate (IPP). This isopentenyl pyrophosphate (IPP) has been proven to be biosynthesized from acetyl CoA via mevalonic acid in eukaryotes such as animals and yeasts.

  In addition to unsaturated hydrocarbons of (C5H8) n, terpenoids have been found in many plants and animals such as redox products (alcohols, ketones, acids, etc.) and compounds from which carbon has been eliminated. Terpenoids are hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40, carotenoids) and other polyterpenes depending on the number of carbon atoms. Can be classified. There are many compounds that exhibit physiological activity such as abscisic acid, juvenile hormone, gibberellin, forskolin, and phorbol. In addition, chlorophyll, vitamin K, ubiquinone, tRNA, and the like are used as complex terpenes having an isoprene structure as a part of the structure, and these also show useful physiological activities.

  For example, ubiquinone, a kind of terpenoid, plays an important role in the body as an essential component of the electron transport system, and is used as a drug effective for heart disease. It is increasing. Vitamin K is an important vitamin involved in the blood clotting system and is used as a hemostatic agent. Recently, it has been suggested to be involved in bone metabolism and is expected to be used for the treatment of osteoporosis. Menaquinone is permitted as a pharmaceutical.

  In addition, ubiquinone and vitamin K have an action of inhibiting the adhesion of shellfish to structures such as hulls and bridge piers, and are expected to be applied to shellfish adhesion prevention paints. Furthermore, carotenoids have an antioxidant action, and some are expected to have cancer prevention and immunostimulatory activities, such as β-carotene, astaxanthin, and cryptoxanthin. As described above, terpenoids contain substances useful for living bodies, and it is known that these compounds are all biosynthesized via isopentenyl pyrophosphate (IPP), which is a basic skeleton unit. .

As described above, the mevalonate pathway exists in eukaryotes such as animals and yeasts, and the production of mevalonate is 3-hydroxy-3-methylglutaryl CoA (hereinafter sometimes referred to as HMG CoA) reductase. Is considered to be a rate-limiting enzyme (Non-Patent Document 2), an attempt has been made to improve the productivity of carotenoids by increasing the level of intracellular mevalonate by highly expressing HMG CoA reductase in yeast. (Non-patent Document 3). In addition, microbial synthesis of ¹³ C-labeled mevalonic acid has been reported (Patent Document 5), and is expected to be used for metabolome research related to terpenoids.

JP-A-2-215389 Japanese Patent Laid-Open No. 3-210174 Japanese Unexamined Patent Publication No. 63-216487 JP-A-2-215389 Japanese Patent Laid-Open No. 2005-269982 Rumer, M. (Rohmer, M.) "In Comprehensive Natural Product Chemistry, Volume 2, Isopreneous Inclusion Carotenoids and Steroids (In Comprehensive Natural Products Chemistry, Vol. 2: Isoprenolids Ind.) (Steroids) "(Netherlands), Burton, D (Barton, D.), Nakanishi, K (Nakanishi, K), Elsvier (Amsterdam) 1999, p. 45-67 "Molecular Biology of the Cell (USA) 1994, Vol. 5, p.655. Misawa et al., "Abstracts of Carotenoid Research Conference" 1997

  An object of the present invention is to provide labeled R-mevalonic acid efficiently and inexpensively.

  As a result of extensive investigations, the present inventors have found that the labeled acetic acid or a salt thereof can be obtained using acetyl CoA synthase, acetoacetyl CoA synthase (ketothiolase), HMG CoA synthase, and HMG CoA reductase. The inventors have found that the above object can be achieved by acting in a cell system, and have reached the present invention.

Therefore, the present invention relates to labeled acetic acid or a salt thereof, acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methylglutaryl CoA. The present invention relates to a method for producing labeled R-mevalonic acid, wherein a reductase is allowed to act in a cell-free system.
In a preferred embodiment of the method for producing labeled R-mevalonic acid according to the present invention, the acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methyl Glutaryl CoA reductase is derived from Neurospora crassa, and in particular, obtained by genetic recombination of a gene cloned from Neurospora crassa.
In a preferred embodiment of the method for producing labeled R-mevalonic acid according to the present invention, the red bread mold (Neurospora crassa) is FGSC2489 strain.
In another preferred embodiment of the method for producing labeled R-mevalonic acid according to the present invention, the cell-free system comprises (a) coenzyme A, (b) reduced nicotinamide adenine dinucleotide phosphate, and / or reduced nicotine. It is characterized by adding amidoadenine dinucleotide and (c) adenosine triphosphate, and further characterized by adding a magnesium salt and / or a manganese salt to the cell-free system.
The present invention also relates to an enzyme cocktail comprising acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methylglutaryl CoA reductase.
In a preferred embodiment of the enzyme cocktail according to the invention, (a) coenzyme A, (b) reduced nicotinamide adenine dinucleotide phosphate, and / or reduced nicotinamide adenine dinucleotide, and (c) adenosine triphosphate, Further, it is characterized by containing a magnesium salt and / or a manganese salt.

  In the present specification, the “cell-free system” means that a reaction in which an enzyme is allowed to act on a substrate to produce a target product is not performed in a cell but in a system in which no cell exists. . However, it does not exclude that cells are mixed so as not to substantially affect the production of the target product.

  In the present specification, the term “enzyme cocktail” means a mixture of enzymes that produce a desired product from a substrate. This enzyme mixture only needs to contain an enzyme necessary for synthesizing a target compound from a substrate. Further, when a cofactor or metal for the enzyme reaction is necessary, salts for providing it may be contained. Furthermore, impurities that do not affect the enzyme reaction may be contained.

  In the present specification, “one to several bases are deleted, substituted, added and / or inserted” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 1. It means that any number of 10 bases, more preferably 1 to 5 bases, has been deleted, substituted, added and / or inserted. In the present specification, “one to several amino acids are deleted, substituted, added and / or inserted” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10. This means that any number of amino acids, more preferably 1 to 5, are deleted, substituted, added and / or inserted.

  Further, in the present specification, the phrase “can hybridize under stringent conditions” uses DNA as a probe, colony hybridization method, plaque hybridization method, Southern blot hybridization method, or the like. Specifically, using a filter in which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, the DNA is high at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter is washed under a condition of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). The DNA which can be identified by this can be mentioned.

  Hybridization is described in Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989. (Hereinafter referred to as “molecular cloning second edition”) and the like. Examples of DNA that can hybridize under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe. The homology is, for example, 60% or more, preferably 70% or more. More preferably, it is 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

According to the present invention, it is possible to provide a method for producing a large amount of labeled R-mevalonic acid from a labeled raw material with high molar efficiency, inexpensively and relatively easily. ^{Further, 1-} 13 _{C 1} - By using acetic acid or a salt thereof ^1,3,5 13 _{C 3} - ^{mevalonate, 2-} 13 _{C 1} - acetic acid or 2,4 by using a salt thereof, (3 By using —CH ₃ ) — ¹³ C ₃ -mevalonic acid and ¹³ C ₂ -acetic acid or a salt thereof, ¹³ C ₆ -mevalonic acid can be selectively prepared.

Hereinafter, the present invention will be described in detail.
As the acetyl CoA synthase, acetoacetyl CoA synthase (ketothiolase), HMG CoA synthase, and HMG CoA reductase used in the present invention, for example, naturally occurring enzymes can be used. For example, an enzyme such as an animal or eukaryote having a mevalonate pathway can be used. Specifically, the above-mentioned enzymes contained in cell extracts of yeast and red bread mold, which are eukaryotes, can be used, and these enzymes can be purified and used. Furthermore, an enzyme obtained by gene recombination obtained by genetic engineering using a gene encoding acetyl CoA synthase, acetoacetyl CoA synthase (ketothiolase), HMG CoA synthase, and HMG CoA reductase It can also be used. For example, an enzyme gene can be incorporated into a vector by genetic recombination, and a host cell such as Escherichia coli can be transformed using the vector to obtain a recombinant. And the enzyme expressed in large quantities by the recombinant can be used. Furthermore, it is possible to express the enzyme outside the cell without transforming the vector into a host cell.

  An enzyme obtained by recombination that can be used in the production method of the present invention can be obtained by the following procedure. First, A) acquisition of DNA encoding acetyl CoA synthase, B) acquisition of DNA encoding acetoacetyl CoA synthase (ketothiolase), C) HMG CoA (3-hydroxy-3-methylglutaryl CoA) synthase And D) acquisition of DNA encoding HMG CoA reductase, and then E) preparation of transformants having DNA encoding the enzyme proteins of A to D) and their total The enzyme protein is expressed in a total of four types of transformants having DNA encoding the four types of the enzyme protein. Using the obtained enzyme, F) Labeled R-mevalonic acid is produced from labeled acetic acid or a salt thereof by the action of an enzyme cocktail containing four kinds of enzymes.

  Specifically, for example, an enzyme that can be used in the present invention can be prepared by the following procedure. That is, in the acquisition of DNA encoding each enzyme, A) to D), total RNA was extracted from a red bread mold (Neurospora crassa) cell using an RNA kit, and then a uniform superparamagnetic polymer polymer bead was used. The mRNA is purified using the formation of a hydrogen bond between the poly (A) + residue present at the 3 ′ end of the mRNA and the residue on the bead surface. As the polymer beads, Dynabeads Oligo (dT) 25 from Dynal can be used.

  Next, cDNA is obtained by reverse transcriptase reaction, and a DNA fragment containing an open reading frame (hereinafter referred to as ORF) encoding the protein of each enzyme is obtained using gene-specific sequence primers and a commercially available PCR kit. As a commercially available PCR kit, Advantage HF2 manufactured by NTECH or the like can be used.

  PCR is performed according to the protocol, and the obtained DNA is incorporated into a TA cloning vector to confirm the sequence.

  Next, a transformant having DNA encoding the four enzyme proteins of E) is prepared, and the enzyme protein is expressed in the transformant having the DNA encoding the enzyme protein. A DNA fragment containing the ORF whose sequence has been confirmed is incorporated into a pQE30 vector and transformed using an E. coli strain or the like. For example, 1 mM (final concentration) of isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as IPTG) is added to the transformed strain, and the promoter is activated to induce production of the target protein. After cell recovery, lysozyme is added, a soluble fraction is prepared by ultrasonic disruption, and production of the target protein is confirmed by SDS-PAGE (CBB staining) of a 10% polyacrylamide gel.

  It is possible to obtain a large excess of enzyme by carrying out genetic recombination using E. coli as a transformant and expressing an enzyme involved in biosynthesis of mevalonic acid. Therefore, sufficient reaction products can be synthesized even when E. coli is disrupted and the soluble fraction is used as a crude enzyme solution and allowed to act on the substrate. Of course, this crude enzyme solution can be purified by a conventional method and allowed to act. Expression of a large excess of these enzymes may be possible only by transforming E. coli with a vector. However, it is usually preferable to add (IPTG) or the like to the transformant to induce protein production. The expression of a large excess of protein can be confirmed visually by electrophoresis with SDS-PAGE of 10% polyacrylamide gel and staining with CBB.

  Using the enzyme cocktail of F) 4 types (acetyl CoA synthase, acetoacetyl CoA synthase (ketothiolase), HMG CoA synthase, HMG CoA reductase) obtained as described above, labeling from labeled acetic acid to R -Production of mevalonic acid.

  The acetyl CoA synthase gene substantially encodes an acetyl CoA synthase, that is, an enzyme protein having an activity of catalyzing the reaction of generating acetyl CoA from acetic acid or acetate and coenzyme A (CoA-SH) and ATP as a substrate. Any DNA sequence may be used. For example, the nucleotide sequence of SEQ ID NO: 2, which is an acetyl-CoA synthase gene derived from Neurospora crassa FGSC 2489, or one to several nucleotide sequences in SEQ ID NO: 2 are deleted, substituted, added and / or inserted. A nucleotide sequence that encodes all sequences necessary for the functioning of acetyl-CoA synthetase, or a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions. And a base sequence encoding all the sequences necessary for the functioning of acetyl-CoA synthase. Furthermore, this invention provides the enzyme protein produced | generated by expressing these genes by a conventional method. In addition, if the enzyme reaction requires a cofactor such as coenzyme A or ATP (adenosine triphosphate), or a salt such as magnesium (Mg) salt and / or manganese (Mn) salt, the necessary amount may be added. Good.

  The acetoacetyl CoA synthase (ketothiolase) gene substantially encodes an acetoacetyl CoA synthase (ketothiolase), that is, an enzyme protein having an activity of catalyzing a reaction for generating acetoacetyl CoA from two molecules of acetyl CoA as a substrate. Any DNA sequence may be used. For example, the nucleotide sequence of SEQ ID NO: 6, which is an acetoacetyl CoA synthetase (ketothiolase) gene derived from the strain of FGSC 2489, or one to several nucleotide sequences in SEQ ID NO: 6 are deleted, substituted, added, and And / or a base sequence that encodes all the sequences necessary for the functioning of the acetoacetyl CoA synthase (ketothiolase) gene, or the base sequence of SEQ ID NO: 6 to hybridize under stringent conditions. It includes a base sequence that can be used for soybeans and that encodes all sequences necessary for functioning the acetoacetyl CoA synthase (ketothiolase) gene. Furthermore, this invention provides the enzyme protein produced | generated by expressing these genes by a conventional method.

  The HMG CoA synthase gene may be a DNA sequence that substantially encodes an HMG CoA synthase, that is, an enzyme protein having an activity of catalyzing the reaction of generating HMG CoA from acetoacetyl CoA and acetyl CoA as substrates. For example, the nucleotide sequence of SEQ ID NO: 10 which is an HMG CoA synthase gene derived from Neurospora crassa FGSC 2489, or one to several nucleotide sequences in SEQ ID NO: 10 are deleted, substituted, added and / or inserted. Or a base sequence that encodes all sequences necessary for the function of the HMG CoA synthase gene, or a base sequence that can hybridize with the base sequence of SEQ ID NO: 10 under stringent conditions. And a base sequence encoding all the sequences necessary for functioning the HMG CoA synthase gene. Furthermore, this invention provides the enzyme protein produced | generated by expressing these genes by a conventional method.

  The HMG CoA reductase gene substantially generates mevalonic acid from HMG CoA reductase, ie, HMG CoA and NADPH (reduced nicotinamide adenine dinucleotide phosphate) or NADH (reduced nicotinamide adenine dinucleotid). Any DNA sequence may be used as long as it encodes an enzyme protein having an activity of catalyzing the reaction. For example, the base sequence of SEQ ID NO: 14 which is an HMG CoA reductase derived from Neurospora crassa FGSC 2489, or one to several base sequences in SEQ ID NO: 14 are deleted, substituted, added and / or inserted. A nucleotide sequence that encodes all of the sequences necessary for functioning HMG CoA reductase, or a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 14 under stringent conditions. , Which contains a base sequence that encodes all sequences necessary for the function of HMG CoA reductase. Furthermore, this invention provides the enzyme protein produced | generated by expressing these genes by a conventional method. In addition, if the enzyme reaction requires a cofactor such as NADPH (reduced nicotinamide adenine dinucleotide phosphate) and / or NADH (reduced nicotinamide adenine dinucleotide), a necessary amount may be added.

  The acetyl CoA synthase gene, acetoacetyl CoA synthase (ketothiolase) gene, HMG CoA synthase gene, and HMG CoA reductase gene that can be used in the present invention may be derived from any organism, but they are eukaryotic. It is preferably derived from the red mold that is an organism. Acquisition of the target gene from red-knot mold is performed by a conventional method, but the outline is as follows.

1) The target gene is extracted from the genome information database of red mold and a primer is synthesized based on the obtained sequence information.
2) A cDNA library (genomic library) is prepared from red mold cells.
3) A sequence containing a target gene is obtained from a cDNA library (genome library) using primers.
The above operation is performed for the following four types of enzyme genes: acetyl CoA synthase gene, acetoacetyl CoA synthase (ketothiolase) gene, HMG CoA synthase gene, and HMG CoA reductase gene.

  The obtained DNA fragments containing the four types of enzyme genes are each incorporated into a plasmid vector (expression vector), and a host (for example, E. coli) is transformed with the obtained plasmid vector. The transformed host is cultured by a conventional method, and the obtained microbial cells are collected by centrifugation or the like and then disrupted to obtain a crude enzyme solution. This crude enzyme solution can also be used in the production method of the present invention as it is. The crude enzyme solution can be purified as necessary. In addition, when a host such as E. coli that expresses an enzyme gene that can be used in the present invention cannot express the target enzyme protein in an active form and forms an inclusion body or the like, It may be expressed in a fused form with a gene encoding a possible peptide.

  The host cell is not particularly limited, but a strain that can be easily handled, such as Escherichia coli, Bacillus subtilis, actinomycetes, baker's yeast, and red mold, is preferable. For example, as a strain of E. coli, E. coli derived from K-12 strain such as JM109 strain, DHα strain, XL1-blue strain, HB101 strain, or B strain such as BL21 can be used. Furthermore, insect cells, plant cells, animal cells and the like can also be used as host cells.

  As the expression vector, those that can replicate autonomously in the host hosts such as Escherichia coli and baker's yeast or can be integrated into the chromosome and have high expression efficiency of the foreign protein are preferable. The expression vector for expressing the DNA is preferably a recombinant vector composed of a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence, as well as being capable of autonomous replication in the microorganism. Moreover, the gene which controls a promoter may be contained.

  Examples of expression vectors include pBTrp2, pBTac1, and pBTac2 (all available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), and pQE-8. (Manufactured by QIAGEN), pQE-30 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48,669 (1984)], pLSA1 [Agri. Biol. Chem. 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK +, pBluescript II SK (-) (manufactured by Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharp, ET) Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 [gene, 33, 103 (1985)], pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Shuzo) Manufactured), pSTV29 (manufactured by Takara Shuzo), pUC118 (manufactured by Takara Shuzo), pPA1 (Japanese Patent Laid-Open No. 63-233798), pEG400 [J. Bacteriol. , 172, 2392 (1990)] and the like.

As the promoter, any promoter can be used as long as it can be expressed in cells such as Escherichia coli and baker's yeast. For example, trp promoter (Ptrp), lac promoter (Plac), _{P L promoter, P R} promoter and _{P SE} promoter, promoters derived from Escherichia coli or phage, such as, SPO1 promoter, SPO2 promoter, and the like penP promoter it can. An artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrpx2), a tac promoter, a letI promoter, and a lacT7 promoter can also be used.

  The ribosome binding sequence may be any sequence that can be expressed in the host actinomycete cells, but an appropriate distance between the Shine-Dalgarno sequence and the initiation codon (eg, It is preferable to use a plasmid adjusted to 6 to 18 bases).

  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into cells such as Escherichia coli and baker's yeast, which are the above hosts. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-2483942), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979), etc. Can be mentioned.

  Each enzyme can be obtained by culturing host cells transformed with the expression vector. For example, when describing the case of Escherichia coli, Escherichia coli may be cultured in a commonly used L medium, YT medium, M9-CA medium or the like. Since an expression vector often has a drug resistance gene, it is desirable to add a corresponding drug at an appropriate concentration. When a gene encoding an enzyme is expressed, expression may be induced by using an upstream promoter in an appropriate manner. For example, expression can be induced by adding IPTG, indoleacrylic acid (IAA), or the like.

  In order to obtain the target enzyme from the cells obtained by culturing, conventional techniques such as lysozyme treatment of cells, ultrasonic disruption, centrifugation, various chromatographies and the like can be used. The target enzyme may be soluble or membrane-bound water-insoluble. When the target enzyme is obtained in the soluble fraction, it can be used as it is as an enzyme solution. The soluble fraction can be purified by various chromatography, for example, affinity chromatography, gel filtration, ion exchange chromatography and the like. When the target enzyme is membrane-bound water-insoluble, for example, HMG CoA reductase is known to be an insoluble enzyme in water having a membrane-bound site in eukaryotes such as mold and mammals. In this case, since the membrane-binding site is not involved in the activity as reported by T. Polakowski, U. Stahl, C. Lang (1998) Appl. Microbiol. Biotechnol. It is also within the scope of the present invention to prepare a water-soluble enzyme having a membrane binding site removed. That is, when the target enzyme becomes insoluble by forming insoluble granules, it can be solubilized with guanidine or urea, purified as it is or purified by various chromatography, refolded and used as an enzyme solution.

  Next, using each of the obtained enzymes, mevalonic acid labeled from labeled acetic acid or a salt thereof is produced in a cell-free system. The reaction volume is not particularly limited, and the reaction volume can be used in a small amount, or the reaction can be carried out in a large amount for industrial use.

  As the enzyme, a soluble fraction obtained from the host cell may be used as it is, or a purified product may be used. As a substrate, labeled acetic acid or a salt thereof is used, and an enzyme solution is allowed to act on the substrate. The enzyme may be allowed to act in the order of acetyl CoA synthase, acetoacetyl CoA synthase (ketothiolase), HMG CoA synthase, and HMG CoA reductase. It is also possible to act as an enzyme cocktail in which all enzymes are mixed, and as a result, the reaction product generated by the enzyme reaction becomes a substrate for the next enzyme reaction, and the reaction proceeds efficiently. It is preferable for accumulation.

In addition, as a cofactor in the enzyme cocktail,
(A) Coenzyme A,
(B) NADPH (reduced nicotinamide adenine dinucleotide phosphate) and / or NADH (reduced nicotinamide adenine dinucleotide), and (c) ATP (adenosine triphosphate),
Is preferably added. These cofactors are contained in cell extracts and the like. When using enzyme cell host extract as an enzyme cocktail, these cofactors do not need to be added, but contain these cofactors. Thus, even when only the substrate is added to the enzyme cocktail, the reaction product can be reliably obtained.

  Furthermore, the enzyme cocktail preferably includes a magnesium (Mg) salt and / or a manganese (Mn) salt. These inorganic salts are contained in cell extracts and the like, and when the enzyme cell-expressing host cell extract is used as an enzyme cocktail, it may not be added. By containing these inorganic salts, the reaction involving ATP can be further advanced.

  Further, since the energy of ATP is utilized for the enzyme reaction by decomposing it into ADP and phosphoric acid, the ATP concentration of the enzyme cocktail decreases and the ADP concentration increases. As a solution to this problem, a high-energy compound that catalyzes the reverse reaction for generating ATP from ADP again, such as phosphoenolpyruvate or polyphosphoric acid, may be added.

  The labeled acetic acid or a salt thereof as a substrate used in the present invention may be any as long as it is labeled with an isotope. Since the labeling rate of the target labeled R-mevalonic acid (ratio of the labeled R-mevalonic acid to the total R-mevalonic acid) is infinitely equal to the labeling rate of acetic acid or acetate used as the raw material, acetic acid Alternatively, if the labeling rate of the salt is substantially 100%, the labeling rate of R-mevalonic acid can be substantially 100%.

The nuclide used for labeling may be any one of carbon (C), hydrogen (H) and oxygen (O) constituting acetic acid, or two or three of them. A double-labeled body or a triple-labeled body may be prepared according to the purpose of use. In addition, a compound labeled with a radioisotope or a compound labeled with a stable isotope may be used, and a compound labeled with both a radioisotope and a stable isotope is prepared. May be. The nuclide used for labeling is a kind selected from the group consisting of ² H (D, deuterium), ³ H (T, tritium), ¹³ C, ¹⁴ C, ¹⁷ O and ¹⁸ O because of its stability. The above is preferable, but other isotopes may be used. However, in the case of stable isotopes (D, ¹³ C, ¹⁷ O, ¹⁸ O, etc.), there are almost no restrictions on use, and industrial use is easy, whereas radioisotopes (T, ¹⁴ C, etc.) In this case, since it is necessary to strictly manage, the cost tends to increase. Conventionally, the detection sensitivity of radioisotopes for measuring radioactivity has been far higher than that of stable isotopes. However, with the recent development of mass spectrometers and nuclear magnetic resonance instruments, stable isotopes are used. Both the detection sensitivity and resolution of the body are improved, and the industrial value of stable isotopes including safety is increasing.

  The substrate concentration is not particularly limited as long as the enzyme can act, but is 0.01 to 50 mg / mL, more preferably 0.03 to 12 mg / mL, and still more preferably. It is 0.1-3.0 mg / mL, Most preferably, it is 0.2-1.5 mg / mL.

  The enzyme concentration is not particularly limited as long as each enzyme can act, but is 0.02 to 50 mg / mL, more preferably 0.05 to 10 mg / mL, still more preferably. 0.1 to 5.0 mg / mL, most preferably 0.2 to 2.5 mg / mL. However, it is preferable to use acetyl-CoA synthetase 1 to 20 times, preferably 2 to 10 times that of other enzymes.

  The cofactor is not particularly limited as long as the enzyme can act, but (a) Coenzyme A is 0.05 to 50 mg / mL, more preferably 0.15 to 20 mg / mL. More preferably 0.5 to 5.0 mg / mL, most preferably 1.0 to 2.5 mg / mL, (b) NADPH (reduced nicotinamide adenine dinucleotide phosphate) and / or NADH (Reduced nicotinamide adenine dinucleotide) is 0.1-100 mg / mL, more preferably 0.3-30 mg / mL, still more preferably 1.0-10 mg / mL, most preferably 2-20 mg / mL. (C) ATP (adenosine triphosphate) is 2.0-100 mg / mL, more preferably 4.0-80 mg / mL, still more preferably .0~50mg / mL, and most preferably 10 to 30 mg / mL.

  The magnesium (Mg) salt and / or manganese (Mn) salt concentration is not particularly limited as long as the enzyme can act, but is more preferably 0.2 to 25 mg / mL. Is 0.5 to 10 mg / mL, more preferably 1.0 to 5.0 mg / mL, and most preferably 1.5 to 3 mg / mL.

  The reaction temperature is not particularly limited as long as the enzyme can act, but is preferably 0 to 80 ° C., more preferably 10 to 60 ° C., and most preferably 20 to 40 ° C.

]
The pH of the reaction is not particularly limited as long as the enzyme can act, but is preferably pH 4-9, more preferably pH 5-8.5, and most preferably pH 6- 8.

  The reaction time is not particularly limited, but is preferably 1 to 48 hours, more preferably 2 to 36 hours, and most preferably 4 to 24 hours. If the reaction time is too long, there is no problem with the reaction itself, but there is a risk of spoilage, so a reaction time of 48 hours or longer is not preferable.

  After the reaction is completed, the produced labeled R-mevalonic acid can be extracted according to a normal extraction method for acidic organic substances. That is, the reaction solution is adjusted to pH 1.0 to 4.0 using an organic acid or an inorganic acid, and then extracted using an organic solvent such as ethyl acetate, methyl ethyl ketone, methyl isobutyl ketone, butyl acetate, or acetone. Further, if necessary, an electrolyte such as sodium chloride may be dissolved in the reaction solution. Furthermore, if it is necessary to reduce the moisture contained in the extract (organic solvent phase), it may be dehydrated with anhydrous sodium sulfate or the like. The extraction solvent is distilled off from the obtained extract under distillation or reduced pressure. In addition, mevalonic acid is easily intramolecularly esterified (lactonized) when extracted with an organic solvent and exists as mevalonolactone. Therefore, mevalonic acid and mevalonic acid can be neutralized with an equimolar amount of an alkali such as sodium hydroxide. can do. However, since mevalonolactone is also a form of mevalonic acid, neutralization is not necessary if it can be used in the form of mevalonolactone.

  In the case of purification, the extract can be adsorbed on column chromatography such as silica gel chromatography and eluted by using a solvent such as hexane / ethyl acetate. It can also be purified directly by distillation. It is also possible to crystallize by using an appropriate solvent.

また、「Ｒ−メバロン酸」は、前記のように有機溶媒中で、脱水して容易に分子内エステル化（ラクトン化）しメバロノラクトンとなるが、メバロノラクトンは、下記の一般式（II）の化合物である。

“R-mevalonic acid” which can be obtained by the production method of the present invention is a compound of the following general formula (I).

In addition, “R-mevalonic acid” is dehydrated in an organic solvent as described above, and is easily esterified intramolecularly (lactonized) to give mevalonolactone. Mevalonolactone is a compound of the following general formula (II): It is.

Examples of the present invention are shown below, but the present invention is not limited to these Examples.
The gene recombination experiments shown in the examples were performed using the method described in Molecular Cloning 2nd edition (hereinafter referred to as a conventional method) unless otherwise specified.

Example 1
(A) Acquisition of DNA encoding acetyl-CoA synthetase (hereinafter sometimes referred to as ACS) Total RNA was extracted from the cells of Neurospora crassa FGSC2489 using the Takara EASYprep RNA kit according to the attached instructions. Using DYNABEADS manufactured by DYNAL, mRNA was purified according to the attached instructions. A cDNA library was obtained from this mRNA by reverse transcriptase reaction.

Gene-specific sequence primer Fwd: 5′-ATGCATGCCATGTCTGGCAGCACCGTTCCT-3 ′ (restriction enzyme site: SphI) [SEQ ID NO: 3]
Rev: 5′-ATCTGCAGTTACTGGCGCTGCCGCGTGGAC-3 ′ (restriction enzyme site: PstI) [SEQ ID NO: 4]

Using a cDNA library as a template, PCR was performed using the above-described primers and an Advantage HF2 kit manufactured by CLONTECH to obtain a DNA fragment containing ORF.
PCR was performed with a reaction volume of 50 μL volume according to the attached instructions. The conditions were 92 ° C. for 3 minutes, 92 ° C. for 45 seconds, 50 ° C. for 45 seconds, 68 ° C. for 2.5 minutes for 30 cycles, and finally 68 ° C. for 5 minutes. The obtained DNA was incorporated into a TA cloning vector and the sequence was confirmed. The sequence is shown in [SEQ ID NO: 2].

Example 2
(B) Acquisition of DNA encoding acetoacetyl CoA synthetase (ketothiolase) (hereinafter sometimes referred to as ACAT) cDNA was obtained from Neurospora crassa FGSC2489 strain by the same procedure as in Example 1.

Gene-specific sequence primer Fwd: 5′-ATGGATCCCATTCTCACCGGTCTTCCCTCC-3 ′ (restriction enzyme site: BamHI) [SEQ ID NO: 7]
Rev: 5′-ATCTGCAGTTTACGCAAGTTTTCGAATAAC-3 ′ (restriction enzyme site: PstI) [SEQ ID NO: 8]

Using a cDNA library as a template, PCR was performed using the above-mentioned primers and an Advantage HF2 kit manufactured by CLONTECH, and a DNA fragment containing ORF was obtained.
PCR was performed with a reaction volume of 50 μL volume according to the attached instructions. The conditions were 92 ° C. for 3 minutes, 92 ° C. for 45 seconds, 50 ° C. for 45 seconds, 68 ° C. for 2.5 minutes for 30 cycles, and finally 68 ° C. for 5 minutes. The obtained DNA was incorporated into a TA cloning vector and the sequence was confirmed. The sequence is shown in [SEQ ID NO: 6].

Example 3
(C) Acquisition of DNA encoding HMG CoA synthase (hereinafter sometimes referred to as HMGS) cDNA was obtained from the cells of Neurospora crassa FGSC 2489 in the same manner as in Example 1.

Gene-specific sequence primer Fwd: 5′-ATGGATCCCATGCTACCCGTCCCCAGAAAC-3 ′ (restriction enzyme site: BamHI) [SEQ ID NO: 11]
Rev: 5′-ATCTGCAGGTTAAGCCCTGATGGAGTATGT-3 ′ (restriction enzyme site: PstI) [SEQ ID NO: 12]

Using a cDNA library as a template, PCR was performed using the above-mentioned primers and an Advantage HF2 kit manufactured by CLONTECH, and a DNA fragment containing ORF was obtained.
PCR was performed with a reaction volume of 50 μL volume according to the attached instructions. The conditions were 92 ° C. for 3 minutes, 92 ° C. for 45 seconds, 50 ° C. for 45 seconds, 68 ° C. for 2.5 minutes for 30 cycles, and finally 68 ° C. for 5 minutes. The obtained DNA was incorporated into a TA cloning vector and the sequence was confirmed. The sequence is shown in [SEQ ID NO: 10].

Example 4
(D) Acquisition of DNA encoding HMG CoA reductase (hereinafter sometimes referred to as ΔHMGR) cDNA was obtained from Neurospora crassa FGSC2489 strain by the same procedure as in Example 1.

Gene-specific sequence primer Fwd: 5′-TTGGATCCCATGGAGTACATGAAGCCCACC-3 ′ (restriction enzyme site: BamHI) [SEQ ID NO: 15]
Rev: 5'-ATCTGCAGTCCAGCGACGCGAACGCTCGAC-3 '(restriction enzyme site: PstI) [SEQ ID NO: 16]

Using a cDNA library as a template, PCR was performed using the above-mentioned primers and an Advantage HF2 kit manufactured by CLONTECH, and a DNA fragment containing ORF was obtained.
PCR was performed with a reaction volume of 50 μL volume according to the attached instructions. The conditions were 92 ° C. for 3 minutes, 92 ° C. for 45 seconds, 50 ° C. for 45 seconds, 68 ° C. for 2.5 minutes for 30 cycles, and finally 68 ° C. for 5 minutes. The obtained DNA was incorporated into a TA cloning vector and the sequence was confirmed. The sequence is shown in [SEQ ID NO: 14].
Note that N. Since the ORF encoding the HMG CoA reductase contained in the total RNA extracted from the cells of the crassa FGSC 2489 strain contains a membrane binding region, the portion encoding the 1 to 663 amino acid residues of the ORF is removed as a membrane binding region, From 664 to 1173 amino acid residues were designed as soluble proteins. The designed gene introduced a translation initiation codon ATG for expression.

Example 5
(E) Transformants having a DNA encoding an enzyme protein, production of a total of four types, and ACS for which the expression sequence of the enzyme protein was confirmed in a total of four types of transformants having a DNA encoding the enzyme protein Using the SphI and PstI sites for the coding DNA fragment, the DNA fragment coding for ACAT using the BamHI and PstI sites, the DNA fragment coding for HMGS using the BamHI and PstI sites, and the DNA fragment coding for ΔHMGR It was integrated into pQE30 vector using BamHI and PstI sites. With these expression vectors, Escherichia coli JM109 was transformed according to a conventional method. The obtained transformed Escherichia coli was cultured in a double concentration of YT medium at 37 ° C. for 4 hours, and then added with 1 mM IPTG (final concentration) and further cultured at 18 ° C. for 20 hours to induce production of the target protein. . After recovering the cells by centrifugation, a soluble fraction was prepared by lysozyme and ultrasonic disruption, and production of the target protein was confirmed by SDS-PAGE (CBB staining) of 10% polyacrylamide gel. As a marker for electrophoresis, Low marker manufactured by Biorad was used. The molecular weights of the markers are 97 kDa, 66 kDa, 45 kDa, 31 kDa, 21 kDa, and 14 kDa from the largest.

The target protein incorporated and expressed in the pQE30 vector can be confirmed in the vicinity of 74 kDa for acetyl CoA synthase, 41 kDa for acetoacetyl CoA synthase (ketothiolase), 50 kDa for HMG CoA synthase, and 54 kDa for HMG CoA reductase. It was.
The obtained target protein has the sequence of acetyl CoA synthase [SEQ ID NO: 1], the sequence of acetoacetyl CoA synthase (ketothiolase) [SEQ ID NO: 5], and the sequence of HMG CoA synthase [SEQ ID NO: 5]. No. 9] shows the sequence of HMG CoA reductase in [SEQ ID No. 13].
Each electrophoretic photograph is shown in FIG.

[Example of analysis]
The analysis of mevalonic acid was carried out using HPLC after extraction by the procedure shown below, followed by dehydration cyclization to form mevalonolactone.
(Extraction) Two drops of 85% phosphoric acid, 0.5 g of anhydrous sodium sulfate, and 2 mL of 2-butanone were added to 1 mL of an aqueous solution containing mevalonic acid, stirred for 30 seconds, and then centrifuged at 3500 rpm for 10 minutes. Next, 1 mL of the upper 2-butanone layer was placed in another test tube and dried with a centrifugal evaporator, and then the oily extract was dissolved in 0.1 mL of isopropanol to obtain a sample solution.
(HPLC) It carried out on the conditions shown below.
Column: Nucleosyl 5N (CH ₃ ) ₂ (M. Nagel, Germany), size diameter 4.6 mm × length 250 mm, 40 ° C.
Moving layer: N-hexane / isopropanol = 9/1, flow rate 1.5 mL / min Detection: differential refractometer (manufactured by Showa Denko KK, SE-61 type), injection amount 0.01 mL

Example 6
Using the enzyme cocktail consisting of 1.64 mg of acetyl CoA synthase obtained in Example 5, 0.115 mg of acetoacetyl CoA synthase (ketothiolase), 0.105 mg of HMG CoA synthase, and 0.135 mg of HMG CoA reductase, as ¹³ C ₂ - sodium acetate 0.25 mg, coenzyme A1.535mg as a cofactor, ATP 9.078mg, NADPH4.4mg, was added 0.5M MgCl ₂ solution 20 [mu] L (final concentration about 20 mM) as an inorganic salt, finally Then, 50 μL of 1M Tris-HCl (pH 7.5) (final concentration: about 100 mM) and 400 μL of water were added as a buffer solution, and the reaction was carried out at a reaction volume of about 0.5 mL and stirred at 28 ° C. for 12 hours. After completion of the reaction, the obtained reaction solution was quantified according to the above analysis example, and it was confirmed that 0.069 mg of mevalonolactone was synthesized (molar yield from sodium acetate 54%). Moreover, it was confirmed by MS, specific rotation, and ¹³ C-NMR that it was ¹³ C ₆ -R-mevalonolactone.
<< Comparative Example 1 >>
A 50 mL medium A consisting of 5 g glucose, 0.25 g polypeptone, 0.125 g yeast extract, 0.05 g monopotassium phosphate, 0.025 g magnesium sulfate heptahydrate, and 0.025 g adecanol LG-294 was prepared. One Saccharomycopsis fibuligera ADK8107 strain (FERM BP-2320) was inoculated into this, and cultured at 26 ° C. and 170 rpm for 4 days to obtain a seed culture solution. Next, instead of the glucose used in the medium A, ¹³ C ₆ -glucose 5 g was used, except that 0.5 g of calcium carbonate was added, a medium B having the same composition was prepared, inoculated with 2 mL of the seed culture solution, and 26 Culturing was performed at 170 ° C. for 5 days. After completion of the culture, the cells were removed by centrifugation to obtain 41 mL of a culture filtrate containing ¹³ C ₆ -R-mevalonic acid. The obtained culture filtrate was quantified according to the analysis example, and it was confirmed that 226 mg of mevalonolactone was synthesized (molar yield from glucose: 6.2%). Moreover, it was confirmed by MS, specific rotation and ¹³ C-NMR that it was ¹³ C ₆ -R-mevalonolactone.

  The labeled R-mevalonic acid obtained by this method is used as a research reagent for elucidating what biosynthetic pathways are used to synthesize and metabolize terpenoids useful in the fields of medicine, cosmetics, foods, etc. It is useful and is also useful as a raw material for synthesizing various labeled compounds.

It is a figure which shows the expression in Escherichia coli of each enzyme of ACS, ACAT, HMGS, and (DELTA) HMGR. Lane MK indicates a marker, and the molecular weight of the marker is 97 kDa, 66 kDa, 45 kDa, 31 kDa, 21 kDa, 14 kDa.

Claims (9)

  Cell-free labeled acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methylglutaryl CoA reductase A method for producing labeled R-mevalonic acid, which is characterized by acting in a system.   The acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methylglutaryl CoA reductase are derived from Neurospora crassa, A method for producing the labeled R-mevalonic acid according to claim 1.   The acetyl-CoA synthase, acetoacetyl-CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and / or 3-hydroxy-3-methylglutaryl CoA reductase is cloned from red bread mold (Neurospora crassa) The method for producing labeled R-mevalonic acid according to claim 1 or 2, which is obtained by genetic recombination of the obtained gene.   The method for producing labeled R-mevalonic acid according to claim 2 or 3, wherein the red bread mold (Neurospora crassa) is FGSC2489 strain. In the cell-free system,
(A) Coenzyme A
(B) reduced nicotinamide adenine dinucleotide phosphate and / or reduced nicotinamide adenine dinucleotide, and (c) adenosine triphosphate,
The method for producing labeled R-mevalonic acid according to any one of claims 1 to 4, wherein:   The method for producing labeled R-mevalonic acid according to any one of claims 1 to 5, wherein a magnesium salt and / or a manganese salt is added to the cell-free system.   An enzyme cocktail comprising acetyl CoA synthase, acetoacetyl CoA synthase, 3-hydroxy-3-methylglutaryl CoA synthase, and 3-hydroxy-3-methylglutaryl CoA reductase. (A) Coenzyme A
(B) reduced nicotinamide adenine dinucleotide phosphate and / or reduced nicotinamide adenine dinucleotide, and (c) adenosine triphosphate,
The enzyme cocktail according to claim 7, characterized in that   The enzyme cocktail according to claim 7 or 8, characterized by containing a magnesium salt and / or a manganese salt.

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