JP2005269982A - Method for producing labeled r-mevalonic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass-producing a labeled R-mevalonic acid relatively easily at low cost. <P>SOLUTION: This method comprises taking a labeling compound into mevalonic acid-productive bacteria to accumulate the objective labeled R-mevalonic acid in the resultant cultured product. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing labeled R-mevalonic acid (hereinafter also referred to as labeled R-mevalonic acid). The labeled R-mevalonic acid obtained by the production method of the present invention is used to elucidate what biosynthetic pathways are used to synthesize and metabolize terpenoids that are useful mainly in the fields of medicine, cosmetics and food. It is useful as a research reagent and also as a raw material for synthesizing various labeled compounds.

  Mevalonic acid is an important bioactive substance group and a biosynthetic intermediate of a group of substances called terpenoids essential for life support. For example, in animals such as mammals and fungi such as mold and yeast, It is a biosynthetic intermediate for terpenoids. As mevalonic acid, natural R-mevalonic acid and non-natural S-mevalonic acid are known. In the production of mevalonic acid, in many cases, a natural form is obtained by the fermentation method, and a racemate which is an equal mixture of a natural form and a non-natural form is obtained by the chemical synthesis method. In the anhydrous state, mevalonic acid is easily dehydrated and intramolecularly esterified (lactonized) to give mevalonolactone, but mevalonolactone can easily react with water in aqueous solution or alkaline aqueous solution to react with mevalonic acid or mevalonic acid salt. It becomes.

  Regarding the fermentative production method of mevalonic acid, a method using Saccharomycopsis fibuligera (see Patent Document 1), a method using casein-derived peptone and corn steep liquor as a medium component (see Patent Document 2), A method using Saccharomycopsis fibuligera having resistance to ML-236B (see Patent Document 3) is known.

  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). In addition to (C5H8) n unsaturated hydrocarbons, their redox products (alcohols, ketones, acids, etc.), carbon-depleted compounds, and the like have been found in many plants and animals. Terpenoids can be hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40, carotenoids) and other terpenoids. Can be classified as polyterpenes. Many terpenoids have physiological activity such as abscisic acid, juvenile hormone, gibberellin, forskolin, phorbol and the like. Moreover, as a complex terpene having an isoprene structure as a part of the structure, there are chlorophyll, vitamin K, ubiquinone, tRNA and the like, and these also show useful physiological activities.

  For example, ubiquinone, a kind of terpenoid compound, 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. doing. 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 applied to osteoporosis treatment. And menaquinone are allowed as pharmaceuticals. In addition, ubiquinone and vitamin K have an inhibitory effect on 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.

  Thus, terpenoids contain substances useful for living organisms, but all these compounds are known to be biosynthesized via the basic skeleton unit isopentenyl pyrophosphate (IPP). Yes.

  IPP, which is the basic skeleton of terpenoid compounds, has been proven to be biosynthesized from acetyl-CoA via mevalonic acid in eukaryotes such as animals and yeasts.

  Recent studies have revealed that there are two completely different terpenoid biosynthetic pathways in living organisms. That is, the mevalonate pathway and the non-mevalonate pathway.

  As already explained, the mevalonate pathway exists in eukaryotes such as animals and yeast, and 3-hydroxy-3-methylglutaryl CoA (hereinafter abbreviated as HMG CoA) reductase is considered to be the rate-limiting enzyme. (See Non-Patent Document 1). From this point of view, attempts have been made to improve carotenoid productivity by increasing the level of intracellular mevalonate by highly expressing HMG CoA reductase in yeast (see Non-Patent Document 2).

  Streptomyces sp. CL190 strain (see Non-Patent Document 7) is known to synthesize IPP via mevalonic acid (see Non-Patent Documents 8 and 9). A gene (hmgr) encoding HMG CoA reductase, an enzyme that catalyzes one reaction on the mevalonate pathway (see Non-Patent Document 10), and all enzymes involved in the mevalonate pathway, namely phosphomevalonate kinase, Diphosphomevalonate decarboxylase, mevalonate kinase, isopentenyl 2-phosphate isomerase, HMG CoA reductase, HMG CoA synthetase, and gene cluster including all have been cloned (Patent Document 4, Non-Patent Document 11 and Non-Patent Document) 12).

On the other hand, in many prokaryotes such as E. coli, 1-deoxy-D-xylulose 5-phosphate (DXP) produced by condensation of non-mevalonic acid pathway, that is, pyruvic acid and glyceraldehyde-3-phosphate is reduced and reduced. MEP pathway IPP via 2-C-methyl -D- erythrose 4-phosphate (MEP) for generating undergoing isomerization is biosynthesized are discovered (see non-Patent Document 3), ¹³ An experiment using a C-labeled substrate proves that it is converted to IPP via 1-deoxy-D-erythritol 4-phosphate (see Non-Patent Documents 4 and 5). In particular, in E. coli, it has been demonstrated that IPP is synthesized only by the non-mevalonate pathway (see Non-Patent Documents 6 and 11 and Non-Patent Document 12).

  Furthermore, in plants, it is known that the mevalonate pathway in cytoplasm and the MEP pathway in plastids such as chloroplasts act on terpenoid biosynthesis (see Non-Patent Document 16). Actinomycetes generally produce the necessary terpenoids by the MEP pathway, but special actinomycetes have been reported to have a separate mevalonate pathway for secondary metabolite production. (Refer nonpatent literature 17). How the terpenoids are biosynthesized in organisms with both of these pathways is largely unclear in terms of quantity and quality. Clarifying this point is one of the very important research subjects because it expands the possibility of using physiologically active substances. In such studies, it is useful to use labeled mevalonic acid as a research reagent.

  In addition, organisms that use R-mevalonate take up mevalonate, convert it to 5-pyrophosphate by the action of both mevalonate kinase and phosphomevalonate kinase, decarboxylate with diphosphomevalonate decarboxylase, and are the basic skeleton of terpenoids Using the production of isopentenyl pyrophosphate (IPP), it is possible to cause R-mevalonic acid-based organisms to incorporate labeled mevalonic acid and produce labeled terpenoids from the mevalonic acid pathway. is there. Labeled terpenoids are useful for elucidating the pathway by which terpenoids are metabolized.

  Synthetic products are available for labeled mevalonic acid. Synthetic products of labeled mevalonic acid, as well as synthetic products of unlabeled mevalonic acid, are almost always a mixture of equal amounts of natural R-mevalonic acid and non-natural S-mevalonic acid ( Racemic). In general, S-mevalonic acid is not used by living organisms, and remains in the culture solution in a microorganism, callus, or cultured cell system. S-mevalonic acid is considered to be excreted as it is when it is orally administered to mammals including humans. However, little information is available on the physiological effects of S-mevalonic acid, and there may be some harmful physiological effects.

  Regarding the chemical synthesis method of labeled mevalonic acid or mevalonolactone, a method of using a labeled raw material (hereinafter also referred to as a labeled compound) as a raw material used for chemical synthesis of mevalonic acid or mevalonolactone has been reported.

  As a method of using a labeled raw material as a raw material used for chemical synthesis of mevalonic acid or mevalonolactone, a synthesis method using dimethoxyphenyl lactone and trimethyl phosphoacetate as starting materials (see Non-Patent Document 13) is known. However, it can be easily analogized to synthesize labeled mevalonic acid by other common mevalonic acid / mevalonolactone chemical synthesis methods. However, mevalonic acid obtained by these chemical synthesis methods is racemic.

  When using a labeled mevalonic acid racemate in the study of biosynthesis and metabolism of terpenoids, not only the content of natural R-mevalonic acid in the racemate is considered to be up to 50% but also physiological effects. Since there is a possibility that an unpredictable physiological action by an unnatural isomer (S-mevalonic acid), which is not obvious, may be affected, there is a possibility that an erroneous result is obtained. Therefore, it is natural that it is not preferable to use a racemate. In addition, when using optically active substances in the pharmaceutical field, it is required to use them in a state that does not contain optical isomers from both safety and physiological functions. Therefore, it is required to clarify the safety and physiological action of both natural and non-natural forms that are optical isomers. As is clear from these results, when using labeled mevalonic acid to investigate the biosynthesis and metabolism of terpenoids, use labeled mevalonic acid with the highest possible content of the natural isomer as much as possible. It is preferable.

For the reasons described above, labeled R-mevalonic acid has been sought. However, until now, labeled R-mevalonic acid has hardly been provided. R- [2- ¹⁴ C] mevalonolactone is provided by Amersham Biosciences, but it is very expensive and is a radioisotope, so it must be equipped with containment equipment and Technology was needed.

  In addition, as a method for chemically synthesizing optically active natural R-mevalonolactone and R-mevalonic acid, a method using diacetone-D-glucose-3-urose as a template (see Non-Patent Document 14), Sharpless asymmetry Although methods using epoxidation have been reported (see Non-Patent Document 15), these methods require advanced techniques and are expensive, so they are inexpensive and can be mass-produced relatively easily. A method was desired. For this reason, it is not practical to apply these methods to the production of labeled R-mevalonic acid.

Japanese Unexamined Patent Publication No. 63-216487 JP-A-2-215389 Japanese Patent Laid-Open No. 3-210174 JP 2001-161370 A Mol. Biol. Cell, 5, 655 (1994) Misawa et al., Proceedings of the Carotenoid Research Conference (1997) Biochem. J, 295, 517 (1993) Tetrahedron Lett. 38, 4769 (1997) Tetrahedron Lett. 39, 4509 (1998) Rohmer, M. In Comprehensive Natural Products Chemistry, Vol. 2: Isoprenoids Including Carotenoids and Steroids; Barton, D. Nakanishi, K. Eds. Elsevier: Amsterdam, 1999; pp. 45-67 J. Antibiot. 43: 444 (1990) Tetrahedron Lett. 31: 6025 (1990) Tetrahedron Lett. 37: 7979 (1996) J. Bacteriol. 181: 1256 (1999) J. Bacteriol. 182: 4153 (2000) Proc. Natl. Acad. Sci., USA, 98: 932 (2001) Journal of the American Chemical Society (1998), 120 (22), 5427-5433 Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1997), (6), 891-895 Tetrahedron (2003), 59 (32), 6035-6038 J. Biol. Chem., 277, 45188-45194, 2002 Biosci. Biotechnol. Biochem., 65 (7), 1627-1635, 2001

  Accordingly, an object of the present invention is to provide a method for producing a large amount of labeled R-mevalonic acid at a low cost and relatively easily.

  The applicant has developed and patented a method for efficiently fermenting and producing R-mevalonic acid by incorporating a gene required for biosynthesis of mevalonic acid using a prokaryotic organism that does not use mevalonic acid. (Japanese Patent Application Nos. 2002-357214 and 2003-31138).

  As a result of intensive studies, the present inventors have found that the above object can be achieved by producing R-mevalonic acid while incorporating a labeled compound into a mevalonic acid-producing bacterium. It came.

  That is, the present invention (invention according to claim 1) is a method for producing a labeled R-mevalonic acid characterized in that a labeled compound is incorporated into a mevalonic acid-producing bacterium and the labeled R-mevalonic acid is accumulated in the culture. A manufacturing method is provided.

  Further, the present invention (invention of claim 2) is a method in which a mevalonate-producing bacterium is first cultured using a medium component for cell growth when the labeled compound is incorporated into the mevalonate-producing bacterium. From the subsequent culture, a culture solution fraction containing unlabeled R-mevalonic acid is removed to prepare a bacterial cell fraction, and then the bacterial cell fraction is added to a medium component containing a labeled compound with 2 components. 2. The method for producing labeled R-mevalonic acid according to claim 1, wherein the labeled R-mevalonic acid is accumulated in the secondary culture by subculturing.

  The present invention (the invention according to claim 3) is the label according to claim 1 or 2, wherein the mevalonic acid-producing bacterium is a strain belonging to Saccharomycopsis fibuligera or a mutant thereof. A method for producing R-mevalonic acid is provided.

  Further, according to the present invention (the invention described in claim 4), the mevalonic acid-producing bacterium is Saccharomycopsis fibuligera ADK 8107 strain (FERM BP-2320) or Saccharomycopsis fibuligera (Saccharomycopsis fibuligera). The present invention provides a method for producing labeled R-mevalonic acid according to any one of claims 1 to 3, which is ADK 8108 strain (FERM BP-2321) or a mutant strain thereof.

  Further, the present invention (invention according to claim 5) is characterized in that the mevalonic acid-producing bacterium has a 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and 3- The method for producing labeled R-mevalonic acid according to claim 1 or 2, which is a strain transformed with a DNA encoding hydroxy-3-methylglutaryl coenzyme A reductase gene or a mutant thereof. is there.

In the present invention (the invention according to claim 6), the labeling compound is selected from the group consisting of ² H (D), ³ H (T), ¹³ C, ¹⁴ C, ¹⁷ O and ¹⁸ O. The method for producing labeled R-mevalonic acid according to any one of claims 1 to 5, which is a compound containing a species or two or more isotopes.

  The present invention (invention according to claim 7) is characterized in that the labeling compound is acetic acid, glycerol, glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate, 1,3-bisphosphoglyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, fructose, fructose 1-phosphate, pyruvic acid, 4 to 22 carbon atoms The fatty acid according to any one of claims 1 to 6, wherein the fatty acid is one or more compounds selected from the group consisting of monoglycerides, diglycerides and triglycerides which are derivatives of the fatty acids, and phospholipids. A method for producing the labeled R-mevalonic acid described herein is provided.

  According to the present invention, it is possible to provide a method for producing a large amount of labeled R-mevalonic acid inexpensively and relatively easily. The labeled R-mevalonic acid obtained by this method is used as a research reagent to elucidate the biosynthetic pathways of terpenoids that are useful in the fields of medicine, cosmetics, food, etc. It is useful and is also useful as a raw material for synthesizing various labeled compounds.

  Hereinafter, the present invention will be described in detail.

  In the method for producing labeled R-mevalonic acid of the present invention, it is essential to use a mevalonic acid-producing bacterium and a labeled compound. Although R-mevalonic acid shows two types of structures, acid type and lactone type, both types are known to convert to each other, and unless otherwise specified in this specification, both types are referred to as R-mevalonic acid. Say.

  Any strain can be used as the above-mentioned mevalonic acid-producing bacterium as long as it is an R-mevalonic acid-producing bacterium. For example, as described in JP-A-63-216487 and JP-A-3-210174 Production bacteria can be used. Use of a strain having high productivity of R-mevalonic acid as the above-mentioned mevalonic acid-producing bacterium can be produced at low cost because of its high production efficiency, and the incorporation of the labeled compound as a labeling raw material into R-mevalonic acid is increased. Therefore, it is preferable. For this reason, Saccharomycopsis fibuligera (Saccharomycopsis fibuligera) belonging to yeast, which is preferably a eukaryote, more preferably Saccharomycopsis fibuligera (Saccharomycopsis fibuligera) ADK 8107 strain (FERM BP-2320) or Saccharomyco Use Saccharomycopsis fibuligera ADK 8108 strain (FERM BP-2321). It is also preferable to use a mutant strain with improved productivity of these R-mevalonic acid-producing bacteria.

  Moreover, it is also preferable to use the following transformant 1 and transformant 2 using actinomycetes, which are prokaryotes characterized by easy handling of the cells and short culture days, as the mevalonic acid-producing bacterium. . In the following paragraphs [0036] to [0052], the transformant 1 will be described, and in the following paragraphs [0053] to [0076], the transformant 2 will be described. The transformant 1 is described in Japanese Patent Application No. 2002-357214, and the transformant 2 is described in Japanese Patent Application No. 2003-31138.

First, the transformant 1 will be described.
Transformant 1 is a 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene against hosts such as actinomycetes that do not have a mevalonate pathway. The two types are transformed.

  The HMG CoA synthase gene used for transformation is a DNA sequence that substantially encodes an HMG CoA synthase protein, that is, an enzyme protein that has the activity of catalyzing the reaction of producing acetoacetyl CoA and acetyl CoA to produce HMG CoA. I just need it. In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

  Further, the HMG CoA reductase gene used for transformation may be a DNA sequence that substantially encodes an HMG CoA reductase protein, that is, an enzyme protein having an activity of catalyzing a reaction for producing mevalonic acid from HMG CoA. . In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

  The HMG CoA synthase gene and the HMG CoA reductase gene are all derived from an organism having a mevalonate pathway, and those derived from microorganisms such as actinomycetes, those derived from animal cells, plant cells Among them, those derived from actinomycetes are preferable, and those derived from Streptomyces sp. CL190 strain are particularly preferable.

  Further, the HMG CoA synthase gene and the HMG CoA reductase gene are preferably those having high expression efficiency of enzyme protein produced from the enzyme gene in cells such as actinomycetes as a host. In addition, it is preferable to select a combination of the HMG CoA synthase gene and the HMG CoA reductase gene and a host so that the expressed enzyme protein can express a high catalytic function. From these points, when a microorganism having no mevalonate pathway is used as a host, HMG CoA synthase genes and HMG CoA reductase genes derived from microorganisms, particularly actinomycetes, are preferable. When actinomycetes that do not have a mevalonate pathway are used as a host, HMG CoA synthase genes and HMG CoA reductase genes derived from actinomycetes are preferable.

  In order to express a DNA fragment containing these two types of enzyme genes in a host cell, first, the target DNA fragment is converted into a DNA fragment of an appropriate length containing the enzyme gene with a restriction enzyme or a DNA degrading enzyme. Thereafter, the expression vector is inserted downstream of the promoter in the expression vector, and then the expression vector into which the DNA has been inserted is introduced into a host cell suitable for the expression vector.

  The host to be used may be any organism that uses the non-mevalonate pathway (without the mevalonate pathway), and examples of organisms that do not have the mevalonate pathway include Gram-positive bacteria such as Alicyclobacillus, Corynebacterium, Brevibacterium, Mycobacterium, Bacillus, Clostridium, Deinococcus, many Streptomyces, Gram-negative bacteria, Escherichia, Salmonella, Agrobacterium, Azotobacter, Methylobacterium, Pseudomonas, Rhodopseudomonas, Zymomonas, Examples include organisms belonging to the genus Chromatium, Rhodospirillum, cyanobacteria, Anabaena, Anacystis, Phormidium, Synnecoccus, green algae, Chlorella, Scenedesmun, etc., which are photosynthetic gram-negative bacteria.

  By the way, organisms using the mevalonate pathway (having the mevalonate pathway) include animals, fungi, yeasts, Gram-positive bacteria, Actinoplanes genus, Lactobacillus genus, Mycoplasma genus, Staphylococcu genus, Enterococcus genus Streptococcus, some Streptomyces, Gram-negative bacteria, Chloropseudomonas, Flavobacterium, Myxococcus, Nannocystis, Borrelia, Archaea , Halobacterium genus, Sulfolobus genus and the like are known.

  Some Streptomyces genera are known to utilize both the mevalonate pathway and non-mevalonate.

  Among the organisms that do not have the mevalonate pathway described above, microorganisms, particularly actinomycetes, are preferable as the host to be used, and examples of the actinomycetes include Streptomyces lividans, Streptomyces chromofuscus, Streptomyces exfoliatus, Streptomyces argenteorus, and the like. lividans is particularly preferred.

  In addition, as the expression vector, a vector which can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the target DNA can be transcribed is used. When a microorganism cell such as a bacterium is used as a host cell, the expression vector for expressing the DNA is capable of autonomous replication in the microorganism cell, and at the same time, a promoter, a ribosome binding sequence, the DNA and the transcription termination. A recombinant vector composed of sequences is preferred. A gene that controls the promoter may also be included.

Examples of the expression vector include pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), 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 [Agric. Biol. Chem. , 53 , 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82 , 4306 (1985)], pBluescriptII SK +, pBluescriptII SK (-) (Stratagene), pTrS30 (FERM BP-5407) , PTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 (gene, 33 , 103 ( 1985)], pUC19 [Gene, 33 , 103 (1985)], pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), pPA1 (Japanese Unexamined Patent Publication No. 63-233798), pEG400 [J. Bacteriol., 172 , 2392 (1990)], pQE-30 (QI AGEN) and the like.

As the promoter, so long as it can be expressed in a host cell may be any, for example, trp promoter (P trp), lac promoter (P lac), P _L promoter, P _R promoter and P _SE promoter, Examples include promoters derived from E. coli and phages, SPO1 promoter, SPO2 promoter, penP promoter and the like. In addition, artificially designed and modified promoters such as a promoter in which two P trp are connected in series (P trp x2), tac promoter, letI promoter, and lacT7 promoter can also be used.

  The ribosome binding sequence may be any sequence that can be expressed in the host cell, but at an appropriate distance (eg, 6 to 18 bases) between the Shine-Dalgarno sequence and the start codon. It is preferred to use a regulated plasmid.

  In the transformation, a plasmid vector is preferably used, and the plasmid vector is particularly preferably a shuttle vector with E. coli.

As a method for introducing an expression vector such as the above-described recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-2483942), or the methods described in Gene, 17 , 107 (1982), Molecular & General Genetics, 168 , 111 (1979), etc. Can be mentioned.

  The transformant 1 can be obtained as described in detail above. As the transformant 1, the actinomycetes Streptomyces lividans 34-1 strain, 34-7 strain or 35-6 strain can be obtained. It can also be used. These strains will be displayed at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary, with the indication of microorganisms, “Streptomyces lividans 34-1” for identification and the accession number “FERM P-19120”. The identification sign “Streptomyces lividans 34-7” given by the depositor, the deposit number “FERM P-19121”; the identification label given by the depositor “Streptomyces lividans 35-6”, the deposit number “FERM” P-19122 "has been deposited.

Next, the transformant 2 will be described.
Transformant 2 is effective against actinomycetes that do not have the mevalonate pathway, acetoacetyl CoA synthase gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and 3-hydroxy-3-methylglutaryl coenzyme. A DNA encoding three types of A reductase genes is transformed.

Hereinafter, acquisition of DNA encoding acetoacetyl CoA synthase protein will be described in detail.
The DNA encoding the obtained acetoacetyl-CoA synthase protein may be any DNA as long as it contains an acetoacetyl-CoA synthase gene. The acetoacetyl-CoA synthase gene is substantially acetoacetyl-CoA synthase, that is, two molecules of acetyl. Any DNA sequence may be used as long as it encodes an enzyme protein having an activity of catalyzing a reaction for producing acetoacetyl CoA from CoA. In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

Specific examples of a method for obtaining DNA encoding an acetoacetyl-CoA synthetase protein include the following methods.
Since several acetoacetyl-CoA synthase genes have already been reported, these genes may be used in the present invention. However, acetoacetyl-CoA synthesis from actinomycetes, particularly Streptomyces sp. It is preferable to obtain an enzyme gene. For example, by cloning an acetoacetyl CoA synthase gene and obtaining a novel acetoacetyl CoA synthase gene from Streptomyces sp. CL190 strain, DNA having the base sequence of SEQ ID NO: 1 can be obtained. . Hereinafter, a method for obtaining a DNA encoding an acetoacetyl-CoA synthase protein will be specifically described with reference to Streptomyces sp. CL190 strain as an example.

  Streptomyces sp. CL190 strain is added to a suitable medium such as 2% glucose, 2.5% soluble starch, 1.5% soy flour, 0.2% dry yeast, and 0.4% calcium carbonate. Incubate for several days at an appropriate temperature (for example, 30 ° C.) with the contained KG medium (pH 6.2). After culturing, microbial cells are obtained from the obtained culture solution by centrifugation, and chromosomal DNA is isolated and purified from the microbial cells according to a conventional method (molecular cloning second edition).

  In order to obtain DNA having the nucleotide sequence of SEQ ID NO: 1 by PCR, chromosomal DNA of Streptomyces sp. CL190 strain was used as a template and designed from the genetic information of known acetoacetyl-CoA synthase Using a pair of primer DNAs, DNA Thermal using TaKaRa LA-PCR (registered trademark) Kit Ver.2 (Takara Shuzo) or Expand (registered trademark) High-Fidelity PCR System (Boehringer Mannheim) Perform PCR with Cycler (manufactured by PerkinElmer Japan). In order to facilitate subsequent cloning operations, it is preferable to add an appropriate restriction enzyme site to the primer.

  As PCR conditions, for example, a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) is performed for 30 cycles, for example. Thereafter, the reaction conditions can be raised at 72 ° C. for 7 minutes. The amplified DNA fragment can then be cloned into an appropriate vector that can be amplified in E. coli. Cloning is performed by a conventional method, for example, Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology). ), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), or a commercially available kit such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies) Or ZAP-cDNA Synthesis Kit (manufactured by Stratagene).

As a cloning vector, any expression vector of E. coli may be used as a cloning vector as long as it can autonomously replicate in E. coli K12. Specifically, ZAP Express (Stratagene, Strategies, 5 , 58 (1992)), pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)], Lambda ZAP II (Stratagene) ), Λgt10, λgt11 [DNA Cloning, A Practical Approach, 1 , 49 (1985)], λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (H. Okayama and P Berg; Mol. Cell. Biol., 3 , 280 (1983)], pMW218 (Wako Pure Chemical Industries), pUC118 (Takara Shuzo), pEG400 [J. Bac., 172 , 2392 (1990)], pQE -30 (manufactured by QIAGEN).

  From the obtained transformant, a plasmid containing the target DNA is prepared by a conventional method, for example, Molecular Cloning, Second Edition, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). By the above method, DNA having the base sequence of SEQ ID NO: 1 can be obtained.

  In addition, in the base sequence of SEQ ID NO: 1, one to several base sequences are deleted, substituted, added and / or inserted, and a sequence necessary for functioning acetoacetyl-CoA synthase A base sequence encoding all or a base sequence that can hybridize with the base sequence of SEQ ID NO: 1 under stringent conditions and that encodes all sequences necessary for the function of acetoacetyl-CoA synthase If it has DNA which has, it can be used for preparation of a transformant.

  Next, a total of three kinds of DNAs encoding the acetoacetyl CoA synthase protein and the DNAs encoding the HMG CoA synthase protein and the HMG CoA reductase protein involved in the biosynthesis of natural R-mevalonate, respectively. The production of a transformant having DNA will be described in detail.

  In transformant 2, three types of genes encoding an enzyme protein involved in the biosynthesis of natural R-mevalonic acid are acetoacetyl CoA synthase gene, HMG CoA synthase gene, and HMG CoA reductase gene. It is essential to transform

  The acetoacetyl CoA synthase gene may be a DNA sequence that substantially encodes an acetoacetyl CoA synthase, that is, an enzyme protein having an activity of catalyzing a reaction for generating acetoacetyl CoA from two molecules of acetyl CoA. In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

  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. In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

  The HMG CoA reductase gene may be a DNA sequence that substantially encodes an HMG CoA reductase, that is, an enzyme protein having an activity of catalyzing a reaction for producing mevalonic acid from HMG CoA. In addition, a cofactor such as NADPH may or may not be required for the enzyme reaction.

  The acetoacetyl CoA synthase gene, the HMG CoA synthase gene, and the HMG CoA reductase gene are all derived from an organism having a mevalonate pathway, and those derived from microorganisms such as actinomycetes, Examples include those derived from animal cells and those derived from plant cells. Among these, those derived from actinomycetes are preferred, and those derived from Streptomyces sp. CL190 are particularly preferred.

  The acetoacetyl CoA synthase gene, the HMG CoA synthase gene, and the HMG CoA reductase gene may be derived from any organism, but are enzymes produced from the enzyme gene in cells of actinomycetes that are hosts. It is preferable that the protein expression efficiency is high. Moreover, the combination with the cell in which the expressed enzyme protein can express a high catalytic function is preferable. From these points, in the method for producing mevalonic acid of the present invention using actinomycetes having no mevalonate pathway as a host, it is preferable to use an enzyme gene derived from actinomycetes, derived from Streptomyces sp. CL190. Enzyme genes are more preferred. In particular, it is preferable that all three types of acetoacetyl CoA synthase gene, HMG CoA synthase gene and HMG CoA reductase gene are derived from actinomycetes, particularly from Streptomyces sp. CL190. A preferred example of DNA used for transformation of the acetoacetyl CoA synthase gene is DNA having the base sequence of SEQ ID NO: 1.

  In order to express the obtained DNA fragment containing the three types of enzyme genes in actinomycetes cells, first, the DNA fragment of interest having a suitable length containing the gene with restriction enzymes or DNA degrading enzymes Then, it is inserted downstream of the promoter in the expression vector. Next, the transformant having the above three types of DNA is obtained by introducing the expression vector into which the DNA has been inserted into actinomycetes cells suitable for the expression vector.

  In the method for producing mevalonic acid of the present invention, actinomycetes having no mevalonic acid pathway are used as a host. As the actinomycetes, Streptomyces lividans, Streptomyces chromofuscus, Streptomyces exfoliatus, and Streptomyces argenteorus are preferable, and among these, Streptomyces lividans is particularly preferable.

  As the expression vector, a vector that can replicate autonomously in a host actinomycete cell or can be integrated into a chromosome and contains a promoter at a position where the target DNA can be transcribed is used. The expression vector for expressing the DNA is preferably a recombinant vector that is capable of autonomous replication in the actinomycetes and is composed of a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence. A gene that controls the promoter may also be included.

Examples of the expression vector include pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), 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 [Agric. Biol. Chem. , 53 , 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82 , 4306 (1985)], pBluescriptII SK +, pBluescriptII SK (-) (Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 (gene, 33 , 103 (1985) )], PUC19 [Gene, 33 , 103 (1985)], pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), pPA1 (Japanese Patent Laid-Open No. 63-233798), pEG400 [ J. Bacteriol., 172 , 2392 (1990)], pQE-30 (QIA GEN) and the like.

Any promoter can be used as long as it can be expressed in cells of actinomycetes as a host. For example, trp promoter (P trp), cited lac promoter (P lac), P _L promoter, P _R promoter and P _SE promoter, promoters derived from Escherichia coli or phage, such as, SPO1 promoter, SPO2 promoter, the penP promoter and the like be able to. In addition, artificially designed and modified promoters such as a promoter in which two P trp are connected in series (P trp x2), tac promoter, letI promoter, and lacT7 promoter can also be used.

  The ribosome binding sequence may be any sequence as long as it can be expressed in the cells of actinomycetes as a host, but an appropriate distance (for example, 6) between the Shine-Dalgarno sequence and the start codon. It is preferable to use a plasmid adjusted to ˜18 bases).

  In the transformation, it is preferable to use a plasmid vector, and as the plasmid vector, a shuttle vector with Escherichia coli is particularly preferable.

As a method for introducing an expression vector such as the above-described recombinant vector, any method can be used as long as it is a method for introducing DNA into cells of actinomycetes as a host. For example, a method using calcium ions [Proc. Natl. Acad Sci. USA, 69 , 2110 (1972)], the protoplast method (JP 63-2483942 A), or Gene, 17 , 107 (1982) or Molecular & General Genetics, 168 , 111 (1979). The method etc. can be given.

  In the method for producing labeled R-mevalonic acid of the present invention, the R-mevalonic acid-producing bacterium may be cultured while incorporating a labeled compound (labeled compound) as a labeling raw material. The labeling compound may be any compound as long as it is a labeling raw material that can be used by R-mevalonic acid-producing bacteria. If the compound containing 1 to 3 of carbon, hydrogen, and oxygen contained in the culture medium at the start of culture is substantially all a labeled compound, it will label all cell components and metabolic components after culture. It is possible. The labeling rate of R-mevalonic acid (ratio of labeled R-mevalonic acid to total mevalonic acid) in the culture broth after culturing is the labeling rate of the labeled compound used as a raw material (ratio of labeled compound to all compounds) Since the labeling rate of the labeling compound is substantially 100%, the labeling rate of R-mevalonic acid can be substantially 100%. However, this method is not only expensive, but also labels unnecessary compounds other than bacterial cells and R-mevalonic acid, so when radioactively labeled compounds are incorporated, bacteria labeled with radioactive isotopes There is a demerit such that a large amount of waste such as body is generated, and the environmental load and processing cost become high. Therefore, the labeling rate of the labeling compound may be selected according to the purpose from the necessity of economical efficiency and high labeling rate.

The nuclide used for labeling may be any one of carbon, hydrogen and oxygen constituting mevalonic 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, there are almost no restrictions on the use and the industrial use is easy, whereas in the case of radioactive isotopes, it is necessary to strictly manage them, so 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.

  In the production method of the present invention, the labeled compound is incorporated into a mevalonic acid-producing bacterium, and the labeled R-mevalonic acid is accumulated in the culture. The biosynthesis of mevalonic acid can be considered in two stages: an acetyl CoA supply stage and a mevalonic acid production stage from acetyl CoA.

  In the supply stage of acetyl-CoA, which is the first half of mevalonic acid biosynthesis, the supply of acetyl-CoA mainly consists of two pathways: glycolysis and β-oxidation of fatty acids. In the glycolysis system, after reaching pyruvic acid from glucose, starch, fructose, sucrose, etc., acetyl-CoA is produced by decarboxylation of pyruvic acid. In β-oxidation of fatty acids, the carbon skeleton is cleaved in units of acetic acid from the oxidation end of fatty acids to produce acetyl CoA.

  In the second half of the biosynthesis of mevalonic acid, acetoacetyl CoA was produced from acetyl CoA 2 molecules by the action of acetoacetyl CoA synthase, and then HMG CoA was produced from acetoacetyl CoA and acetyl CoA by the action of HMG CoA synthase. Subsequently, mevalonic acid is produced from HMG CoA by the action of HMG CoA reductase.

  The labeling compound to be incorporated into the mevalonic acid-producing bacterium used in the present invention may be any substance that is related to mevalonic acid biosynthesis, but can be directly introduced into the glycolysis system or β-oxidation system described above. Such a substance is preferable because it can be easily taken into mevalonic acid, and among these compounds, compounds that are inexpensive and easily available are more preferable.

  Preferred examples of substances that can be directly introduced into the glycolysis include acetic acid, glycerol, glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3 -Phosphoric acid, 1,3-bisphosphoglyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, fructose, fructose 1-phosphate, pyruvate, among these, glycerol, Acetic acid, glucose and fructose are particularly preferred.

  Preferable examples of substances that can be directly introduced into β-oxidation include various fatty acids having 4 to 22 carbon atoms, monoglycerides, diglycerides and triglycerides that are derivatives of these fatty acids, and various phospholipids.

  In addition, since organic acids such as acetic acid and phosphate esters such as glucose 6-phosphate are acidic substances, the culture solution becomes acidic when added as it is, whereas when added as a salt such as sodium salt, it depends on the cells. By absorption of these compounds, alkalis such as sodium remain in the culture solution, and the culture solution tends to become basic. For this reason, when these acidic substances are used, it is necessary to strictly control the pH of the culture medium during culture, which may increase management factors and increase labor and cost. The labeling compound used as a raw material is preferably an inexpensive and stable compound.

  In the production method of the present invention, mevalonic acid is cultured by culturing mevalonic acid-producing bacteria using a culture solution containing one or more of these labeled compounds, and mevalonic acid fermentation is performed by a known method. The labeling compound is incorporated into the producing bacteria, and the labeled R-mevalonic acid is accumulated in the culture. The concentration of the labeled compound in the culture solution is preferably 0.1 to 10% by weight.

  When the labeled compound is added to the culture solution, when the R-mevalonate-producing bacterium is used to perform the mevalonic acid fermentation, the labeled compound is incorporated into the R-mevalonate-producing bacterium in the culture solution. If possible, it may be added to the culture solution before culturing, or may be added to the culture solution during the culturing. When added during culture, when the biosynthesis rate of mevalonic acid by the R-mevalonic acid-producing bacterium is high, it is possible to add a label for the intermediate of mevalonic acid biosynthesis to obtain R-mevalonic acid. This is preferable because the labeling rate can be increased. However, the method of adding a labeling compound in the middle of culturing includes R-mevalonic acid biosynthesized by a mevalonic acid-producing bacterium before adding the labeling compound and labeled R biosynthesized after adding the labeling compound. Since -mevalonic acid is mixed, the labeling rate of the obtained labeled R-mevalonic acid is lowered.

  The medium used in the present invention contains a carbon source that can be assimilated by R-mevalonic acid-producing bacteria, a nitrogen source, inorganic salts, etc., and any medium that can efficiently culture mevalonic acid-producing bacteria, Any synthetic medium may be used.

  Any carbon source may be used as long as it can be assimilated by R-mevalonic acid-producing bacteria. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic substances such as acetic acid and propionic acid Alcohols such as acid, ethanol and propanol can be used.

  Nitrogen sources include ammonium salts of various inorganic and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep, Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof, and the like can be used.

  Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 14 days. During the culture, the pH is preferably maintained at 3.0 to 9.0. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

  Moreover, you may add antibiotics, such as an ampicillin, tetracycline, and thiostrepton, to a culture medium during culture | cultivation as needed.

  In general, since a labeled compound is often expensive, a method with high incorporation efficiency into the obtained labeled R-mevalonic acid is economically advantageous. In addition, the higher the ratio of the labeled R-mevalonic acid to the total mevalonic acid (labeling rate), the higher the detection sensitivity and the higher the utility value. Moreover, it is preferable from a viewpoint of production efficiency to use a microbial cell with high mevalonic acid production ability. As a result of studying these points, the inventors of the present invention first cultivated mevalonic acid-producing bacteria using medium components for cell growth, and then incorporated the labeled compound into mevalonic acid-producing bacteria. A culture broth fraction containing unlabeled R-mevalonic acid is removed from the culture to prepare a bacterial cell fraction, and then the bacterial cell fraction is subjected to secondary treatment using a medium component containing a labeled compound. It was found that, by culturing, labeled R-mevalonic acid was accumulated in the secondary culture, whereby it was possible to prepare R-mevalonic acid with high economic efficiency and high labeling rate.

  The medium component used in the primary culture may be any medium such as those described above as long as the mevalonate-producing bacterium has a high mevalonate fermentation ability. For example, a medium containing a nitrogen source or inorganic salts is used. Can do. Examples of the nitrogen source include yeast extract, peptone, corn steep liquor, and combinations thereof, and examples of the inorganic salts include phosphates such as monopotassium phosphate, magnesium sulfate, and combinations thereof. It is done. In the primary culture, the same conditions as described above can be adopted as conditions such as culture temperature, culture time, and pH. In the case of Saccharomycopsis fibuligera whose mevalonic acid-producing bacterium is a eukaryote, it can be cultured by the method described in JP-A-2-215389. When the bacterial cell concentration was sufficiently increased in primary culture and the mevalonic acid fermentability increased, the culture solution and unlabeled R-mevalonic acid in the culture solution were removed from the primary culture by centrifugation or filtration. Thereafter, the cells are collected, washed thoroughly with a phosphate buffer or the like, and subjected to secondary culture. The bacterial cells prepared in the primary culture are mixed with the medium components for secondary culture containing the labeled compound, and the culture is continued aerobically to produce labeled R-mevalonic acid from the labeled compound. . Conditions such as the culture temperature in the secondary culture can be the same as described above. Further, as a method for determining the specific culture time of the primary culture, a method in which mevalonic acid-producing bacteria are preliminarily subjected to mevalonic acid fermentation and the time-lapse data obtained by monitoring the concentration of mevalonic acid to be generated is preferable. Thereby, a microbial cell with high mevalonic acid production ability is obtained by obtaining the microbial cell immediately before the mevalonic acid production rate becomes the maximum.

Medium components used for secondary culture include, for example, inorganic salts such as phosphates and magnesium salts, labeled compounds used as raw materials for labeled R-mevalonic acid, and the minimum amino acids required by the cells in mevalonic acid fermentation A nitrogen source containing nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), coenzyme A (CoA), adenosine 3-phosphate (ATP), thiamine pyrroline acid (TPP), can be composed of a flavin mononucleotide (FADH ₂₎ and the required amount of 0-7 kinds of auxiliary components selected from the group consisting of lipoic acid (0 to 100 mM) added ingredients.

  In addition, it is possible to store cells having high mevalonic acid-producing ability in advance and use them. However, since the activity of the cells decreases due to storage, immediately after the primary culture as described above. A secondary culture method is preferred. Of course, the primary culture may be omitted if the cells are stored under the condition that the activity does not decrease.

  In addition, in order to isolate and purify labeled R-mevalonic acid from the culture after culturing, the usual isolation and purification methods of mevalonolactone, which is an intramolecular ester of mevalonic acid or mevalonic acid, that is, cell separation, extraction, distillation , Chromatography, recrystallization and the like can be obtained by appropriately combining them.

Since labeled R-mevalonic acid has a low molecular weight (for example, the mass number of ¹³ C ₆ H ₁₂ O ₄ is 154), in many cases, it can permeate the cell membrane of mevalonic acid-producing bacteria and accumulates in the culture filtrate. There is relatively little mevalonic acid detected in the body. For this reason, in order to obtain a culture filtrate, it is preferable to remove a microbial cell using centrifugation or filtration. However, when R-mevalonic acid accumulates in the cells of the transformant, it may be extracted from cells or culture filtrate containing cells.

  The extraction of the labeled R-mevalonic acid can be performed according to a normal extraction method for acidic organic substances. That is, the culture can be extracted with an organic solvent such as ethyl acetate, methyl ethyl ketone, methyl isobutyl ketone, butyl acetate and acetone after the culture is adjusted to pH 1.0 to 4.0 using an organic acid or an inorganic acid. Moreover, you may dissolve electrolytes, such as sodium chloride, in a culture as needed. 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. An extract containing labeled mevalonolactone can be obtained by distilling the extraction solvent from the obtained extract under distillation or under reduced pressure. However, mevalonic acid is easily esterified intramolecularly (lactonization, mevalonolactone) when extracted with an organic solvent. When it is desired to use mevalonolactone as mevalonic acid, it can be neutralized with an alkali such as sodium hydroxide in an equimolar amount with mevalonolactone.

  The extract containing labeled mevalonolactone can be purified by adsorbing to a column chromatography such as silica gel chromatography and eluting with a solvent such as hexane / ethyl acetate. It is also possible to purify mevalonolactone directly by distillation. It is also possible to crystallize by using an appropriate solvent.

  Examples of the present invention are shown below, but the present invention is not limited to these Examples. In the following Examples and Comparative Examples, “%” is “% by weight” unless otherwise specified.

[Example 1-1] Cultivation of mevalonic acid-producing bacterium Saccharomycopsis fibrigera (S. fibuligera) ADK8107 strain (FERM BP-2320) and cell preparation (primary culture)
S. fibuligera ADK8107 strain (FERM BP-2320) stored in YM agar medium is inoculated into one platinum loop in 50 ml of sterilized culture solution having the following composition, and is used at 26 ° C and 200 rpm using a rotary incubator. Pre-cultured for 4 days. 2 ml was taken from the culture solution after the preculture, inoculated into 50 ml of the culture solution having the same composition as the preculture, and cultured for 4 days under the same conditions as the preculture. The obtained culture solution is centrifuged at 20 ° C. and 3500 rpm for 15 minutes, the supernatant is removed by decantation, 20 ml of potassium phosphate buffer (pH 6.5) is added, the cells are well suspended, and again Centrifugation was performed under the same conditions, and the supernatant was removed (bacterial cell washing step). The washing of the cells was repeated twice.
<Composition of culture solution>
Polypeptone: 0.5%
Yeast extract: 0.25%
MgSO ₄ · 7H ₂ O: 0.05%
KH ₂ PO ₄ : 0.10%
Adecanol LG-294: 0.05%
D-glucose: 10.0%
Water (no pH adjustment): the rest

[Example 1-2] Production of labeled R-mevalonic acid by cells of S. fibuligera ADK8107 strain (FERM BP-2320) producing mevalonic acid (secondary culture)
Glucose was removed from the culture solution composition used in Example 1-1, and 10 ml of a culture solution (no glucose) in which the concentration of each component was doubled was prepared. Next, 1 g of D-glucose-U- ¹³ C ₆ (Chlorella Kogyo Co., Ltd., Lot No. 3052, [ ¹³ C]: 98.2 atom%) was dissolved in 10 ml of water and filtered through a 0.45 μm filter. It mixed with the said culture solution and obtained the labeled compound containing culture solution. The obtained labeled compound-containing culture solution (20 ml) and the bacterial cells prepared in Example 1-1 were mixed and cultured for 14 days. The culture solution after the culture was centrifuged (3500 rpm, 10 minutes) to obtain 18 ml of a supernatant (culture filtrate). When this culture filtrate was analyzed by the following method, the mevalonic acid concentration was 0.91 mg / ml.

<Analysis method>
Add 2 drops of phosphoric acid, 1 g of anhydrous sodium sulfate and 2 ml of ethyl acetate to 1 ml of the culture filtrate, stir for 30 seconds, then centrifuge at 3500 rpm for 10 minutes, and take the separated ethyl acetate layer in a separate test tube . Add 2 ml of ethyl acetate again to the remaining aqueous layer, stir for 30 seconds, and then centrifuge at 3500 rpm for 10 minutes. Further, this operation is performed again to obtain a total of about 6 ml of extract. After this extract is dried by a centrifugal evaporator to obtain an extract, the extract is dissolved in 0.1 ml of isopropanol and analyzed using HPLC under the analysis conditions shown below.
(Analysis conditions)
Column: Nucleosyl 5N (CH3) 2 (M. Nagel, Germany), size diameter 4.6 mm x length 250 mm, 40 ° C
Moving bed: N-hexane / isopropanol = 9/1, flow rate 2.0 ml / min Detection: differential refractometer (manufactured by Showa Denko KK, SE-61 type), injection amount 0.005 ml

[Example 1-3] Purification of labeled R-mevalonolactone After the culture filtrate obtained in Example 1-2 was adjusted to pH 2.0 with 10% phosphoric acid, 36 ml of methyl ethyl ketone was added, and AC distribution was performed. The extraction was performed by 36 ml of methyl ethyl ketone was again added to the aqueous layer, and AC distribution was performed again, and the aqueous layer was extracted three times in total. Thereafter, 4 g of sodium chloride was added to the aqueous layer and stirred. 36 ml of ethyl acetate was added thereto for AC extraction, and the aqueous layer was extracted 3 times in total. The three methyl ethyl ketone layers (108 ml) and the ethyl acetate layer (108 ml) obtained were combined and dried on a rotary evaporator to obtain 96 mg of labeled crude crude mevalonolactone.
The obtained crude mevalonolactone was developed on an analytical silica gel plate containing no fluorescent agent (developing solvent: hexane / ethyl acetate = 1/9), then the mevalonolactone fraction was scraped off, and mevalonolactone was extracted from the scraped silica gel with acetone. did. Acetone was removed from the extract and dried to obtain 11.9 mg of labeled purified mevalonolactone.
When the obtained purified mevalonolactone was subjected to mass spectrometry by GC-MS, 136 which was a molecular ion (M ⁺ ) was detected. Further, when ¹³ C-NMR (JEOL JMN-AL400 FT-NRM System) in which protons were decoupled was measured, a spectrum showing coupling between carbons shown in Table 1 below was obtained.

In addition, since this product showed levorotation by optical rotation measurement, it was confirmed that it was a natural type R-mevalonolactone.
From the above, it is clear that this product is R-mevalonolactone with all carbon atoms labeled with ¹³ C.

[Example 2] Labeled R-mevalonate fermentation by mevalonate-producing bacterium S. fibuligera ADK8108 strain (FERM BP-2321) Mevalon used in Examples 1-1 and 1-2 The same procedure as in Examples 1-1 to 1-3 was performed except that the acid-producing bacterium S. fibuligera ADK8107 strain (FERM BP-2320) was changed to the mevalonic acid-producing bacterium S. fibuligera ADK8108 strain (FERM BP-2321). Thus, 11.2 mg of labeled purified mevalonolactone was obtained. When the obtained R-mevalonolactone was subjected to mass spectrometry by GC-MS, 136 as a molecular ion (M ⁺ ) was detected, and it was confirmed that all carbon atoms were R-mevalonolactone labeled with ¹³ C.

[Comparative Example 1]
Purified mevalonolactone was obtained in the same manner as in Examples 1-1 to 1-3 except that D-glucose was used instead of D-glucose-U- ¹³ C ₆ used in Example 1-2. Mass analysis of this mevalonolactone by GC-MS detected 130, which is a molecular ion (M ⁺ ). Further, when ¹³ C-NMR obtained by decoupling protons was measured, a spectrum showing no coupling was obtained (Table 2). The optical rotation showed levorotatory. From the above, it is clear that this product is an unlabeled natural R-mevalonolactone.

[Example 3] Labeled R-mevalonic acid fermentation by mevalonic acid-producing bacterium Streptomyces lividans 35AS-3 strain Transformed strain Streptomyces lividans 35AS- obtained according to the following paragraphs [0113] to [0158] Three strains (which are described in Japanese Patent Application No. 2003-31138) were added to SK-II liquid medium (2% soluble starch, 0.5% glucose) containing thiostrepton 5 μg / ml. %, Yeast extract 0.5%, Bacto ^™ Peptone 0.3%, meat extract 0.3%, KH ₂ PO ₄ 0.02%, MgSO ₄ 0.006%) The cells were cultured for 2 days at 30 ° C. and 120 rpm in a shaking incubator. KG liquid medium (5% glucose, 2.5% soluble starch, 1.5% soy flour, 0.2% dry yeast, calcium carbonate, 1 ml each of the obtained culture solution containing 5 μg / ml thiostrepton 0.4%, pH 6.2) The solution was dispensed into baffled Erlenmeyer flasks containing 50 ml, and the culture was further continued for 2 days. The obtained culture solution was centrifuged at 3500 rpm for 10 minutes to obtain about 2.5 ml of bacterial cells. To the obtained cells, 20 ml of potassium phosphate buffer (pH 6.5) was added to suspend the cells well, and centrifuged again under the same conditions to remove the supernatant (bacterial cell washing step). The washing of the cells was repeated twice.
Medium containing thiostrepton 5 μg / ml, yeast extract 0.1%, KH ₂ PO ₄ 0.02%, MgSO ₄ 0.006% and D-glucose-U- ¹³ C ₆ 5% Using 20 ml, the cells were cultured for 7 days at 30 ° C. and 120 rpm in a shaking incubator.
After completion of the culture, mevalonolactone was purified from this culture solution in the same manner as in Example 1-3, and it was confirmed that all carbon atoms were R-mevalonolactone labeled with ¹³ C.

The gene recombination experiment in the preparation of the transformed strain Streptomyces lividans 35AS-3 described below was carried out using the method described in Molecular Cloning 2nd edition (hereinafter referred to as a conventional method) unless otherwise specified.
(1) Cloning of acetoacetyl-CoA synthase gene from Streptomyces sp. CL190
Streptomyces sp. CL190 strain 1 platinum loop, 100 ml KG liquid medium (glucose 2%, soluble starch 2.5%, soy flour 1.5%, dry yeast 0.2%, calcium carbonate 0.4%, pH 6.2 ) And cultured at 30 ° C. for 3 days.
Chromosomal DNA was isolated and purified from the cells according to a conventional method.

A sense primer and an antisense primer having a combination of the base sequence of SEQ ID NO: 2 and the base sequence of SEQ ID NO: 3 were synthesized using a DNA synthesizer. In the base sequence of SEQ ID NO: 2, the site “gaattcc” from the 4th base to the 9th base indicates an EcoRI cleavage site, s is g or c, h is a, c or t, and r is a or g is shown. In the base sequence of SEQ ID NO: 3, the site “aagctt” from the 4th base to the 9th base indicates a HindIII cleavage site, s indicates g or c, and v indicates a, c or g.
Using chromosomal DNA as a template, these primers and TaKaRa LA-PCR (registered trademark) Kit Ver.2 (Takara Shuzo), Expand (registered trademark) High-Fidelity PCR System (Roche) or Taq DNA polymerase (Promega) PCR was performed using a DNA amplification apparatus (manufactured by MJ Research).
PCR was performed under the conditions of 30 cycles at 95 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by 30 cycles followed by reaction at 72 ° C. for 10 minutes.
After completion of PCR, the reaction solution was subjected to agarose gel electrophoresis to obtain a DNA fragment of about 800 bp.

The DNA fragment obtained above was mixed with pGEM-T Easy vector (Promega), ethanol precipitation was performed, the resulting DNA precipitate was dissolved in 5 μl of distilled water, and ligation reaction was performed for recombination. Body DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
By determining the base sequence of the plasmid isolated by this method, it was confirmed that the plasmid was inserted with a DNA fragment containing a part of the target acetoacetyl-CoA synthase gene, and this plasmid was designated as pAAS-PCR1. Named.

  After digesting pAAS-PCR1 with EcoRI and HindIII, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain a DNA fragment of about 0.8 kb. Using this fragment as a probe, Southern hybridization with Streptomyces sp. Strain CL190 chromosomal DNA was carried out by a conventional method. As a result, the fragment hybridized with a BamHI fragment of about 3.0 kb.

After digesting Streptomyces sp. CL190 strain chromosomal DNA with BamHI, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain a DNA fragment of about 3.0 kb.
The approximately 3.0 kb BamHI-treated DNA fragment obtained above was mixed with the BamHI-treated pUC118 (purchased from Sakai Shuzo) fragment, followed by ethanol precipitation. The resulting DNA precipitate was dissolved in 5 μl of distilled water and ligated. Recombinant DNA was obtained by carrying out the reaction.

Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
The colony of the grown ampicillin resistant transformant was transplanted to a nylon membrane filter by a conventional method, the above pAAS-PCR1 was digested with EcoRI and HindIII, and then purified by agarose gel electrophoresis, about 0.8 kb acetoacetyl-CoA synthase Colony hybridization was performed by a conventional method using a DNA fragment containing a part of the gene as a probe.
A colony of a transformant harboring pUC118 into which a DNA fragment containing an acetoacetyl CoA synthase gene was identified, was cultured with shaking in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin at 37 ° C. for 16 hours. .

The cells were obtained by centrifuging the culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The DNA having the base sequence 2949 bp described in SEQ ID NO: 1 was named pAAS2.

(2) Construction of HMG CoA synthase gene and HMG CoA reductase gene derived from Streptomyces sp. CL190 strain, and plasmid pSEMV34 for transformation of Streptomyces genus containing the upstream base sequence of these genes
1-1) Construction of shuttle vector pSE101 that can replicate in E. coli and actinomycetes:
After digesting pUC19 (Takara Shuzo) with the restriction enzyme AatII, a BglII linker (Takara Shuzo) was ligated to create a plasmid vector pUC19B in which the AatII site was changed to the BglII site.
After digesting pUC19B with restriction enzymes BglII and KpnI, agarose gel electrophoresis was performed to obtain a BglII-KpnI-treated pUC19B fragment.
Actinomycetes vector pIJ702 (John Innes Institute) was digested with restriction enzymes BglII and KpnI and then subjected to agarose gel electrophoresis to obtain a BglII-KpnI-treated pIJ702 fragment.

By mixing the BglII-KpnI-treated pUC19B fragment obtained above and the BglII-KpnI-treated pIJ702 fragment, ethanol precipitation was performed, and the resulting DNA precipitate was dissolved in 5 μl of distilled water, followed by ligation reaction. Recombinant DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was a shuttle vector that could replicate in both E. coli and actinomycetes and was named pSE101.

1-2) Construction of pUMV33 containing Streptomyces sp. CL190 strain-derived mevalonate pathway gene cluster and promoter region upstream of the cluster:
PUMV19 described in JP-A-2001-161370 is a plasmid containing a mevalonate pathway gene cluster derived from Streptomyces sp. Strain CL190, and contains an HMG CoA synthase gene and an HMG CoA reductase gene (PCT / JP00 / 08620, Genes of enzymes involved in the mevalonate pathway derived from actinomycetes). The base sequence of a DNA fragment consisting of 6798 bp cloned in pUMV19 is shown in SEQ ID NO: 4.

Streptomyces sp. CL190 strain 1 platinum loop, 100 ml KG liquid medium (glucose 2%, soluble starch 2.5%, soy flour 1.5%, dry yeast 0.2%, calcium carbonate 0.4%, pH 6.2 ) And cultured at 30 ° C. for 3 days.
Chromosomal DNA was isolated and purified from the cells according to a conventional method.

A sense primer and an antisense primer having a combination of the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 6 were synthesized using a DNA synthesizer.
Using chromosomal DNA as a template, these primers and TaKaRa LA-PC® (registered trademark) Kit Ver.2 (Takara Shuzo), Expand (registered trademark) High-Fidelity PCR System (Roche) or Taq DNA polymerase (Promega) PCR was performed using a DNA amplification apparatus (manufactured by MJ Research).
PCR was performed under the conditions of 30 cycles at 95 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by 30 cycles followed by reaction at 72 ° C. for 10 minutes.

After completion of PCR, the reaction solution was subjected to agarose gel electrophoresis to obtain a DNA fragment having an EcoRI site of about 2.3 kb at both ends.
The DNA fragment obtained above was mixed with pGEM-T Easy vector (Promega), ethanol precipitation was performed, the resulting DNA precipitate was dissolved in 5 μl of distilled water, and ligation reaction was performed for recombination. Body DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin-resistant transformants that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The DNA having the base sequence of 2370 bp described in SEQ ID NO: 7 was named pCL33016.

After digesting pCL33016 with EcoRI, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain an about 2.3 kb EcoRI-treated DNA fragment.
After digesting pUMV19 with the restriction enzyme EcoRI, agarose gel electrophoresis was performed to obtain an EcoRI-treated pUMV19 fragment.

The about 2.3 kb EcoRI-treated DNA fragment obtained above was mixed with the EcoRI-treated pUMV19 fragment, and then ethanol precipitation was performed. The resulting DNA precipitate was dissolved in 5 μl of distilled water and subjected to a ligation reaction. Recombinant DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin-resistant transformants that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The DNA having the base sequence 8719 bp described in SEQ ID NO: 8 was named pUMV33.

1-3) Construction of pUMV34 and pUMV35 containing HMG CoA reductase gene, HMG CoA synthase gene derived from Streptomyces sp. Strain CL190 and promoter regions of different base lengths:
pUMV33 was completely digested with the restriction enzyme Aor51HI and then partially digested with the restriction enzyme SnaBI to recover about 7.5 kb and about 6.4 kb of DNA. Recombinant DNA was obtained by dissolving each of the obtained DNA fragments in 5 μl of distilled water and performing self-ligation reaction.

Using each of the recombinant DNAs, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin. Cultured overnight.
Several colonies of each ampicillin-resistant transformant that had grown were cultured with shaking in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin at 37 ° C. for 16 hours.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The DNA having the base sequence shown in SEQ ID NO: 9 was named pUMV34.
The DNA having the base sequence shown in SEQ ID NO: 10 was named pUMV35.

1-4) Construction of pBMV34 (vector exchange to blue script to give restriction enzyme sites at both ends):
After digesting pUMV34 with restriction enzymes EcoRI and HindIII, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain about 3.2 kb EcoRI-HindIII-treated pUMV34.
pBluescript II SK (+) (Stratagene) was digested with restriction enzymes EcoRI and HindIII and then subjected to agarose gel electrophoresis to obtain an EcoRI-HindIII-treated pBluescript II SK (+) fragment.

About 3.2 kb EcoRI-HindIII-treated pUMV34 obtained above was mixed with EcoRI-HindIII-treated pBluescript II SK (+), followed by ethanol precipitation, and the resulting DNA precipitate was dissolved in 5 μl of distilled water. Recombinant DNA was obtained by performing a ligation reaction.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was named pBMV34.

1-5) Construction of actinomycete expression plasmid pSEMV34 (including promoter region 0.8 kb):
After digesting pBMV34 with restriction enzymes XbaI and HindIII, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain about 3.2 kb of XbaI-HindIII-treated pBMV34.
After digesting pSE101 with restriction enzymes XbaI and HindIII, agarose gel electrophoresis was performed to obtain an XbaI-HindIII-treated pSE101 fragment.

After mixing the approximately 3.2 kb XbaI-HindIII-treated pBMV34 obtained above with XbaI-HindIII-treated pSE101, ethanol precipitation is performed, and the resulting DNA precipitate is dissolved in 5 μl of distilled water to perform a ligation reaction. The recombinant DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was named pSEMV34.

(3) Transformation of Actinomyces Streptomyces lividans TK23 strain with plasmid pSEMV34 Transformation of Actinomyces S. lividans TK23 strain with plasmid pSEMV34 was performed using Practical Streptomyces Genetics (ISBN 0-7084-0623-8, Tobias Kieser et al., The John Innes Foundation).

About several colonies of the obtained transformants, 10 ml of SK-II liquid medium containing 2 μg / ml thiostrepton (soluble starch 2%, glucose 0.5%, yeast extract 0.5%, Bacto ^™ Peptone 0 3%, meat extract 0.3%, KH ₂ PO ₄ 0.02%, MgSO ₄ 0.006%) at 30 ° C. for 3 days.
The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
One strain of the transformant of S. lividans TK23 obtained above was named S. lividans 34-1 strain. This strain is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number “FERM P-19120”.

(4) Construction of Streptomyces sp. CL190-derived HMG CoA synthase gene and HMG CoA reductase gene and a plasmid vector for transformation of Streptomyces genus (pSEMV35) containing the base sequence of the upstream region of these genes
3-1) Construction of pBMV35 (vector exchange to blue script to give restriction enzyme sites at both ends):
After digesting pUMV35 with restriction enzymes EcoRI and HindIII, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain about 4.3 kb EcoRI-HindIII-treated pUMV35.
pBluescript II SK (+) (Stratagene) was digested with restriction enzymes EcoRI and HindIII and then subjected to agarose gel electrophoresis to obtain an EcoRI-HindIII-treated pBluescript II SK (+) fragment.

About 4.3 kb EcoRI-HindIII-treated pUMV35 obtained above was mixed with EcoRI-HindIII-treated pBluescript II SK (+), followed by ethanol precipitation, and the resulting DNA precipitate was dissolved in 5 μl of distilled water. Recombinant DNA was obtained by performing a ligation reaction.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was named pBMV35.

3-2) Construction of actinomycete expression plasmid pSEMV35 (including promoter region 1.9 kb):
After digesting pBMV35 with E restriction enzymes XbaI and HindIII, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain about 4.3 kb of XbaI-HindIII-treated pBMV35.
After digesting pSE101 with restriction enzymes XbaI and HindIII, agarose gel electrophoresis was performed to obtain an XbaI-HindIII-treated pSE101 fragment.

After mixing the approximately 4.3 kb XbaI-HindIII-treated pBMV35 obtained above with XbaI-HindIII-treated pSE101, ethanol precipitation is performed, and the resulting DNA precipitate is dissolved in 5 μl of distilled water to perform a ligation reaction. The recombinant DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was named pSEMV35.

(5) Transformation of Actinomyces S. lividans TK23 strain with plasmid vector pSEMV35 Transformation of Actinomyces S. lividans TK23 strain with plasmid pSEMV35 was performed using Practical Streptomyces Genetics (ISBN 0-7084-0623-8, Tobias Kieser et al., The John Innes Foundation).

Several colonies of the obtained transformants were cultured with shaking in 10 ml of SK-II liquid medium containing 5 μg / ml of thiostrepton at 30 ° C. for 3 days.
The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
One of the transformants of the S. lividans TK23 strain obtained above was named S. lividans 35-6 strain. This strain is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number “FERM P-19122”.

(6) Streptomyces sp. Strain CL190-derived acetoacetyl-CoA synthase gene, HMG CoA synthase gene and HMG CoA reductase gene, and a plasmid pSEMV34AS for transformation of the Streptomyces genus containing the base sequence upstream of these genes And transformation of Actinomyces S. lividans TK23 strain with pSEMV34AS A sense primer and an antisense primer having a combination of the nucleotide sequence of SEQ ID NO: 11 and the nucleotide sequence of SEQ ID NO: 12 were synthesized using a DNA synthesizer . In the base sequence of SEQ ID NO: 11, the site from the 5th base to the 10th base “tctaga” indicates the Xba I cleavage site, and in the base sequence of SEQ ID NO: 12, the 4th base to the 9th base The site “gaattc” up to the base indicates the EcoRI cleavage site.
Using pAAS2 obtained in (1) above as a template, these primers and TaKaRa LA-PCR (registered trademark) Kit Ver. 2 (Takara Shuzo), Expand (registered trademark) High-Fidelity PCR System (Roche) or PCR was performed using Taq DNA polymerase (Promega) with a DNA amplification device (MJ Research).
PCR was performed under the conditions of 30 cycles at 95 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by 30 cycles followed by reaction at 72 ° C. for 10 minutes.
After completion of PCR, the reaction solution was subjected to agarose gel electrophoresis to obtain a DNA fragment having an XbaI site and an EcoRI site of about 1.3 bp at both ends.

The DNA fragment obtained above is mixed with pUC118 vector (manufactured by Sakai Shuzo Co., Ltd.) cleaved with XbaI and EcoRI, followed by ethanol precipitation, and the resulting DNA precipitate is dissolved in 5 μl of distilled water to perform a ligation reaction. As a result, recombinant DNA was obtained.
Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
By determining the base sequence of the plasmid isolated by this method, it was confirmed that the plasmid was inserted with a DNA fragment containing the target acetoacetyl-CoA synthase gene.
The DNA having the nucleotide sequence of 1308 bp of SEQ ID NO: 13 was named pAAS3.

(7) Streptomyces sp. CL190-derived acetoacetyl-CoA synthase gene, HMG CoA synthase gene and HMG CoA reductase gene, and the plasmid pSEMV35AS for transformation of Streptomyces genus containing the base sequence of the upstream region of these genes Construction and transformation of Actinomyces S. lividans TK23 strain with pSEMV35AS After digesting pAAS3 obtained in (5) above with XbaI and EcoRI, the restriction enzyme-treated DNA fragment was subjected to agarose gel electrophoresis to obtain a DNA fragment of about 1.3 kb did.
The pSEMV35 constructed in the above (4) was digested with restriction enzymes XbaI and EcoRI, and then subjected to agarose gel electrophoresis and treated with XbaI and EcoRI to obtain DNA fragments.
Two DNA fragments derived from 1.3 kb and pSEMV35 obtained above were mixed, followed by ethanol precipitation. The obtained DNA precipitate was dissolved in 5 μl of distilled water, and ligation reaction was performed to obtain recombinant DNA. I got it.

Using this recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed according to a conventional method, and then the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. did.
Several colonies of ampicillin-resistant transformants that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
The resulting plasmid was named pSEMV35AS.

Transformation of Actinomyces S. lividans TK23 strain with plasmid pSEMV35AS was performed using the method described in Practical Streptomyces Genetics (ISBN 0-7084-0623-8, Tobias Kieser et al., The John Innes Foundation).
Several colonies of the obtained transformants were cultured with shaking in 10 ml of SK-II liquid medium containing 5 μg / ml of thiostrepton at 30 ° C. for 3 days.

The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein.
One of the transformants of S. lividans TK23 obtained above was named S. lividans 35AS-3.

[Examples 4 and 5] Uptake of ¹³ C-labeled acetic acid and glycerol into mevalonic acid Instead of D-glucose-U- ¹³ C ₆ used in Example 1-2, acetic acid-U- ¹³ C Purified mevalonolactone was obtained in the same manner as in Examples 1-1 to 1-3 except that ₂ (Example 4) or glycerol-U- ¹³ C ₃ (Example 5) was used. Mass analysis of these mevalonolactones by GC-MS detected 136, which is a molecular ion (M ⁺ ), and all of the mevalonolactones obtained in Examples 4 and 5 were ¹³ C. It was confirmed that it was labeled R-mevalonolactone.

[Example 6] Incorporation of acetic acid labeled with D into mevalonic acid As in Example 4, except that CD ₃ COOH was used instead of acetic acid-U- ¹³ C ₂ used in Example 4. And purified mevalonolactone was obtained. When this mevalonolactone was subjected to mass spectrometry by GC-MS, 137 was detected as the molecular ion (M ⁺ ). The obtained mevalonolactone was confirmed to be R-mevalonolactone labeled with D at 7 positions of the 2-position methylene hydrogen, the 3-methyl group hydrogen, and the 4-position methylene hydrogen.

[Example 7] Uptake of ¹³ C-labeled D-glucose into mevalonic acid S. fibuligera strain ADK8107 (FERM BP-2320) stored in potato sucrose agar medium, D-glucose as D-glucose Except for changing to glucose-U- ¹³ C ₆ , 1 platinum ear was inoculated into 20 ml of a sterilized culture solution having the same composition as the culture solution used in Example 1-1, and 26 ° C using a rotary incubator. Culture was performed at 200 rpm for 7 days. R-mevalonolactone was purified from the obtained culture solution in the same manner as in Example 1-3. When the obtained R-mevalonolactone was subjected to mass spectrometry by GC-MS, 136 as a molecular ion (M ⁺ ) was detected, and it was confirmed that all carbon atoms were R-mevalonolactone labeled with ¹³ C.

Claims (7)

  A method for producing labeled R-mevalonic acid, comprising incorporating a labeled compound into a mevalonic acid-producing bacterium and accumulating the labeled R-mevalonic acid in a culture.   When incorporating a labeled compound into a mevalonic acid-producing bacterium, the mevalonic acid-producing bacterium is first cultured using the culture medium components for cell growth, and then unlabeled R-mevalonic acid is removed from the primary culture. The culture broth fraction is removed to prepare a bacterial cell fraction, and then the bacterial cell fraction is subjected to secondary culture using a medium component containing a labeled compound to thereby label R in the secondary culture. The method for producing labeled R-mevalonic acid according to claim 1, characterized in that -mevalonic acid is accumulated.   The method for producing labeled R-mevalonic acid according to claim 1 or 2, wherein the mevalonic acid-producing bacterium is a strain belonging to Saccharomycopsis fibuligera or a mutant thereof.   The mevalonic acid-producing bacterium is Saccharomycopsis fibuligera ADK 8107 (FERM BP-2320), or Saccharomycopsis fibuligera ADK 8108 (FERM BP-2321) or a mutation thereof The method for producing labeled R-mevalonic acid according to any one of claims 1 to 3, which is a strain.   The mevalonate-producing bacterium encodes 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene against actinomycetes without mevalonate pathway The method for producing labeled R-mevalonic acid according to claim 1 or 2, which is a strain transformed with DNA to be transformed or a mutant thereof. The labeled compound is a compound containing one or more isotopes selected from the group consisting of ² H (D), ³ H (T), ¹³ C, ¹⁴ C, ¹⁷ O and ¹⁸ O. The method for producing labeled R-mevalonic acid according to any one of claims 1 to 5, wherein:   The labeled compound is acetic acid, glycerol, glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphospho Glyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, fructose, fructose 1-phosphate, pyruvate, fatty acids having 4 to 22 carbon atoms, monoglycerides, diglycerides and triglycerides which are derivatives of these fatty acids And a method for producing labeled R-mevalonic acid according to any one of claims 1 to 6, wherein the compound is one or more compounds selected from the group consisting of phospholipids.