WO2001042476A1 - Actinomycetes-origin genes of enzymes participating in mevalonate pathway - Google Patents

Abstract

Actinomycetes-origin DNA encoding a plural number of enzymes participating in the mevalonate pathway; DNAs encoding respective enzymes contained in the above DNA; proteins encoded thereby; vectors and transformants containing these DNAs; and a process for producing isoprenoid compounds by using these DNAs. Isoprenoid compounds can be produced by culturing the transformants constructed by transferring these genes into a host such as Escherichia coli.

Description

 Specification

 Genes of enzymes involved in the baronic acid pathway from actinomycetes

 The present invention provides a DNA encoding a plurality of enzymes involved in the actinomycete-derived mevalonate pathway, a DNA encoding each enzyme contained in the DNA and a protein encoded thereby, a vector containing the DNA, and a plasmid. The present invention relates to a transformant and a method for producing an isoprenide compound using the DNA. Background art

 Isoprenide (also referred to as terpene) is a general term for a group of organic compounds having a carbon skeleton of isoprene units in the basic skeleton, and isopentenyl pyrophosphate (IP

Biosynthesized by polymerization of P). In addition to (C ₅ H ₈ ) _n unsaturated hydrocarbons, their redox products (alcohols, ketones, acids, etc.) and compounds with carbon eliminated have been found in many plants and animals. Isoprenoids can be classified into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesame terpenes (C25), and triterpenes (C30) according to the number of carbon atoms , Tetraterpenes (C40, carotenoids), and other polyterpenes.

 Many compounds show physiological activity, such as abscisic acid, juvenile hormone, gibberellin, forskolin, and phorbol. In addition, chlorophyll, vitamin K, ubiquinone, tRNA, and the like as complex terpenes having an isoprene structure as a part of the structure include useful physiological activities.

 For example, ubiquinone, an isoprenide 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. As demand increases.

Vitamin K is an important vitamin involved in the blood coagulation system and is used as a hemostatic agent. In addition to its use in recent years, its involvement in bone metabolism has recently been suggested, and its application in the treatment of osteoporosis is expected. Filoquinone and menaquinone have been approved as pharmaceuticals.

 In addition, ubiquinone and vitamin K have an inhibitory effect on shellfish adhesion, and are expected to be applied to shellfish adhesion prevention paints.

 In addition, carotenoid has an antioxidant effect, and some carotenoids, such as carotene, astaxanthin and cryptoxanthin, are expected to have cancer-preventing and immunostimulatory activities.

 As described above, isoprenide compounds contain substances useful for living organisms, and if these inexpensive production methods are established, it will be of great social and medical benefit. .

 The production of isoprenide compounds by fermentation has been studied for some time, and attempts have been made to examine the culture conditions, breed strains by mutagenesis, and improve the production by genetic engineering techniques. However, its effect is limited to individual compound species, and no effective method is known for isoprenide compounds in general.

 Isopentenyl biphosphoric acid (IPP), the basic skeleton unit of isoprenide compounds, is biosynthesized from acetyl CoA via mevalonic acid (evalonic acid pathway) in eukaryotes such as animals and yeast. Has been proven.

 In the mevalonate pathway, 3-hydroxy-13-methylglucuryl-CoA (HMG-CoA) reductase is considered to be the rate-limiting enzyme (Mol. Biol. Cell, 5, 655 (1994)). Attempts have been made to increase the expression of HMG-CoA reductase and increase carotenoid productivity (Mizawa et al., Proceedings of the Carotenoid Research Symposium (1997)).

In prokaryotes such as Escherichia coli, IPP is biosynthesized through another pathway, namely, 1-deoxy D-xylose 5-phosphate generated by condensation of pyruvate and glyceraldehyde 3-phosphate. A non-mevalonate pathway has been discovered in many prokaryotes (Biochem. J., 295, 517 (1993)), and experiments using ¹³ C-labeled substrates have Deoxy-D-xylulose 5-phosphate has been demonstrated to be converted to IPP via 2-C-methyl-D-erythritol 4-monophosphate (Tetrahedron Lett. 38, 4769 (1997). ); Tetrahedron Lett. 39, 4509 (1998)). In particular, in E. coli, IPP has been demonstrated to be synthesized only through the non-mevalonate pathway (Rohmer,. In Comprehensive Natural Products Chemistry, Vol. 2: Isoprenoids including carotenoids and steroids; Barton, D. Nakanisni, K. Eds. Elsevier: Amsterdam, 1999; pp. 45-67).

 On the other hand, it is known that the actinomycetes Streptomyces sp. Strain CL190 (J. Antibiot. 43: 444, 1990) synthesizes IPP via menosulfonic acid (Tetrahedron Lett. 31: 6025, 1990. and Tetrahedron Lett. 37: 7979, 1996.) The present inventors have previously reported from an actinomycete Streptomyces sp. CL190 strain, an enzyme that catalyzes one reaction on the mevalonic acid pathway, 3-hydroxy-13-methylgluuryl. The gene (hmgr) encoding the CoA (HMG-CoA) reductase has already been cloned (J. Bacteriol. 181: 1256, 1999). However, genes encoding other enzymes involved in the mevalonate pathway, which are considered to be present in this actinomycete strain, have not been cloned yet. Disclosure of the invention

 One of the objects of the present invention is to provide a biosynthetic pathway for isoprenide compounds useful for pharmaceuticals, health foods, and shellfish anti-adhesive paints for heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation, etc. One object of the present invention is to provide a DNA containing a group of genes involved in the mevalonate pathway. Yet another object of the present invention is to provide a method for producing an isoprenide compound by culturing a transformant obtained by introducing the above DNA into a host cell.

The present inventors have conducted intensive studies to solve the above problems, and as a result, obtained a gene involved in the mevalonate pathway of Streptomyces sp. Strain CL190 and transformed it into Escherichia coli. Was cultured, the isoprenoid compound The present inventors have found that the production of ubiquinone, one of the products, has been improved, and have completed the present invention.

 That is, according to the present invention, a DNA having any of the following:

 (1) the nucleotide sequence of SEQ ID NO: 1;

 (2) a nucleotide sequence in which one to several nucleotides have been deleted, substituted, added and / or inserted in SEQ ID NO: 1, and which encodes all the enzymes necessary for the function of the mevalonate pathway An array; or

 (3) a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and that encodes all enzymes necessary for the function of the mevalonate pathway:

Is provided.

 Preferably, the enzymes necessary for the functioning of the mevalonate pathway are at least phosphomevalonate kinase, diphosphomevalonate decarboxylase, mevalonate kinase, HMG-CoA reductase and HMG-CoA. This is Shin-Yuse. According to another aspect of the present invention, there is provided a protein encoded by the above DNA.

 According to yet another aspect of the present invention, DNA having any of the following:

 (1) a nucleotide sequence of SEQ ID NO: 2 in which one to several nucleotides have been deleted, replaced, added and / or inserted in SEQ ID NO: 2, which encodes phosphomevalonate kinase A sequence or a base sequence capable of hybridizing under stringent conditions to the base sequence of SEQ ID NO: 2, which base sequence encodes phosphomevalonate kinase;

(2) the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence in which one to several nucleotides have been deleted, substituted, added and / or inserted in SEQ ID NO: 3, which encodes diphosphomevalonate decarboxylase Or a base sequence capable of hybridizing with the base sequence of SEQ ID NO: 3 under stringent conditions, wherein the base sequence encodes diphosphomevalonate decarboxylase; (3) the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence in which one to several nucleotides are deleted, substituted, added and / or inserted in SEQ ID NO: 4, which encodes a mevalonate kinase; Or a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 4 under stringent conditions, and that encodes mevalonate kinase;

 (4) the nucleotide sequence of SEQ ID NO: 5; or

 (5) a nucleotide sequence of SEQ ID NO: 7, in which one to several nucleotides are deleted, substituted, added and / or inserted in SEQ ID NO: 7, and are HMG-CoA synthases. Or a base sequence capable of hybridizing with the base sequence of SEQ ID NO: 7 under stringent conditions, wherein the base sequence encodes HMG-CoA synthase;

Is provided.

 According to still another aspect of the present invention, there is provided a protein encoded by the above DNA.

 According to yet another aspect of the invention, a protein having any of the following:

(1) an amino acid sequence of SEQ ID NO: 8, an amino acid sequence in which one to several amino acids have been deleted, substituted, added, or inserted in SEQ ID NO: 8, having an amino acid sequence having phosphomevalonate kinase activity, or An amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 8, and an amino acid sequence having phosphomevalonate kinase activity;

 (2) the amino acid sequence of SEQ ID NO: 9; an amino acid sequence in which one to several amino acids are deleted, substituted, added, and / or inserted in SEQ ID NO: 9, and which has diphosphomevalonate decarboxylase activity An amino acid sequence having 60% or more homology with the sequence or the amino acid sequence of SEQ ID NO: 9 and having an activity of diphosphomevalonate decarboxylase;

(3) an amino acid sequence of SEQ ID NO: 10, wherein one or more amino acids in SEQ ID NO: 10 are deleted, substituted, added, Z- or inserted, and An amino acid sequence having a lonate kinase activity, or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence having a mevalonate kinase activity;

 (4) the amino acid sequence of SEQ ID NO: 11; or

 (5) the amino acid sequence of SEQ ID NO: 13; the amino acid sequence of SEQ ID NO: 13 in which one to several amino acids are deleted, substituted, added and / or inserted; An amino acid sequence having an HMG-CoA synthase activity, which is an amino acid sequence having an activity of 60% or more with the amino acid sequence of SEQ ID NO: 13;

Is provided.

 According to still another aspect of the present invention, there is provided a vector comprising the above DNA of the present invention. According to still another aspect of the present invention, there is provided a transformant having the above vector. Preferably, the transformant is E. coli.

According to still another aspect of the present invention, a step of culturing a transformant produced by transforming a vector containing the DNA of the present invention into a host to produce an isoprenoid compound in a culture, and There is provided a method for producing an isoprenide compound, comprising a step of collecting an isoprenide compound from a culture. Preferably, Isopurenoi de compound Yubikinon an I Sopurenoi de compound selected from vitamin K ₂ or carotenoids. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the structure of the gene of the present invention, the structure of the deletion mutant, and the results of the presence or absence of growth of each transformant under various culture conditions. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, a method and an embodiment of the present invention will be described in detail.

In the present specification, "1 to several bases are deleted, substituted, added and / or inserted. `` Is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5 bases of any number of deletions, substitutions, It means addition and no or inserted.

 As used herein, "one to several amino acids are deleted, substituted, added and / or inserted" means, for example, 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 15 amino acids. It means that any number of 10 amino acids, more preferably 1 to 5 amino acids, is deleted, substituted, added and / or inserted.

As used herein, the phrase "can be hybridized under stringent conditions" means that the DNA is used as a probe and the colony hybridization method, the plaque hybridization method, or the Southern blot hybridization method is used. Means DNA obtained by using a method such as colony or plaque-derived DNA or a DNA fragment immobilized with a fragment of the DNA. After hybridization at 65 ° C in the presence of NaCl, ◦.Approximately 1 to 2 times SSC solution (The composition of a 1x concentration SSC solution is 15 mM OCI, 15 mM DNA that can be identified by washing the filters with sodium citrate) at 65 ° C can be mentioned. Hybridization is described in Molecular Cloning: A laboratory Manual, 2 ^{n <1} Ed "Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989." It can be done according to the method that is used.

 Examples of the DNA that can hybridize under stringent conditions include DNAs having a certain degree of homology with the base sequence of the DNA used as the probe.The homology is, for example, 60% or more, preferably 7% or more. It is at least 0%, more preferably at least 80%, further preferably at least 90%, particularly preferably at least 95%, most preferably at least 98%.

The present invention also relates to an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 8, 9, 10, or 13 and having an amino acid having a desired activity. Regarding protein. The homology with the amino acid sequence of SEQ ID NO: 8, 9, 10, or 13 is not particularly limited as long as it is 60% or more, for example, 60% or more, preferably 70% or more, more preferably 80% or more, further preferably It is at least 90%, particularly preferably at least 95%, most preferably at least 98%.

 As used herein, the “mevalonate pathway”

 (1) Step of converting acetoacetyl CoA into HMG-CoA (catalyzed by HMG-CoA synthase);

 (2) HMG-CoA conversion to mevalonic acid (catalyzed by HMG-CoA reductase);

 (3) converting mevalonic acid to pyrophosphomevalonate (catalyzed and regulated by mevalonate (MVA) kinase, phosphomevalonate (PMVA) kinase); and

 (4) The process by which pyrophosphomevalonic acid is converted to isopentenyl biphosphate (IPP) (catalyzed by diphosphomevalonic acid (PMVA) decarboxylase): This is the pathway by which IPP is biosynthesized.

 Enzymes required to function the mevalonate pathway include at least PMVA kinase, PMVA decarboxylase, MVA kinase, 11010- (08-reductase and HMG-CoA sinkase).

 Next, a method for acquiring DNA and a method for using the DNA according to the present invention will be described.

(A) Acquisition of DNA encoding an enzyme involved in the actinomycete mevalonate pathway As described above, the present inventors have previously performed one reaction on the mevalonate pathway from the actinomycete Streptomyces sp. We have cloned the gene (hmgr) encoding the enzyme that catalyzes 3-hydroxy-13-methylglutylyl CoA (HMG-CoA) reductase (J. Bacteriol. 181: 1256, 1999). The DNA having the nucleotide sequence of SEQ ID NO: 1 of the present invention can be obtained by using this hmgr gene as a probe. The nucleotide sequence of hmgr gene includes the nucleotide sequence of SEQ ID NO: 6. Yeast involved in the mevalonic acid pathway Specific examples of the method for obtaining the DNA region encoding the element include the following methods.

Actinomycetes, for example Streptomyces sp. And CL190 strain suitable medium, for example GPY medium (1% glucose, 0.4% polypeptone, 0.4% yeast E custo _{Lactobacillus, 0. 5% MgS0 4 · 7H} 2 0, 0. 1 % K ₂ HP0 ₄₎ at a suitable temperature (e.g., cultured for several days at 3 0 ° C). After culturing, cells are obtained from the obtained culture by centrifugation, and chromosomal DNA is isolated and purified from the cells according to a standard method (Molecular Cloning, 2nd edition). After cutting the obtained chromosomal DNA with an appropriate restriction enzyme (for example, SnaB I or the like), Southern hybridization (Molecular Cloning 2nd Edition) using the hmgr gene as a probe is performed. Southern hybridization—The result of the probe is that the signal of the probe at a specific location (for example, at 6.7 kb when SnaBI is used as a restriction enzyme for digestion of chromosomal DNA) is detected. Is detected.

Next, the chromosomal DNA of Streptomyces sp. CL190 strain was cut again with the same restriction enzymes as above (for example, SnaB I), agarose gel electrophoresis was performed, and the position at which the signal was detected as a result of Southern hybridization (S If naBI is used, the DNA fragment corresponding to (6.7 kb position) is extracted from the agarose gel and recovered. The recovered DNA fragment is blunt-ended using T4 DNA polymerase (purchased from Takara Shuzo), inserted into an appropriate plasmid (for example, pUC118, etc.), and the chromosomal DNA library of Streptomyces sp. Is prepared. Using this chromosomal DNA library, a suitable host (for example, E. coli strain JM109) is transformed according to a standard method (Molecular Cloning, 2nd edition), and the transformant is used as a probe with the hmgr gene as a probe. By screening using the colony hybridization method, a transformant of Escherichia coli having a plasmid containing the hmgr gene can be isolated. By extracting a plasmid from the isolated transformant according to a conventional method, a DNA fragment containing the hmgr gene can be isolated. An example of the DNA that can be isolated by the above method is a DNA having the base sequence of SEQ ID NO: 1. Also, a DNA having the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7, which is a partial sequence of the nucleotide sequence of SEQ ID NO: 1, is within the scope of the present invention. DNA having the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: can be appropriately treated with restriction enzyme treatment or PCR based on the nucleotide sequence information of SEQ ID NO: 1. It can be obtained by a conventional method known to a trader.

 In order to obtain DNA having the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7 by PCR, the chromosomal DNA of Streptomyces sp. A pair of primers designed to amplify the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7 using DNA having the nucleotide sequence of PCR using DNAThermal Cycler (manufactured by Pachinkin Elmer Japan) using TaKaRa LA PCR ™ Kit Ver.2 (Takara Shuzo) or Expand ™ High-Fidelity PCR System (manufactured by Boehringer Mannheim). I do. In order to facilitate the subsequent cloning operation, it is preferable to add an appropriate restriction enzyme site to the primer.

The reaction conditions for PCR were as follows: a reaction process consisting of 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds to 1 minute (aniring), and 72 ° C for 2 minutes (extension). For example, after 30 cycles, the reaction may be carried out at 72 ° C. for 7 minutes. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in E. coli. Cloning is carried out by a conventional method, for example, Molecular Protocol, 1st Edition, Current Protocols in Molecular Biology, supplements 1-38, John Wiley & Sons (1987-1997) (hereinafter, `` Current Protocols in Molecular Biology ''). DNA Clonin 1: CoreTechniques, A Practical Approach, Second Edition, the method described in Oxford University Press (1995), or a commercially available kit, for example, Superscript Plasmid System for cDNA It can be performed using Synthesis and Plasmid Cloning (manufactured by Life'Technologies Corporation) or ZAP 'cDNA Synthesis Kit (manufactured by Stratagene).

 As a cloning vector, either a phage vector or a plasmid vector can be used as long as it can replicate autonomously in E. coli K12 strain. Good. Specifically, ZAP Express (Stratagene, Strategies, 5, 58 (1992)), pBluescrlpt II SK (+) (Nucleic Acids Research, 17, 9494 (1989)), Lambda ZAP II (Stratagene) Gtl0, Agtll CDNA Cloning, A Practical Approach, 1, 49 (1985) λ riplEx (Clontech), ExCell (Pharmacia), pT7T318U (Pharmacia), cD2 CMol.Cen Biol., 3, 280 (1983)], pMW218 (manufactured by Wako Pure Chemical Industries), pUCll8 (manufactured by Takara Shuzo), pEG400 (Bac 172, 2392 (1990)), pQE-30 (manufactured by QIAGEN), etc. Can be given.

 From the obtained transformant, a plasmid containing the target DNA can be prepared by a conventional method, for example, Molecular Cloning, 2nd edition, Current 'Protocols', 'Molecularity, DNA Cloning, DNA Cloning'. 1: Can be obtained by the method described in Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) and the like.

 According to the above method, DNA having the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7 can be obtained.

Also, a nucleotide sequence in which one to several nucleotides are deleted, substituted, added and / or inserted in the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7. A base sequence encoding an enzyme protein having a specific activity, or hybridizing under stringent conditions to the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 7. A nucleotide sequence that encodes an enzyme protein having a specific activity is also within the scope of the present invention. For example, using the nucleotide sequence of a DNA fragment derived from an actinomycete having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7 Can be isolated by screening under appropriate conditions.

 Alternatively, the mutant DNA as described above can be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, a DNA having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7 can be used to obtain a mutant DNA by introducing a mutation into these DNAs. it can.

 For example, a method of bringing a DNA having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 7 into contact with a mutagenic agent, irradiating with ultraviolet rays, genetic engineering, It can be performed using a dynamic method or the like.

 Site-directed mutagenesis, which is one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning Second Edition, Current 'Protocols' 'Molecular Biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Proc. Natl. Acad. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sci. USA, 82, 488, 1985, Gene, 34, 315, 1985, Gene, 102, 67, 1991. It can be carried out according to.

(B) Preparation of a transformant having DNA encoding an enzyme of the actinomycete mevalonate pathway and expression of the protein of the enzyme

In order to express the DNA obtained as described above in a host cell, first, the DNA fragment of interest is digested with a restriction enzyme or a DNA degrading enzyme to obtain a DNA fragment of an appropriate length containing the gene. After that, insert the expression vector downstream of the promoter in the expression vector, and then insert the expression vector into which the above DNA has been inserted into the expression vector. Into the combined host cells.

 As the host cell, any cell that can express the desired gene can be used. For example, bacteria belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc .; Yeasts, animal cells, insect cells, etc. belonging to the genus, Trichosporonus, Schizinomyces and the like.

 Although the DNA of the present invention encodes an enzyme involved in the mevalonate pathway, even a bacterium which does not originally have a mevalonate pathway as a host cell may be a yeast, animal cell, or insect having the mevalonate pathway. Any of cells may be used.

 As the expression vector, those which can replicate autonomously in the above-mentioned host cells or can be integrated into a chromosome, and which contain a promoter at a position where the above-mentioned target DNA can be transcribed are used.

 When a bacterium or the like is used as a host cell, an expression vector for expressing the DNA is capable of autonomous replication in the bacterium, and is composed of a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence. It is preferably a recombinant vector. A gene that controls the promoter may be included.

Examples of expression vectors include pBTrP2, pBTacl, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (lnvitrogen), pGEMEX-l (Promega), pQE-8 (QIAGEN Co.), pQE-30 (QIAGEN, Inc.), pKYPlO (JP-A-58- 110600), pKYP ² 00 [Agrc.Biol.Chem., 48, 669 (l984 ) PLSAl Agrc. Biol. Chem., 53, 277 (1989)), pGELl [Pro Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescrlptll SK10, pBluescriptll SK (-) (Stratagene), pTrS30 (FERMBP-5407) ), _P TrS32 (FERM BP-5408), pGEX (Pharmacia), pET ^ Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pCl94, pUCl8 (Gene, 33, 103 ( 1985)), pUCl9 (Gene, 33, 103 (1985)), pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), Examples thereof include pPAl (Japanese Patent Application No. 63-233798), pEG400 [J. Bacteriol., 172, 2392 (1990)], pQE-30 (manufactured by QIAGEN), and the like.

Any promoter may be used as long as it can be expressed in the host cell. For example, tr promoter Isseki one (P trp), lac promoter (P lac), PL promoter, P _R promoter Isseki one, promoter Isseki one derived from the P _SE promoter evening one such as Escherichia coli or phage, such as, SP01 promoter, SP02 promoter, penP promoter, etc. Also, artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp X 2), a tac promoter, a letl promoter, and a lacT7 promoter can be used.

 The liposome-binding sequence may be any as long as it can be expressed in the host cell. An appropriate distance (for example, 6 to 18 bases) between the Shine-Dalgamo sequence and the initiation codon It is preferable to use a plasmid that is regulated to a certain degree. C Although a transcription termination sequence is not always necessary for expression of a target DNA, it is desirable to arrange a transcription termination sequence immediately below a structural gene.

The host cells include Escherichia) U, Corynebacterium J¾ Brevibacterium, Bacillus, Microbacterium, Serratia, Pseudomonas, Agrobacterium, Alicyclob acillus, Anabaena rot, Anacystis, Arthrobacter 厲, Azobacter, Chromatium genus, Methyl ob acterium rot, Phormidium rot, Rhodobacter rot, Rhodopseudomonas genus, Rhodospirillum aside, Scenedesmun厲, Streptomyces sp., Synnecoccus genus, can Rukoto force ^s raised a microorganism belonging to the Zymomonas genus and the like, preferably, Escherichia spp., Corynebacterium Jffi , Brevibacterium rot, aculus yuu, Pseudomonas wu, Agrobacterium wu, Alicyclob aculus exhibition, Anabaena wu, Anacystis genus, Arthrobacter 胰, Azobacter genus, Chromatium ¾ Erwinia rot, Methylob acterium J uu, Phormidium genus, Rhodobacter genus, Rhodopudomonas Rhodospirillum rot, Scenedesmun) U ヽ Streptomyces, Synnecoccus, Zymomonas It can gel. Specific examples of the microorganism include, for example, Escherichia coli XL 1 Blue, Escherichia coli XL2 "Blue _s Escherichia coli DH1, Escherichia coli DH5, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No49 _N Escherichia Eschercher Escherichia coli W3110 coli NM522, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium flavum ATCC 14067,

Br evib acterium lactofermentum ATCC 13869, Coryneb acterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 14297, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Serratia ficaria Serratia fonticola, Serratia liquefaciers, Serratia liquefaciers, Serratia liquefaciers, Serratia liquefaciers rhizogeness Agrob acterium rubi, Anabaena cylindrica, Anabaena doliolum, Anabaena flosaquae, Arthrobacter aurescens, Arthrobacter ci reus,

Arthrobacter globformis Arthrobacter hydrocarboglutamicus Arthrobacter mysorens _N Arthrobacter nicotianae _s Arthrobacter paraffineus _x Arthrobacter protophormiae _N Arthrobacter roseoparaffinus Arthrobacter sulfureus

Arthrobacter ureafaciens, Chromatium buderi, Chromatium tepidum

Chromatium vinosum, Chromatium warmingii, Chromatium fluviatile,

Erwinia uredovora, Erwinia carotovora _s Erwinia ananas Erwnia herbicola Erwinia punctata, Erwinia terreus, Methylob acterium rhodesianum,

Methylobacterium extorquens, Phormidium sp.ATCC29409, Rhodobacter capsulatus ヽ Rhodobacter sphaeroides, Rhodopseudomonas blastica

Rhodopseudomonas marina Rhodopseudomonas palustris Rhodospirillum rubrum, Rhodospirillum salexigens, Rh o do spir ill illum salinarum Streptomyces ambofaciens, Streptomyces aureofaciens _x Streptomyces aureus, Streptomyces fungicidicus, Streptomyces griseochromogenes Streptomyces griseochromogenes Streptomyces lividans Streptomyces olivogriseus, Streptomyces rameus, Strep tomyces tanashiensis, Streptomyces vinaceus, Zymomonas mobilis and the like.

 Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.For example, a method using calcium ions !: Pro Natl. Acad. SCI. USA, 69, 2110 ( 1972)], the protoplast method (JP-A-63.2483942), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).

When yeast is used as a host cell, examples of the expression vector include YEpl3 (ATCC37115), YEp24 (ATCC37051), Ycp5O (ATCC37419) _s pHS19, and PHS15.

 Any promoter can be used as long as it can be expressed in yeast.For example, PHO5 promoter overnight, PGK promoter, GAP promoter, ADH promoter, gall promoter evening, gallO promoter evening, heat shock protein Promoters such as Quality Promo One Night, MF Hi One Promo One, and CUP1 Promo One Night can be listed.

 As host cells, Saccharomyces cerevisae (Saccharomyces cerevisae), Nzosaccharoses sembi (Schizosaccharomyces pombe), and Kluyveromice saccharomyces (Kluyveromyces lactis containing Trichosporon. And so on.

As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. Examples of the method include the electroporation method (Methods.Enzymol, 194, 182 (1990)) and spheroplast. Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], or Pro Natl. Acad. Sci. USA, 75, 1929 (1978). When animal cells are used as host cells, as an expression vector, for example, pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)], pAS3_3 (JP-A-2-227075), pCDM8 [Nature, 329, 840, (1987) )], PcDNAI / AmPd itrogen), pREP4 (lnvitrogen), pAGE103 CJ-Blochem., 101, 1307 (1987)], pAGE210 and the like.

 Any promoter can be used as long as it can be expressed in animal cells. For example, the promoter of the IE (immediate early) gene of cytomegalovirus (human CMV), the early promoter of SV40, and the retrovirus Promo One, Meta Mouth Chainone Promoter, Heat Shock Promo One, SR Hi Promo One, etc. Alternatively, the enhancer of the IE gene of human CMV may be used together with the promoter.

 Examples of the host cell include Namalba cell, HBT5637 (JP-A-63-299), COS1 cell, COS7 cell, CHO cell and the like.

 As a method for introducing a recombinant vector into animal cells, any method capable of introducing DNA into animal cells can be used, for example, the electrification method (Cytotechnology, 3, 133 (1990)), calcium phosphate Method (Japanese Patent Application Laid-Open No. 2-227075), the lipofusion method (Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)), the method described in virology, 52, 456 (1973), and the like can be used. The transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.

 When an insect cell is used as a host, for example, baculovirus expression vectors, a laboratory manual (Baculovirus Expression Vectors, A Laboratory Manual), current 'Protocols in Morexura'-biology, Bio / Technology , 6, 47 (1988), etc., and the protein can be expressed.

That is, the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the culture supernatant of the insect cells, and then the recombinant virus is further transferred to the insect cell. It can infect insect cells and express proteins.

 Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrodin) and the like. Examples of the baculovirus include, for example, Atographa californica, nuclei, polyhedrosis, and virus (Autographa californica nuclear polyhedrosis virus), which is a virus that infects insects of the night moth family. Insect cells include Spodoptera frugiperda ovarian cells Sf9, S £ 21 (baculovirus 'Expression Vectors, A. Laboratory.Manual, W.H.'Freeman' and Campaign (WH Freeman and Company) New York, (1992)], and High5 (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.

 Methods for co-transferring the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include, for example, a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

 As a method for expressing the gene, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, etc., in addition to direct expression.

 When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added protein can be obtained.

 Culturing a transformant having the recombinant DNA into which the DNA has been incorporated in a medium, producing and accumulating enzymes involved in the mevalonate pathway in the culture, and collecting the protein from the culture; Thus, an enzyme protein involved in the mevalonate pathway can be produced. Proteins thus produced (enzymes involved in the actinomycetes mevalonate pathway) are also within the scope of the present invention.

The method for culturing the transformant carrying the DNA of the present invention in a medium can be performed according to a usual method used for culturing a host. When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the microorganism. However, either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

 Any carbon source can be used as long as each microorganism can assimilate it, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol are used.

 Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., various inorganic and organic acid ammonium salts, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, etc. 1. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof are used.

 As the inorganic substance, potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

 The culture is 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 7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urine, calcium carbonate, ammonia and the like.

 If necessary, an antibiotic such as ampicillin / tetracycline may be added to the medium during the culture.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, culturing a microorganism transformed with an expression vector using the trp promoter or isoprovir-1 /?-D-thiogalactoviranoside (IPTG), etc. When culturing For example, indoleacrylic acid (IAA) may be added to the medium.

 As a medium for culturing a transformant obtained using animal cells as host cells, commonly used RPM11640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium (Science, 122) , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum etc. Medium or the like is used.

The cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO ₂ . If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

 As a medium for culturing transformants obtained using insect cells as host cells, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400 And ExCell405 [all manufactured by JRH Biosciences], Grace's Insect Medium Grace, TCC, Nature, 195,788 (1962)] and the like.

 Cultivation is usually carried out under conditions of pH 6 to 7, 25 to 30 ° C, etc. for 1 to 5 days.

 If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.

In order to isolate and purify the protein of the present invention (enzyme involved in the mevalonate pathway) from the culture of the transformant of the present invention, a conventional enzyme isolation and purification method may be used. For example, when the protein of the present invention is expressed in a lysed state in cells, the cells are recovered by centrifugation after cell culture, suspended in an aqueous buffer, and then sonicated, a french press, a Menton-Gaulin homogenizer. The cells are disrupted using a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting out method with ammonium sulfate, a desalting method, a precipitation method with an organic solvent, Anion-exchange chromatography using resins such as getylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (Mitsubishi Kasei) Cation exchange chromatography using a resin such as S-Sepharose T (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenylsepharose, and molecular sieve. A purified sample can be obtained by using the gel filtration method, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing or the like used alone or in combination. When the protein is expressed in an insoluble form in the cells, the cells are similarly recovered, crushed, and the protein is recovered by a usual method from the precipitate fraction obtained by centrifugation. Thereafter, the insoluble form of the protein is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution to a solution containing no protein denaturing agent or a diluting concentration of the protein denaturing agent such that the protein is not denatured, the protein is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method.

 When the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.

 Examples of the protein thus obtained include a protein having an amino acid sequence of SEQ ID NOS: 8 to 13.

The protein expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Also, Kuwawa Trading (US Advanced Chem Tech), Perkin Elmer Japan (US Perkin-Elmer), Pharmacia Biotech (Sweden Pharmacia Biotech), Aroca (US Protein Technology Instrument), Syntheses can also be carried out using peptide synthesizers such as Kurabo Industries (US Synthecell-Vega), Japan Perceptive-Limited (US PerSeptive) and Shimadzu Corporation. (C) Production of isoprenide compounds

 The transformant obtained in the above (B) is cultured according to the method in the above (B), an isoprenide compound is produced and accumulated in the culture, and the isoprenoid compound is collected from the culture. Thus, an isoprenide compound can be produced.

By this culture, isoprenide compounds such as ubiquinone, vitamin K ₂ , carotenoid and the like can be produced. Specific examples include, for example, production of ubiquinone-8 and menaquinone-18 using a microorganism belonging to the genus Escherichia as a transformant, production of ubiquinone-10 using a microorganism belonging to the genus Rhodobacter as a transformant, production of ubiquinone-10 belonging to the genus Arthrobacter Production of Bimin 1 < _{2 using} a microorganism as a transformant, production of astaxanthin using a microorganism belonging to the genus Agrobacterium as a transformant, production of a microorganism belonging to the genus Erwinia as a transformant, copen, Ichirotin, Production of zeaxanthin can be mentioned.

 After completion of the culture, the isoprenoid compound is extracted by adding a suitable solvent to the culture solution, and the precipitate is removed by centrifugation, etc., and then various is chromatographies are used to isolate and purify the isoprenoid compound. Can be.

 The present invention will be more specifically illustrated by the following examples, but the present invention is not limited by these examples. The genetic recombination experiments described in the Examples were performed using the method described in Molecular Cloning, 2nd Edition (hereinafter, referred to as ordinary method) unless otherwise specified. Example

Example 1: Acquisition of genes related to the mevalonate pathway of actinomycete Streutomvces sp. Strain CL190

First, a DNA fragment containing the gene (hmgr) encoding 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, an enzyme that catalyzes one reaction on the mevalonate pathway, was transformed into Streptomyces sp. from CL190 strain We obtained and analyzed the nucleotide sequence of the DNA fragment, and clarified that genes involved in the mevalonate pathway existed in a class-unique manner around the hmgr gene. The details are shown below.

Streptomyces sp. CL190 strain was cultured in a 15 ml GPY medium (1% glucose, 0.4% polypeptone (Wako Pure Chemical Industries, Ltd.), 0.4% yeast extract (Difco), 0.5% MgS0 ₄ · 7H ₂ 0, and cultured for 2 days at 30 ° C in _{0. 1% K 2 HP 0 4} ). After the culture, cells were obtained from the resulting culture by centrifugation. Chromosomal DNA was isolated and purified from the obtained cells according to a standard method (Molecular Cloning, 2nd edition). After digestion of l〃g of the obtained DNA with the restriction enzyme SnaB I (Takara Shuzo), Southern Hybridization (molecular cloning second edition) using the hmgr gene as a probe was performed. A probe signal was detected at 7 kb. From this result, it was found that the hmg r gene was contained in a 6.7 kb DNA fragment derived from actinomycete chromosome.

 Next, the chromosomal DNA of Streptomyces sp. Strain CL190 was cut again with SnaBI, followed by agarose gel electrophoresis, and a DNA fragment of l〃g near 6.7 kb was extracted from the agarose gel and recovered. The recovered DNA fragment of about 6.7 kb was blunt-ended using T4 DNA polymerase (purchased from Takara Shuzo), and inserted into a plasmid site of plasmid pUC118 (purchased from Takara Shuzo). Then, a chromosomal DNA library of Streptomyces sp. Strain CL190 was prepared. Using this chromosomal DNA library, Coli JM109 strain (purchased from Takara Shuzo) was transformed according to a standard method (Molecular Cloning, 2nd edition). Transformants were screened by the colony hybridization method (molecular cloning second edition) using the hmgr gene (J. Bacteriol. 181: 1256, 1999) as a probe, and the plasmid containing the hmgr gene was screened. A transformant of Escherichia coli was isolated. Plasmids were extracted from the isolated transformants using a plasmid extraction kit QIAprep Spin Miniprep Kit (Qiagen).

The nucleotide sequence of the extracted plasmid is determined by a standard method (Molecular cloning No. 2 Version). At that time, a thermosequenase cycle sequencing gate (manufactured by Amersham Pharmacia Biotech) as a sequencing reaction reagent and a DNA sequencer model 4000L (manufactured by Li-cor) as a sequence analyzer were used. The determined nucleotide sequence is shown in SEQ ID NO: 1. Example 2: Analysis of the structure of the gene involved in the mevalonate pathway of Streptomvces sp.

From the nucleotide sequence of SEQ ID NO: 1, an open reading frame (hereinafter abbreviated as 0 rf) was predicted using the genetic information processing software GENETYX-MAC (manufactured by Software Development Corporation). As a result, in addition to the hmgr gene, five orfs were named as orf A, orf B _s orf C, orf D, or rf E, for convenience, in ascending order of the nucleotide sequence number of SEQ ID NO: 1. The positions of orf A to E and hmgr gene in SEQ ID NO: 1 are as follows.

o r f A (base number 132—1 169 of SEQ ID NO: 1)

o r f B (base number 1 159—22 11 1 of SEQ ID NO: 1)

0 r f C (base numbers 2208—3332 in SEQ ID NO: 1)

o r f D (base numbers 3335—4426 in SEQ ID NO: 1)

hmgr gene (base numbers 4423-5484 in SEQ ID NO: 1)

0 r f E (base No. 5488-6657 of SEQ ID NO: 1)

 The base sequences of o rf A, o rf B, orf C, orf D, hm gr and orf E are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The amino acid sequences are shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

Homology searches for these orfs were performed using the National Institute of Genetics FAS TA program (Pro Natl. Acad. Sci. USA, 85: 2444, 1998). As a result, orf A is phosphomevalonate kinase (accession number P24521) and orf B is diphosphomevalonate decarboxylase (accession number P32377) and orf C was found to have significant homology to mevalonate kinase (accession number P46086) and orf E was to HMG-CoA synthase (accession number P54873).

 Furthermore, orfD was found to be homologous to a protein of unknown function (hereinafter referred to as protein X) (accession number P46086). These five orf-encoding novel genes form a cluster with the hmgr gene and are transcribed in the same direction. Based on the results of the homology search, these genes were estimated to be genes involved in the mevalonate pathway in the same manner as the hmgr gene. Figure 1 shows the positional relationship between these genes. Example 3: Analysis of the function of genes involved in the mevalonate pathway of Streptomvces sp. Strain CL190

 (1) Functional analysis of orfA, orfB, orfC, orfD and orfE Both ends of the above 6.7 kb DNA fragment were made blunt-ended using T4 DNA polymerase (purchased from Takara Shuzo), Plasmid with the N-terminus of phosphomevalonate (PMVA) kinase inserted so that the N-terminus of the phosphomevalonate (PMVA) kinase is close to the 1 ac promoter of pUC118 is inserted into pUCV18 (purchased from Takara Shuzo). Named. pUMV 19 contains phosphomevalonate kinase (orf A), difoshomevalonate decarboxylase (orf B), mevalonate kinase (orf C), protein X (orf D) ヽ HMG—CoA reductase (or hmgr) ヽ HMG—contains a total of six genes encoding CoA synthase (orf E). (Figure Next, pUMV 19 was introduced into E. coli JM109 (p In the E. coli JM109 (pUMV 19) strain, the above six genes were expressed as IP.

It is designed to be transcribed and expressed from 1 ac promoter overnight by adding TG.

Fosmidomycin is known to specifically inhibit the non-mevalonate pathway (Tetrahedron Lett. 39: 7913, 1998.). Therefore, it has only a non-mevalonate pathway E. coli strain JM109 cannot grow in a medium containing fosmidomycin because it cannot biosynthesize IPP essential for growth. Therefore, if the E. coli JM109 (pUMV19) strain prepared above can grow with the addition of IPTG even in the presence of fosmidomycin, this E. coli JM109 (pUMV19) strain can be grown by the mevalonate pathway. It can prove that IPP is biosynthesized. That is, it can be proved that the above-mentioned 6.7 kb DNA fragment contains all the genes involved in the mevalonate pathway.

 In addition, pUMV 19AE, pUMV 19ΔS, and pUMV 19ΔΜ were constructed as deletion mutants of pUMV 19. pUMV 19AE and pUMV 1 pU MV 19AM are plasmids prepared by digesting pUMV 19 with restriction enzymes EcoRI, Sse8387I or M1uI and performing self-ligation, and pUMV 19ΔΕ. In phosphomevalonate kinase, HUM-CoA synthase in pUMV 19AS, and protein X in pUMV 19ΔΜ (see Figure 1). These deletion mutants are also transformed into E. coli JM109 in the same manner as pUMV19, and E. coli JM109 (p UMV19 ΔΕ), E. coli JM109 (UMV19AS) and E. coli JM109 ( p UMV 19 ΔΜ) was prepared. Phosmidomycin was synthesized according to (Chem. Pharm. Bull. 30: 111, 1982.).

 E. coli JM109 (pUMV19), E. coli JM109 (pUMV19), E. coli JM109 (pUMV19 AS), E. coli JM109 (pUMV19ΔΜ) and E E. coli JM109 (pUC118), LB medium (trypton (Difco)) 1% containing 50 / g / ml antibiotic ambicilin (Sigma) to retain plasmid; 5 ml of a struct (manufactured by Difco) and 0.5% of NaCl were cultured at 37 ° C. with shaking at 5 ° C. (culture conditions 1 to 3 below were used). Then their growth was observed. E. coli JM109 (pUC118) was used for control experiments.

Culture condition 1: 0. ImM I PTG (Wako Pure Chemical Industries) and 50 g / ml The above five types of culture solutions were added in an amount of 1/1000 to 5 ml of an LB medium containing picillin, and cultured at 37 ° C for 12 hours.

Culture conditions 2: 0.11111 ^ 1 Ding 0, 20〃G / ml of Hosumi Domaishin, and 50〃 / ^/ 1111 of the 1 ^ 8 medium 51111 of the five kinds of culture solution was added 1/1000 amount 37 The cells were cultured at ° C for 12 hours.

 Culture condition 3: 0. ImM IPTG, 0.02% mevalonic acid (Wako Pure Chemical Industries), 20 μg / ml fosmidomycin, and 50 zg / ml ampicillin in LB medium 5 ml above Five types of culture solutions were added in 1/1000 volume and cultured at 37 ° C for 12 hours.

 The results of growth after cultivation are shown in Figure 1 (+ indicates normal growth,-indicates normal growth).

In culture condition 1, E. coli JM109 (pUMV 19), E. coli JM109 (pUMV 19 AE) _S E.coK JM109 (pUMV 19AS), E. coli JM109 (p UMV 19ΔΜ) ヽ E. coH JM109 (pUC All of the strains (1) and (8) were viable.

 Under culture condition 2, as expected, only E. coli JM109 (pUMV 19) was able to grow. In other words, under the condition that the non-mevalonate pathway is blocked by the addition of fosdomycin, if you have pUMV 19 which has all six genes integrated on the above 6.7 kb DNA fragment, Only in this case, the mevalonate pathway functioned normally and E. coli JM109 was viable. Therefore, it became clear that six genes including protein X are involved in the mevalonate pathway.

Under culture condition 3, E. coli JM109 (pUMV 19) and E. coli JM109 (pUMV 19AS) were able to grow as expected. Since E. coli JM109 (pUMV 19AS) has normal functions except for HMG-CoA synthase, mevalonic acid added to the medium can be converted to IPP. Therefore, it is considered that E. coli JM109 (pUMV 19.S) was able to grow even in the presence of fosmidomycin if mevalonic acid and IPTG were added. On the other hand, E.coH JM109 (pUMV 19ΔΕ), E. coli JM109 (pUMV 19ΔΜ), and E. coli JM109 (pUC118) were added to the medium. Since each gene for converting mevalonate to IPP is lacking, it is considered that growth was impossible even in the presence of fosmidomycin even when mevalonate and IPTG were added.

 From the above results, (1) all of the above six genes are essential for synthesizing IPP by the mevalonate pathway in E. coli JM109; (2) lacking HMG-C0A synthase In this case, it was shown that the addition of mevalonic acid enables the synthesis of IPP by the mevalonic acid pathway in E. coli JM109. 7 o Example 4: E. coli JM109 (pUMVl 9 ) Production of ubiquinone by strains

 The production of ubiquinone (one of the isoprenide compounds) by the E. coli JM109 (pUMV 19) strain was compared with that of the E. coli JM109 (pUC118) strain having only a vector not containing a mevalonate pathway gene.

The determination of ubiquinone was performed as follows. The E. coH JM109 (p UMV 19) and E. coli JM109 (pUC 118) strains were cultivated at 37 ° C. for 14 hours in a 501111 1 ^ 8 medium containing 50 μg / ml ampicillin, respectively. Cells were collected by centrifugation. The recovered cells were freeze-dried, and the weight of the dried cells was weighed. Then, 60 ml of a solution of black-mouthed form: methanol = 2: 1 was added, and the mixture was left for 1 hour to extract ubiquinone. Then, the methanol solution of chloroform at the outlet was evaporated overnight at the evaporator, and 1 ml of acetone was added to the remaining solid to dissolve it. The amount of ubiquinone in this acetone solution was measured by high performance liquid chromatography (purchased from JASCO) using a Sensiupak PEGAS IL 0DS column (manufactured by JASCO Corporation) using isopropyl alcohol: methanol = 1: 1. Eluted and quantified. Under these conditions, ubiquinone elutes at 9.8 minutes. The amount of ubiquinone was represented by the relative value of the area of the peak corresponding to ubiquinone in the above chromatogram. The results are shown in Table 1 below. table 1 :

 Bacterial cell amount Ubiquinone amount Ubiquinone amount Body amount

E. coU JM109 (pUC 1 1 3) 0.12g 12135

E. coli JM109 (pUMV 19) 0.057 ^ 25684 450000

The production of ubiquinone per culture volume (in this case, 50 ml) in E. coli JM109 (pUMV19) was 2.1 times that of E. coli JM109 (pUC118) (see table). 25 684/1 2 1 35 = 2.1). In addition, the production of ubiquinone per cell mass in E. coli JM109 (pUMV 19) was 4.5 times that of E. coli JM109 (pUC 118) (in the table, 4500 00/1 00 000 = 4.5). These results indicated that the mevalonate pathway gene from Streptomyces sp. Strain CL190 was effective in improving isoprenoid productivity. Industrial applicability

 The gene of the present invention is a novel gene. By culturing a transformant in which the gene of the present invention is introduced into a host such as Escherichia coli, an isoprenide compound can be produced.

As organisms in which the entire length of the gene involved in the mevalonate pathway has been completely revealed, other than the actinomycetes Streptomyces sp.CL190 strain disclosed in the present invention, yeast Saccharomyces cerevisiae can be mentioned (Nature 387: 5, 199 97) ₀ However, the genes involved in the mevalonate pathway in yeast have not created a class. Therefore, in order to express the mevalonate pathway gene of Escherichia coli in Escherichia coli so that the Escherichia coli can use the mevalonate pathway, the genes of the mevalonate pathway must be converted into yeast cells one by one. It is necessary to perform a complicated operation of obtaining such a vector and integrating it into each vector suitable for expression in E. coli. In addition, the genome of eukaryotes such as yeast has a region called intron, which does not encode a protein. Because of its high content, the mevalonate pathway gene can be obtained by extracting yeast messenger RNA, converting it into type I DNA using reverse transcriptase, and then encoding the mevalonate pathway enzyme. It is necessary to adopt a complicated method of acquiring only the area where the data exists.

 On the other hand, the genes of the mevalonate pathway of the actinomycete Streptomyces sp. Strain CL190, which is a prokaryote, are clustered on the genome as described above, and are arranged so that they are transcribed in the same direction. It can be expressed in Escherichia coli without any special operations.

Claims

The scope of the claims

1. DNA having any of the following:

 (1) the nucleotide sequence of SEQ ID NO: 1;

 (2) a base sequence in which one to several bases have been deleted, substituted, added and / or inserted in SEQ ID NO: 1, and which encodes all enzymes necessary for the function of the mevalonate pathway Array; or

 (3) A nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and that encodes all enzymes necessary for the function of the mevalonate pathway.

 2. The enzymes required for the mevalonate pathway to function are at least phosphomevalonate kinase, diphosphomevalonate decarboxylase, mevalonate kinase, HMG-CoA reductase and HMG-CoA synthase. The DNA of claim 1, wherein the DNA is present.

 3. A protein encoded by the DNA of claim 1 or 2.

 4. DNA with any of the following:

 (1) a base sequence of SEQ ID NO: 2 in which one to several bases have been deleted, replaced, added and / or inserted in SEQ ID NO: 2, which encodes phosphomevalonate kinase A sequence or a base sequence capable of hybridizing under stringent conditions to the base sequence of SEQ ID NO: 2, which base sequence encodes phosphomevalonate kinase;

 (2) SEQ ID NO: 3, a nucleotide sequence in which one to several bases are deleted, substituted, added and / or inserted in SEQ ID NO: 3, and encodes diphosphomevalonate decarboxylase Or a base sequence capable of hybridizing with the base sequence of SEQ ID NO: 3 under stringent conditions, wherein the base sequence encodes diphosphomevalonate decarboxylase;

(3) base sequence of SEQ ID NO: 4; one to several bases are deleted and replaced in SEQ ID NO: 4 A base sequence that has been added and / or inserted, and that can hybridize under stringent conditions to the base sequence encoding mevalonate kinase or the base sequence of SEQ ID NO: 4. A base sequence encoding mevalonate kinase;

 (4) the nucleotide sequence of SEQ ID NO: 5; or

 (5) The nucleotide sequence of SEQ ID NO: 7, which is a nucleotide sequence in which one to several nucleotides have been deleted, replaced, added, or inserted in SEQ ID NO: 7, wherein HMG-CoA synthase is A base sequence which encodes a base sequence which can hybridize with the base sequence of SEQ ID NO: 7 under stringent conditions, and which codes for HMG-CoA synthase.

 5. A protein encoded by the DNA of claim 4.

 6. A protein having any of the following:

 (1) an amino acid sequence of SEQ ID NO: 8, an amino acid sequence in which one to several amino acids have been deleted, substituted, added and / or inserted in SEQ ID NO: 8, which has phosphomevalonate kinase activity Or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 8, wherein the amino acid sequence has phosphomevalonate kinase activity;

 (2) the amino acid sequence of SEQ ID NO: 9; an amino acid sequence in which one to several amino acids are deleted, substituted, added, and / or inserted in SEQ ID NO: 9, and which has diphosphomevalonate decarboxylase activity A sequence or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 9, and an amino acid sequence having diphosphomevalonate decarboxylase activity;

(3) an amino acid sequence of SEQ ID NO: 10 wherein the amino acid sequence of SEQ ID NO: 1 has one to several amino acids deleted, substituted, added and / or inserted, and has a mevalonate kinase activity. An amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 10 and having an activity of mevalonate kinase; (4) the amino acid sequence of SEQ ID NO: 11; or

 (5) an amino acid sequence of SEQ ID NO: 13, in which one to several amino acids are deleted, substituted, added and / or inserted in SEQ ID NO: 13, An amino acid sequence having an enzyme activity, or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 13, wherein the amino acid sequence has an HMG-C0A synthase activity.

 7. A vector comprising the DNA according to claim 1, 2 or 4.

8. A transformant having the vector according to claim 7.

9. The transformant according to claim 8, which is Escherichia coli.

 10. A step of culturing a transformant prepared by transforming a vector containing the DNA according to claim 1 or 2 into a host to produce an isoprenide compound in the culture, and isoprenoid from the culture. A method for producing an isoprenide compound, comprising a step of collecting a compound.

11. Isopurenoi de compound, Yubikinon a Isopurenoi de compound selected from vitamin kappa ₂ or Karoteno I de, method of isoprene Noi de The compound according to claim 10.