JP2001161370A - Gene of enzyme involved in actinomyces-derived mevalonate pathway - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA containing a group of genes involved in mevalonate pathway, one of biosynthesis pathways for isoprenoid compounds useful for medicaments and health foods each intended for cardiopathy, osteoporosis, hemostasis, cancer prevention, immununopotentiation, etc., and for shellfish stickproofing coatings, etc., and to provide a method for producing an isoprenoid compound by culturing a transformant obtained by transferring the above DNA into host cells. SOLUTION: This DNA is such as to have either one of the following: (1) a specific base sequence; (2) such a base sequence that one to several bases are deleted, substituted, added and/or inserted in the base sequence and as to encode all of the enzymes necessary for functioning the mevalonate pathway; or (3) such a base sequence as to be hybridizable with the above specific base sequence (1) under stringent conditions and to encode all of the enzymes necessary for functioning the mevalonate pathway.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA encoding a plurality of enzymes involved in the mevalonate pathway derived from actinomycetes,
The present invention relates to a DNA encoding each enzyme contained in the DNA, a protein encoded thereby, a vector and a transformant containing the DNA, and a method for producing an isoprenoid compound using the DNA.

[0002]

2. Description of the Related Art Isoprenoids (also referred to as terpenes) are a general term for a group of organic compounds having an isoprene unit having 5 carbon atoms as a basic skeleton, and are biosynthesized by polymerization of isopentenyl pyrophosphate (IPP). (C ₅ H _{8)
In addition to n} unsaturated hydrocarbons, their redox products (alcohols, ketones, acids, etc.), compounds from which carbon has been eliminated, and the like have been found in many plants and animals. Isoprenoids are classified into hemiterpenes (C
5), monoterpene (C10), sesquiterpene (C1)
5), diterpenes (C20), sesterterpenes (C2)
5), triterpene (C30), tetraterpene (C4
0, carotenoids), and other polyterpenes. There are many compounds that 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 in a part of the structure include useful physiological activities.

[0003] For example, ubiquinone, which is one of the isoprenoid compounds, plays an important role in the living 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. Recently, it is suggested that vitamin K is involved in bone metabolism, and is expected to be applied to a therapeutic drug for osteoporosis. Philoquinone and menaquinone have been approved as pharmaceuticals.

[0004] Ubiquinone and vitamin K have an inhibitory effect on the adhesion of shellfish, and are expected to be applied to paints for preventing adhesion of shellfish. In addition, carotenoids have an antioxidant effect,
Some of them, such as β-carotene, astaxanthin, and cryptoxanthin, are expected to have cancer prevention and immunostimulatory activities. As described above, isoprenoid compounds include substances useful for living organisms,
The establishment of these inexpensive manufacturing methods would have significant social and medical benefits.

[0005] Production of isoprenoid compounds by fermentation has been studied for some time, and attempts have been made to examine culture conditions, breed strains by mutagenesis, and to improve production by genetic engineering techniques. However, its effect is limited to individual compound species, and no effective method is known for isoprenoid compounds in general. Isopentenyl pyrophosphate (IPP), a basic skeleton unit of isoprenoid compounds, has been proven to be biosynthesized from acetyl-CoA via mevalonic acid in eukaryotes such as animals and yeasts (mevalonic acid pathway). .

In the mevalonate pathway, 3-hydroxy-3-
Methylglutaryl-CoA (HMG-CoA) reductase is considered to be the rate-limiting enzyme (Mo1. Bio1. Cell, 5, 655 (19
94)), attempts have been made to increase the expression of HMG-CoA reductase in yeast and to improve the productivity of carotenoids (Mizawa et al., Proceedings of the Carotenoid Research Symposium (199)
7)).

[0007] In prokaryotes such as Escherichia coli, IPP is biosynthesized via another pathway, namely, 1-deoxy-D-xylulose 5-phosphate generated by the condensation of pyruvate and glyceraldehyde 3-phosphate. Non-mevalonate pathways have been discovered in many prokaryotes (Biochem. J., 2
95, 517 (1993)), from experiments using ¹³ C-labeled substrates.
Deoxy-D-xylulose 5-phosphate has been shown to be converted to IPP via 2-C-methyl-D-erythritol 4-phosphate (Tetrahedron Lett.
38, 4769 (1997); Tetrahedron Lett. 39, 4509 (199
8)). In particular, it has been demonstrated that IPP is synthesized only in the non-mevalonate pathway in E. coli (Rohmer, M. In Co.).
mprehensive Natural Products Chemistry, Vol. 2: Is
oprenoidsincluding carotenoids and steroids; Barto
n, D. Nakanishi, K. Eds. Elsevier: Amsterdam, 199
9; pp. 45-67).

On the other hand, the actinomycete Streptomyces sp. Strain CL190 (J. Antibiot. 43: 444, 1990) is used for the transfer of I via mevalonic acid.
It is known that PP is synthesized (Tetrahedron
Lett. 31: 6025, 1990. and Tetrahedron Lett. 37: 7979,
1996.), the present inventors have previously reported that actinomycetes Streptomyc
The gene (hmgr) encoding 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, an enzyme that catalyzes one reaction on the mevalonate pathway, has already been cloned from es sp. strain CL190 (J . Bac
teriol. 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.

[0009]

One of the objects of the present invention is to provide isoprenoids useful for medicines, health foods, and shellfish adhesion preventive paints for the purpose of heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation, etc. An object of the present invention is to provide a DNA containing a group of genes involved in the mevalonate pathway, which is one of the biosynthetic pathways of compounds. Still another object of the present invention is to provide the above-mentioned D
An object of the present invention is to provide a method for producing an isoprenoid compound by culturing a transformant obtained by introducing NA into a host cell.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that Streptomyces
A gene involved in the mevalonate pathway of ces sp. strain CL190 was obtained and transformed into Escherichia coli. The resulting transformant was cultured. As a result, the production of ubiquinone, one of the isoprenoid compounds, was improved. And completed the present invention.

That is, according to the present invention, a DNA having any one of the following: (1) a base sequence of SEQ ID NO: 1; (2) a deletion, substitution, addition, And / or an inserted nucleotide sequence encoding all of the enzymes necessary for the function of the mevalonate pathway; or (3) hybridizing with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions. A base sequence that encodes all of the enzymes necessary for the functioning of the mevalonate pathway. Preferably, the enzymes necessary for functioning the mevalonate pathway are at least phosphomevalonate kinase, diphosphomevalonate decarboxylase, mevalonate kinase, HMG-CoA reductase and HMG-CoA synthase. According to another aspect of the present invention, there is provided a protein encoded by the DNA.

According to still another aspect of the present invention, a DNA having any one of the following: (1) a nucleotide sequence of SEQ ID NO: 2; And / or a base sequence that is inserted and is a base sequence that encodes phosphomevalonate kinase or a base sequence that can hybridize under stringent conditions to the base sequence of SEQ ID NO: 2; (2) the base sequence of SEQ ID NO: 3, which is a base sequence in which one to several bases have been deleted, substituted, added and / or inserted in SEQ ID NO: 3, wherein diphosphomevalonate decarboxylase is SEQ ID NO: 3, or the nucleotide sequence encoding
A base sequence capable of hybridizing under stringent conditions with the base sequence of SEQ ID NO: 4; a base sequence encoding diphosphomevalonate decarboxylase; (3) a base sequence of SEQ ID NO: 4; Is a nucleotide sequence having deletion, substitution, addition and / or insertion of a base, which is capable of hybridizing under stringent conditions to the nucleotide sequence encoding mevalonate kinase or the nucleotide sequence of SEQ ID NO: 4. A nucleotide sequence encoding mevalonate kinase; (4) a nucleotide sequence of SEQ ID NO: 5; or (5) a nucleotide sequence of SEQ ID NO: 7; , A base sequence that has been substituted, added and / or inserted and that codes for HMG-CoA synthase, A nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions of SEQ ID NO: 7, the nucleotide sequence encoding HMG-CoA synthase; is provided. According to still another aspect of the present invention, there is provided a protein encoded by the DNA.

According to yet another aspect of the invention, a protein having any of the following: (1) the amino acid sequence of SEQ ID NO: 8, wherein one to several amino acids are deleted, substituted, added and An amino acid sequence having phosphomevalonate kinase activity, which is 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 Sequence; (2) an amino acid sequence of SEQ ID NO: 9, in which one to several amino acids are deleted, substituted, added and / or inserted in SEQ ID NO: 9, wherein the amino acid has diphosphomevalonate decarboxylase activity Sequence or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 9 An amino acid sequence having diphosphomevalonate decarboxylase activity; (3) an amino acid sequence of SEQ ID NO: 10, in which one to several amino acids are deleted, substituted, added and / or inserted in SEQ ID NO: 10 An amino acid sequence having mevalonate kinase activity, or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 10 and having an mevalonate kinase activity; (4) SEQ ID NO: 11 Or (5) the amino acid sequence of SEQ ID NO: 13, wherein the amino acid sequence of SEQ ID NO: 13 in which one to several amino acids are deleted, substituted, added, and / or inserted, wherein HMG-C
an amino acid sequence having oA synthase activity or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 13 and having HMG-CoA synthase activity;

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 yet another aspect of the present invention, the above D of the present invention
A method for producing an isoprenoid compound, comprising the steps of: culturing a transformant produced by transforming a vector containing NA into a host to produce an isoprenoid compound in a culture; and collecting the isoprenoid compound from the culture. Is provided. Preferably, the isoprenoid compound is a isoprenoid compound selected ubiquinone, vitamin K ₂ or carotenoid.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and an embodiment of the present invention will be described in detail. In this specification, “1”
"In which several bases are deleted, substituted, added and / or inserted", for example, from 1 to 20, preferably from 1 to 1
5, more preferably 1 to 10, even more preferably 1
It means that any number of bases from 5 to 5 have been deleted, substituted, added and / or inserted. In the present specification, "1 to several amino acids are deleted, substituted, added and / or inserted" means, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, More preferably, 1 to 5 amino acids are deleted, substituted, added and / or inserted. As used herein, `` can hybridize under stringent conditions '' means that DNA is used as a probe and colony hybridization, plaque hybridization, or Southern blot hybridization is used. Obtained DN
A, specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA or a fragment of the DNA was immobilized. Thereafter, DNA that can be identified by washing the filter at 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of the SSC solution having a 1 × concentration is 150 mM sodium chloride and 15 mM sodium citrate) is used. I can give it. Hybridization is performed by Mole
cular Cloning: A laboratory Mannual, 2 ^nd Ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY.,
1989. Hereinafter, abbreviated as "Molecular Cloning 2nd Edition") and the like. DNAs that can hybridize under stringent conditions include the D
DNA having a certain degree of homology with the base sequence of NA is mentioned, and the homology is, for example, 60% or more, preferably 70% or more.
% Or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

The present invention also relates to a protein having 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. SEQ ID NO: 8, 9, 10 or 1
The homology with the amino acid sequence of No. 3 is not particularly limited as long as it is 60% or more, and is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more.
% Or more, particularly preferably 95% or more, most preferably 9% or more.
8% or more.

The term "mevalonate pathway" as used herein means: (1) a step of converting acetoacetyl-CoA to HMG-CoA (catalyzed by HMG-CoA synthase); (2) a step of converting HMG-CoA to mevalonate. Step of converting (H
(3) the step of converting mevalonate to pyrophosphomevalonate (catalyzed and regulated by mevalonate (MVA) kinase, phosphomevalonate (PMVA) kinase); and (4) pyro Step of converting phosphomevalonic acid to isopentenyl pyrophosphate (IPP) (diphosphomevalonic acid (P
MVA) decarboxylase catalyzed):
This is the pathway by which PP is biosynthesized. Enzymes required for the functioning of the mevalonate pathway include at least PMVA kinase, PMVA decarboxylase, MVA kinase, HMG-CoA reductase and HMG-CoA synthase.

Next, a method for obtaining the DNA of the present invention and a method for using the same will be described. (A) Acquisition of DNA Encoding Enzyme Involved in Actinomycete Mevalonate Pathway As described above, the present inventors have previously described Streptomyces strep
A gene (hmgr) encoding 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, an enzyme that catalyzes one reaction on the mevalonate pathway, has been cloned from tomyces sp. strain CL190 (J. Bac
teriol. 181: 1256, 1999). The DNA having the nucleotide sequence of SEQ ID NO: 1 of the present invention can be obtained by using the hmgr gene as a probe. h
As the nucleotide sequence of the mgr gene, the nucleotide sequence of SEQ ID NO: 6 can be mentioned. Specific examples of a method for obtaining a DNA region encoding an enzyme involved in the mevalonate pathway include the following methods.

Actinomycetes, such as Streptomyces sp. CL190
The strain is placed in a suitable medium, such as GPY medium (1% glucose,
0.4% polypeptone, 0.4% yeast _{extract, 0.5% MgSO 4 · 7H 2} O, 0.1% K 2 HP
Incubate for several days at an appropriate temperature (eg, 30 ° C.) at O ₄ ). After culturing, cells are obtained from the resulting 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, SnaBI), Southern hybridization (molecular cloning second edition) using the hmgr gene as a probe is performed. As a result of Southern hybridization, a specific position (for example, when SnaBI is used as a restriction enzyme for digestion of chromosomal DNA,
The probe signal is detected at 6.7 kb).

Next, the chromosomal DNA of Streptomyces sp. Strain CL190 was ligated to the same restriction enzyme (for example, SnaBI) as described above.
After re-cutting, agarose gel electrophoresis was performed, and the position at which a signal was detected as a result of Southern hybridization (when SnaBI was used as a restriction enzyme,
DNA fragment corresponding to 6.7 kb) is extracted from the agarose gel and collected. The recovered DNA fragment is blunt-ended using T4 DNA polymerase (purchased from Takara Shuzo), and an appropriate plasmid (for example, pUC118) is used.
) To produce a chromosomal DNA library of Streptomyces sp. CL190 strain. This chromosome DN
Using a suitable host (eg, E. coli)
JM109 strain, etc.) (Molecular cloning second
Version), and by screening the transformant by a colony hybridization method using the hmgr gene as a probe, 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, D
The NA fragment can be isolated.

An example of a DNA that can be isolated by the above method is a DNA having the nucleotide 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. The 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 is obtained by appropriately treating the DNA with the nucleotide sequence of SEQ ID NO: 1 with restriction enzyme treatment or PCR. It can be obtained by a conventional method known to a trader.

In order to obtain 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 by PCR, the actinomycetes Streptomyces ssp.
Using the chromosomal DNA of the p.CL190 strain or a DNA having the nucleotide sequence of SEQ ID NO: 1 as a template, 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 amplified. using a pair of primers designed using the TaKaRa LA-PCR ^TM Kit Ver.2 (Takara Shuzo Co., Ltd.) or Expand ^TM High-Fidelity PCR System (base made one Ringer Mannheim) or the like, DNAThermal Cycl
Perform PCR using er (PerkinElmer Japan). In order to facilitate the subsequent cloning operation, it is preferable to add an appropriate restriction enzyme site to the primer.

As a reaction condition of PCR, a reaction process 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 defined as one cycle, for example, 30 cycles. After the reaction, conditions for reacting at 72 ° C. for 7 minutes can be exemplified. Next, the amplified DNA fragment is
It can be cloned into an appropriate vector that can be amplified in E. coli. Cloning is performed by a conventional method, for example, Molecular Cloning, 2nd edition, Current Protocols in M
olecular Biology, Supplement 1-38, John Wiley & S
ons (1987-1997) (hereafter, Current Protocols
In Molecular Biology), DNA Clon
ing 1: CoreTechniques, A Practical Approach, Secon
d Edition, Oxford University Press (1995), or a commercially available kit such as SuperScrip
t Plasmid System for cDNA Synthesis and Plasmid Cl
oning (manufactured by Life Technologies) and ZAP-cDNASynthe
It can be performed using sis Kit (manufactured by Stratagene).

As a cloning vector, Escherichia coli K1
Escherichia coli expression vectors, which can be used as phage vectors or plasmid vectors, as long as they can autonomously replicate in the two strains, may be used as cloning vectors. Specifically, ZAPExpress (Stratagies, 5, 58 (1992)), pBluescrlpt IISK
(+) (Nuclelc Acids Research, 17, 9494 (1989)), Lam
bda ZAP II (Stratagene), λgt10, λgt11 (DN
A Cloning, A Practical Approach, 1, 49 (1985)), λ
TriplEx (Clontech), λExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mo1.Cen.
Bio1, 3., 280 (1983)), pMW218 (manufactured by Wako Pure Chemical Industries), pUC
118 (Takara Shuzo), pEG400 (J. Bac., 172, 2392 (199
0)], pQE-30 (manufactured by QIAGEN) and the like.

From the obtained transformant, the target DNA
Can be prepared by a conventional method, for example, Molecular Cloning 2nd Edition, Current Protocols in
Molecular biology, DNA Cloning 1: Core Te
chniques, A Practical Approach, Second Edition, Ox
It can be obtained by the method described in ford 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.

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 in which several bases have been deleted, substituted, added and / or inserted, wherein the base sequence encodes an enzyme protein having a specific activity, or 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 can hybridize with a nucleotide sequence under stringent conditions, and 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. Or,
The mutant DNA as described above can also be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, it is possible to obtain a mutant DNA by using 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 and 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, a method of irradiating ultraviolet rays, It can be performed using a genetic engineering technique 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. Molecular cloning, 2nd edition, Current Protocols in Molecular Co., Ltd. Biology, NucleicAcids Research, 1
0, 6487, 1982, Nucleic Acids Research, 12, 9441, 1
984, Nucleic Acids Research, 13, 4431, 1985, Nucle
ic Acids Research, 13, 8749, 1985, Proc. Natl. Aca
d. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sc
i. USA, 82, 488, 1985, Gene, 34, 315, 1985, Gene,
102, 67, 1991 and the like.

(B) Preparation of a Transformant Having DNA Encoding the Actinomycete Mevalonate Pathway Enzyme and Expression of the Enzyme Protein In order to express the DNA obtained as described above in host cells, First, the DNA fragment of interest is converted into a DNA fragment of an appropriate length containing the gene with a restriction enzyme or a DNA degrading enzyme, and then inserted downstream of a promoter in an expression vector. The obtained expression vector is introduced into a host cell suitable for the expression vector.

As the host cell, any cell that can express the gene of interest can be used. For example, Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus,
Bacteria belonging to the genus Microbacterium, Kluyveromyces, Saccharomyces, Schizosaccharomyces,
Examples include yeast, animal cells, insect cells, and the like belonging to the genus Trichosporon, Siwaniomyces 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 can be used for yeast, animal cells, and insect cells having the mevalonate pathway. Either is acceptable.

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 above DNA is capable of autonomous replication in the bacterium and, at the same time, has a recombination composed of a promoter, a ribosome binding sequence, the above DNA and a transcription termination sequence. It is preferably a vector. A gene that controls the promoter may be included.

As an expression vector, for example, pBTrP2,
pBTac1, pBTac2 (all commercially available from Behringer Ringermanheim), pKK233-2 (Pharmacia), pSE280 (Invitroge
n), pGEMEX-1 (Promega), pQE-8 (QIAGEN),
pQE-30 (manufactured by QIAGEN), pKYP10 (JP-A-58-110600), pKYP2
00 (Agrc. Biol. Chem., 48, 669 (1984)), PLSA1 (Agrc.
Blo1.Chem., 53, 277 (1989)], pGEL1 (Proc. Natl.
Acad. Sci. USA, 82,4306 (1985)], pBluescrlptII SK
+, PBluescriptII SK (-) (Stratagene), pTrS30 (FER
MBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US49390)
94, US5160735), pSupex, pUB110, pTP5, pC194, pUC18
(Gene, 33, 103 (1985)), pUC19 (Gene, 33, 103 (198
5)), pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC1
18 (manufactured by Takara Shuzo Co., Ltd.), pPA1 (JP-A-63-233798), pEG400 (J.
Bacterio 1., 172, 2392 (1990)), pQE-30 (QIAGEN)
And the like.

As the promoter, any promoter can be used as long as it can be expressed in a host cell. For example, tr
p promoter (P trp), lac promoter (P lac), P _L promoter, P _R promoter and P _SE promoter,
Promoters derived from Escherichia coli, phage and the like, SP01 promoter, SP02 promoter, penP promoter and the like can be mentioned. Further, artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp × 2), a tac promoter, a letl promoter, and a lacT7 promoter can also be used.

The ribosome binding sequence is not particularly limited as long as it can be expressed in a host cell, but an appropriate distance (for example, 6 to 18) is used between the Shine-Dalgamo sequence and the initiation codon. (Base) is preferably used. Although a transcription termination sequence is not always necessary for expression of the target DNA, it is desirable to arrange the transcription termination sequence immediately below the structural gene.

As the host cell, Escherichia, Coryn
genus ebacterium, genus Brevibacterium, genus Bacillus, Microb
acterium, Serratia, Pseudomonas, Agrobacteri
um, Alicyclobacillus, Anabaena, Anacystis
Genus, Arthrobacter, Azobacter, Chromatium, Erw
genus inia, Methylobacterium, Phormidium, Rhodobac
Genus ter, Rhodopseudomonas, Rhodospiri11um, Scene
genus desmun, genus Streptomyces, genus Synnecoccus, Zymomonas
Microorganisms belonging to the genus and the like can be given, preferably,
Escherichia, Corynebacterium, Brevibacterium
Genus, Bacillus, Pseudomonas, Agrobacterium, Al
genus icyclobacillus, genus Anabaena, genus Anacystis, Arthrob
acter, Azobacter, Chromatium, Erwinia, Met
hylobacterium, Phormidium, Rhodobacter, Rhod
genus opseudomonas, genus Rhodospirillum, genus Scenedesmun, S
Microorganisms belonging to the genus treptomyces, the genus Synnecoccus, the genus Zymomonas and the like can be mentioned.

Specific examples of the microorganism include, for example, Escher
ichia coli XL1-Blue, Escherichiacoli XL2-Blue, Esc
herichia coli DH1, Escherichia coli DH5α, Escheri
chia coli MC1000, Escherichia coli KY3276, Escheri
chia coli W1485, Escherichia coli JM109, Escherich
ia coli HB101, Escherichia coli No49, Escherichia
coli W3110, Escherichia coli NY49, Escherichia col
iMP347, Escherichia coli NM522, Bacillus subtili
s, Bacillus amyloliquefacines, Brevibacterium ammo
niagenes, Brevibacterium immariophilum ATCC14068,
Brevibacteriumsaccharolyticum ATCC14066, Brevibact
erium flavum ATCC14067, Brevibacterium lactofermen
tum ATCC13869, Corynebacterium glutamicum ATCC1303
2, Corynebacterium glutamicum ATCC14297, Corynebac
terium acetoacidophilum ATCC13870, Microbacterium
ammoniaphilum ATCC15354, Serratia ficaria, Serrati
afontico1a, Serratia liquefaciens, Serratia marces
cens, Pseudomonas sp.D-0110, Agrobacterium radioba
cter, Agrobacterium rhizogenes, Agrobacterium rub
i, Anabaena cylindrica, Anabaena do1iolum, Anabaen
a flosaquae, Arthrobacter aurescens, Arthrobacter
citreus, Arthrobacter globformis, Arthrobacter hyd
rocarboglutamicus, Arthrobacter mysorens, Arthroba
cter nicotianae, Arthrobacter paraffineus, Arthrob
acter protophormiae, Arthrobacter roseoparaffinu
s, Arthrobacter sulfureus, Arthrobacter ureafacien
s, Chromatium buderi, Chromatium tepidum, Chromati
um vinosum, Chromatium warmingii, Chromatium fluvi
atile, Erwinia uredovora, Erwinia carotovora, Erwi
nia ananas, Erwnia herbicola, Erwinia punctata, Er
winia terreus, Methylobacterium rhodesianum, Methy
lobacterium extorquens, Phormidium sp.ATCC29409,
Rhodobacter capsulatus, Rhodobacter sphaeroides, R
hodopseudomonasblastica, Rhodopseudomonas marina,
Rhodopseudomonas palustris, Rhodospirillum rubru
m, Rhodospirillum salexigens, Rhodospirillum salin
arum, Streptomyces ambofaciens, Streptomyces aureo
faciens, Streptomyces aureus, Streptomyces fungici
dicus, Streptomyces griseochromogenes, Streptomyce
s griseus, Streptomyces lividans, Streptomyces oli
vogriseus, Streptomyces rameus, Streptomyces tanas
hiensis, Streptomyces vinaceus, Zymomonas mobilis
Etc. can be given.

As a method for introducing a recombinant vector, any method for introducing DNA into the above host cells can be used. For example, a method using calcium ions [Proc. Nat1. Acad. SCl. USA, 69 , 2110 (1972)), the protoplast method (JP-A-63-2483942), or Gene, 17,
107 (1982) and Molecular & General Genetics, 168, 111
(1979).

When yeast is used as a host cell, examples of expression vectors include YEp13 (ATCC37115) and YEp24.
(ATCC37051), Ycp5O (ATCC37419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast.For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MF
Promoters such as α1 promoter and CUP1 promoter can be mentioned.

As the host cells, Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullonias saccharomyces. Schwanniomyces a11uvius) and the like.

As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, electroporation [Methods. Enz.
ymol, 194, 182 (1990)), spheroblast method (Proc.
Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], or Pro
c. Nat1. Acad. Sci. USA, 75, 1929 (1978).

When an animal cell is used as a host cell, examples of the expression vector include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979;
ology, 3, 133, (1990)), pAS3-3 (Japanese Patent Application Laid-Open No. 2-227075), pCD
M8 (Nature, 329, 840, (1987)), pcDNAI / AmP (Invitrog
en), pREP4 (Invitrogen), pAGE103 (J. Bloche
m., 101, 1307 (1987)], pAGE210, and the like.

Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (human CMV) IE (immediate ea) can be used.
rly) gene promoter, SV40 early promoter,
Retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters and the like can be mentioned. Further, an enhancer of the IE gene of human CMV may be used together with the promoter.
As the host cell, Namalva cell, HBT5637 (Japanese Patent Laid-Open No. 63-2
99), COS1 cells, COS7 cells, CHO cells 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, an electroporation method [Cytotechnology, 3, 133 (1990)], a calcium phosphate method. (Japanese Patent Application Laid-Open No. 2-227075), lipofection method (Proc. Nat.
1. Acad. Sci., USA, 84, 7413 (1987)], virology, 5
2, 456 (1973). Acquisition and culture of the transformant can be performed according to the method described in JP-A-2-227075 or JP-A-2-25791.

When insect cells are used as hosts, for example, Baculovirus Expression Vectors A Laboratory Manual (Baculovirus Expre
ssion Vectors, A Laboratory Manua1), Current Protocols in Molecular Biology, Bi
The protein can be expressed by the method described in o / Technology, 6, 47 (1988). That is, after the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, the recombinant virus is further infected into insect cells to express the protein. it can. The gene transfer vector used in the method includes, for example, pVL139
2, pVL1393, pBlueBacIII (both from Invitrogen) and the like.

Examples of the baculovirus include, for example, Autographa californica nuclea polyhedrosis virus which is a virus that infects night insects.
(Autographa californica nuclear polyhedrosis viru
s) and the like can be used. As insect cells, Spodop
Tera frugiperda ovary cells Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company,
New York, (1992)], Trichoplusia ni
Ovarian cell High5 (Invitrogen) or the like can be used.

As a method for preparing a recombinant virus, the above-described recombinant gene transfer vector and the above baculovirus are co-transfected into insect cells by, for example, a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method [Proc. Na
t1. 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.

A transformant having the recombinant DNA into which the above DNA has been incorporated is cultured in a medium, an enzyme involved in the mevalonate pathway is produced and accumulated in the culture, and the protein is collected from the culture. As a result, 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 carried out 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 that can be assimilated by the microorganism. Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently culture the transformant. The carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, and ethanol. And alcohols such as propanol.

Examples of the nitrogen source include ammonium salts of various inorganic acids and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn corn. Chip liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used. As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, 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 culturing temperature is preferably 15 to 40 ° C, and the culturing time is usually 16 hours to 7 days. PH during culture
Is kept at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin or 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 as necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is trp.
When culturing a microorganism transformed with an expression vector using a promoter, indoleacrylic acid (IAA) or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host cell, generally used RP
M11640 medium (The Journal of the American Medical As
sociation, 199, 519 (1967)), Eagle's MEM medium (Scienc
e, 122, 501 (1952)), DMEM medium (Virology, 8, 396 (19
59)), 199 medium (Proceeding of the Society for the B
iological Medicine, 73, 1 (1950)], or a medium obtained by adding fetal bovine serum or the like to such a medium. Culture is usually pH6~8,30~40 ℃, 1 under conditions such as 5% C0 ₂ presence
Do for ~ 7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
As a medium for culturing a transformant obtained using insect cells as host cells, a commonly used TNM-FH medium (Ph
armingen), Sf-900 II SFM medium (Gibco BRL),
ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium (Grace, TCC, Natur
e, 195, 788 (1962)]. Culture is
Usually, it is carried out for 1-5 days under conditions of pH 6-7, 25-30 ° C. If necessary, an antibiotic such as gentamicin 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 dissolved state in cells, after completion of the culture, the cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, and Mentongaulin homogenizer. The cells are crushed with a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) Chromatography using resin such as butyl sepharose, phenyl sepharose, etc., gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Alternatively, it can be used in combination to obtain a purified sample.

When the protein is expressed in an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to obtain a precipitate fraction obtained by a conventional method. After recovering the protein, the insoluble form of the protein is solubilized with a protein denaturant. After diluting the lysate into a solution containing no protein denaturing agent or a solution in which the concentration of the protein denaturing agent is so small that the protein is not denatured, or dialyzed to form the protein 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, the culture is treated by a method such as centrifugation as described above to obtain a soluble fraction, 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 proteins having the amino acid sequences 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 Javan (US Perkin-Elmer), Pharmacia Biotech (Pharmacia Biotech Sweden), Aloka (US
Protein Technology Instrument), Kurabo Industries (US S
ynthecell-Vega>, Nippon Perceptive Limited (US PerSeptive), Shimadzu Corp., etc.

(C) Production of isoprenoid compound The transformant obtained in the above (B) is cultured according to the method of the above (B) to produce and accumulate the isoprenoid compound in the culture. The isoprenoid compound can be produced by collecting the isoprenoid compound. By this cultivation, isoprenoid compounds such as ubiquinone, vitamin K ₂ and carotenoid can be produced. As specific examples, for example, production of ubiquinone-8 and menaquinone-8 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, belonging to the genus Arthrobacter microbial production of vitamin K ₂ in which the transformants, a
Production of astaxanthin using a microorganism belonging to the genus grobacterium as a transformant, production of a microorganism belonging to the genus Erwinia as a transformant, production of copen, β-carotene, and zeaxanthin can be exemplified. After completion of the culture, an isoprenoid compound is extracted by adding a suitable solvent to the culture solution, and the precipitate is removed by centrifugation or the like, and then the isoprenoid compound can be isolated and purified by performing various chromatography.

The present invention will be more specifically illustrated by the following examples, but the present invention is not limited to these examples. Genetic recombination experiments shown in the examples,
Unless otherwise specified, the method was performed using the method described in Molecular Cloning, 2nd Edition (hereinafter, referred to as a conventional method).

[0058]

EXAMPLES Example 1: Actinomyces Streptomyces sp. CL190
Acquisition of Gene Related to Mevalonate Pathway of Strain First, 3-hydroxy-3-methylglutaryl CoA (H, an enzyme that catalyzes one reaction on the mevalonate pathway)
MG-CoA) gene encoding reductase (hmg
r) The DNA fragment containing the gene was transformed into Streptomyces sp.
The nucleotide sequence of the DNA fragment obtained from the CL190 strain was analyzed, and it was revealed that genes involved in the mevalonate pathway were present in a cluster around the hmgr gene. The details are shown below.

The actinomycete Streptomyces sp.
ml GPY medium (1% glucose, 0.4% polypeptone (Wako Pure Chemical Industries, Ltd.), made 0.4% yeast extract (Difco _{Co.), 0.5% MgSO 4 · 7H} 2 O,
(0.1% K ₂ HPO ₄ ) at 30 ° C. for 2 days. 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 1 μg of the obtained DNA was digested with the restriction enzyme SnaBI (Takara Shuzo), Southern hybridization (molecular cloning, 2nd edition) using the hmgr gene as a probe was performed. As a result, the probe signal was detected at a position of 6.7 kb. was detected. From this result, it was found that the hmgr gene was contained in the 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 1 μg of a DNA fragment of about 6.7 kb was extracted from the agarose gel and recovered. The recovered 6.7 kb DNA fragment was blunt-ended using T4 DNA polymerase (purchased from Takara Shuzo), inserted into the HincII site of plasmid pUC118 (purchased from Takara Shuzo), and a chromosomal DNA library of Streptomycessp. CL190 strain was prepared. did. Using this chromosomal DNA library, E. coli JM109 strain (purchased from Takara Shuzo) was transformed according to a conventional method (Molecular Cloning, 2nd edition). The transformant was transformed with the hmgr gene (J. Bacteriol. 18).
1: 1256, 1999) by a colony hybridization method (molecular cloning second edition) using a probe, and a transformant of Escherichia coli having a plasmid containing the hmgr gene was isolated. From the isolated transformant, use the plasmid extraction kit QIAprep Spin Mini
Plasmids were extracted using a prep Kit (Qiagen).

The nucleotide sequence of the extracted plasmid was determined according to a standard method (Molecular Cloning, 2nd edition). At that time, Thermo Seque
nase cycle sequencing kit (Amersham Pharmacia Bi
otech) as a sequence analyzer
er model 4000L (manufactured by Li-cor) was used. The determined nucleotide sequence is shown in SEQ ID NO: 1.

Example 2: Actinomyces Streptomyces sp. CL190
Analysis of the structure of the gene related to the mevalonate pathway of the strain An open reading frame (hereinafter abbreviated as orf) was predicted from the nucleotide sequence of SEQ ID NO: 1 using the genetic information processing software GENETYX-MAC (manufactured by Software Development Co., Ltd.). As a result, in addition to the hmgr gene, 5
For convenience, the names of two orfs are given in ascending order of the base sequence number of SEQ ID NO: 1, orfA, orfB, orfC, or
fD and orfE. orfA to E and hmgr
The positions of the genes in SEQ ID NO: 1 are as follows. orfA (base numbers 132-1169 of SEQ ID NO: 1) orfB (base numbers 1159-2211 of SEQ ID NO: 1) orfC (base numbers 2208-3332 of SEQ ID NO: 1) orfD (base numbers 3335-4426 of SEQ ID NO: 1) hmgr gene (Base numbers 4423-54 of SEQ ID NO: 1
84) orfE (base numbers 5488-6657 of SEQ ID NO: 1)

OrfA, orfB, orfC, orf
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6
And the amino acid sequence is 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. The homology search of these orfs was performed using the FASTA program (Proc.
Acad. Sci. USA, 85: 2444, 1998). As a result, orfA was phosphomevalonate kinase (accession number P24521) and orfB was diphosphomevalonate decarboxylase (accession number P323).
77) and orfC are mevalonate kinase (accession num
ber P46086) and orfE were found to have significant homology 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 or
The novel gene coding for f forms a cluster with the hmgr gene and is transcribed in the same direction. From the result of the homology search, it was presumed to be a gene involved in the mevalonate pathway similarly to the hmgr gene. FIG. 1 shows the positional relationship between these genes.

Example 3 Actinomyces Streptomyces sp. CL190
Analysis of the functions of genes related to the mevalonate pathway in strains (1) orfA, orfB, orfC, orfD and o
Functional analysis of rfE Both ends of the 6.7 kb DNA fragment were made blunt using T4 DNA polymerase (purchased from Takara Shuzo), and Hincl of plasmid pUC118 (purchased from Takara Shuzo) was used.
A plasmid in which the N-terminus of phosphomevalonate (PMVA) kinase was inserted in the l-site so that it was close to the lac promoter of pUC118 was named pUMV19.
pUMV19 includes phosphomevalonate kinase (orf
A), diphosphomevalonate decarboxylase (orf)
B), mevalonate kinase (orfC), protein X
(OrfD), HMG-CoA reductase (hmg
r), a total of six genes encoding HMG-CoA synthase (orfE) are included (FIG. 1). next,
pUMV19 was introduced into E. coli JM109 strain, and E. coli JM1 was introduced.
09 (pUMV19) strain. E. coli JM109 (pU
In the MV19) strain, the above six genes are designed to be transcribed and expressed from the lac promoter by the addition of IPTG.

Fosmidmycin is known to specifically inhibit the non-mevalonate pathway (Tetrahedron Let
t. 39: 7913, 1998.). Therefore, the E. coli JM109 strain having only the non-mevalonate pathway cannot grow in a medium containing fosmidycin because it cannot biosynthesize IPP essential for growth. Therefore, E. coli JM10 prepared above
9 (pUMV19) is not affected by fosmidycin.
If growth is possible by adding PTG, this E. coli JM1
It can be demonstrated that the 09 (pUMV19) strain biosynthesizes IPP through the mevalonate pathway. That is, it can be proved that the 6.7 kb DNA fragment contains all genes related to the mevalonate pathway.

Further, as a deletion mutant of pUMV19,
pUMV19 △ E, pUMV19 △ S, pUMV19 △
M was constructed. pUMV19 @ E, pUMV19 @ S,
pUMV19ΔM is a plasmid prepared by self-ligation by digesting pUMV19 with restriction enzymes EcoRI, Sse8387I or MluI, respectively. At ΔM, protein X is missing (see FIG. 1). These deletion mutants also have pU
E. coli JM109 was transformed in the same manner as MV19, and E. coli JM109 was transformed.
JM109 (pUMV19 △ E), E. coli JM109 (pUMV1
9 △ S) and E. coli JM109 (pUMV19 △ M). In addition, fosmidomycin (Chem. Pharm. Bul
l. 30: 111, 1982.).

E. coli JM109 (pUMV19), E. coli J
M109 (pUMV19 △ E), E. coli JM109 (pUMV19 △ E)
ΔS), E. coli JM109 (pUMV19ΔM) and E. coli
JM109 (pUC118) was added to an LB medium (tryptone (Difco)) 1 containing 50 μg / ml antibiotic ampicillin (Sigma) to retain the plasmid.
%; Yeast extract (manufactured by Difco) 0.5
And 5 ml of NaCl) at 37 ° C. overnight with shaking culture (the following culture conditions 1 to 3 were used as culture conditions). Then their growth was observed. E. coli JM109
(PUC118) was used for control experiments.

Culture condition 1: L containing 0.1 mM IPTG (Wako Pure Chemical Industries) and 50 μg / ml ampicillin
The above five types of culture solutions were added in an amount of 1/1000 to 5 ml of B medium and cultured at 37 ° C for 12 hours. Culture condition 2: 0.1 mM IPTG, 20 μg / ml
Of fosmidycin and 50 μg / ml of LB medium in an amount of 1/1000,
The cells were cultured at 7 ° C for 12 hours. Culture condition 3: LB containing 0.1 mM IPTG, 0.02% mevalonic acid (Wako Pure Chemical Industries), 20 μg / ml fosmidomycin, and 50 μg / ml ampicillin
The above five kinds of culture solutions were added in 1/1000 amount to 5 ml of the medium, and cultured at 37 ° C. for 12 hours.

The results of growth after the culture are shown in FIG. 1 (+ indicates normal growth,-indicates non-normal growth). In culture condition 1, E. coli JM109 (p
UMV19), E. coli JM109 (pUMV19 △ E), E. coli
coli JM109 (pUMV19ＶS), E. coli JM109 (p
UMV19 △ M) and E. coli JM109 (pUC118). Under culture condition 2, only E. coli JM109 (pUMV19) was able to grow as expected. In other words, under the conditions in which the non-mevalonate pathway is blocked by the addition of fosmidomycin, mevalon is limited to those having pUMV19 in which all six genes on the 6.7 kb DNA fragment are incorporated. The acid pathway functioned normally and E. coli JM109 was viable. Therefore, it was revealed that six genes including protein X are involved in the mevalonate pathway.

In culture condition 3, as expected, E. coli JM10
9 (pUMV19) and E. coli JM109 (pUMV19 △)
S) was viable. E. coli JM109 (pUMV1
9 △ S) has normality except for HMG-CoA synthase, so that mevalonic acid added to the medium can be converted to IPP. For this purpose, E. coli JM109 (pU
It is considered that MV19 (S) could grow even in the presence of fosmidomycin if mevalonic acid and IPTG were added. On the other hand, E. coli JM109 (pUMV19 △ E),
li JM109 (pUMV19 △ M), E. coli JM109 (pUMV19 △ M)
C118) lacks one gene each for converting mevalonic acid added to the medium to IPP, so that in the presence of fosmidomycin, mevalonic acid and IPTG
It is considered that growth was impossible even with the addition.

From the above results, (1) all of the above six genes are indispensable for synthesizing IPP in E. coli JM109 by the mevalonate pathway;
(2) When HMG-CoA synthase was lacking, it was shown that IPP can be synthesized by the mevalonate pathway in E. coli JM109 by adding mevalonic acid.

Example 4 E. coli JM109 (pUMV1
9) Production of ubiquinone by strain The production of ubiquinone (one of the isoprenoid compounds) produced by E. coli JM109 (pUMV19) was measured using only E.coli J, which has no mevalonate pathway gene.
This was compared with the M109 (pUC118) strain. Ubiquinone was quantified as follows. E. coli JM109 (pUMV1
9) Each of the strain and E. coli JM109 (pUC118) was mixed with 50 ml of L containing 50 μg / ml of ampicillin.
The cells were cultured in a B medium at 37 ° C. for 14 hours, and the cells were collected by centrifugation. The collected cells were freeze-dried, the weight of the dried cells was measured, 60 ml of a chloroform: methanol = 2: 1 solution was added, and the mixture was allowed to stand for 1 hour to extract ubiquinone. Next, the chloroform-methanol solution was evaporated by an evaporator, and the remaining solid was dissolved by adding 1 ml of acetone. The amount of ubiquinone in the acetone solution was quantified by high-performance liquid chromatography (purchased from JASCO Corporation) using a Senshupak PEGASIL ODS column (manufactured by Senshu Kagaku), eluting with isopropyl alcohol: methanol = 1: 1. did. Under these conditions, ubiquinone is eluted at 9.8 minutes. The amount of ubiquinone was represented by the relative value of the peak area corresponding to ubiquinone in the above chromatogram. The results are shown in Table 1 below.

[0073]

[Table 1]

The production amount of ubiquinone per culture volume (in this case, 50 ml) in E. coli JM109 (pUMV19) was higher than that of E. coli JM109 (pUC118).
2.1 times (in the table, 25684/121
35 = 2.1). In addition, E. coli JM109 (pUMV1
The production amount of ubiquinone per cell mass in 9) is E. coli.
It was 4.5 times that of E. coli JM109 (pUC118) (450,000 / 100000 in the table).
= 4.5). From these results, Streptomyces s
It was found that the mevalonate pathway gene derived from p.CL190 strain was effective in improving isoprenoid productivity.

[0075]

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

Organisms in which the full length of the genes involved in the mevalonate pathway have been completely clarified include Streptomyces sp.
Yeast Saccharomyces cerevisiae (Natu
re 387: 5, 1997). However, genes involved in the mevalonate pathway of yeast have not formed a cluster. 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, obtain the mevalonate pathway genes one by one from the yeast. However, it is necessary to perform a complicated operation of incorporating each of them into a vector suitable for expression in E. coli. In addition, since the genome of a eukaryote such as yeast contains many regions that do not encode a protein called an intron, the yeast messenger RN is required to obtain a mevalonate pathway gene.
After extracting A and converting it into DNA using reverse transcriptase as a template, a complicated method of obtaining only the region encoding the enzyme of the mevalonate pathway has to be adopted.
On the other hand, the genes of the mevalonate pathway of the actinomycete Streptomyces sp.CL190 strain, which is a prokaryote, are clustered on the genome as described above, and are arranged so that they are transcribed in the same direction, which is complicated. No action required,
It can be expressed in E. coli.

[0077]

[Sequence List] SEQUENCE LISTING <110> SETO Haruo and KUZUYAMA Tomohisa <120> Genes of the enzymes involved in mevalonate pathway which are deri ved from inomycetes <130> 99422A <160> 13

[0078] <210> 1 <211> 6798 <212> DNA <213> Streptomyces <400> 1 tacgtacttc cctggcgtgt gcagcggttg acgcgccgtg ccctcgctgc gagcggcgcg 60 cacatctgac gtcctgcttt attgctttct cagaactcgg gacgaagcga tcccatgatc 120 acgcgatctc catgcagaaa agacaaaggg agctgagtgc gttgacacta ccgacctcgg 180 ctgagggggt atcagaaagc caccgggccc gctcggtcgg catcggtcgc gcccacgcca 240 aggccatcct gctgggagag catgcggtcg tctacggagc gccggcactc gctctgccga 300 ttccgcagct cacggtcacg gccagcgtcg gctggtcgtc cgaggcctcc gacagtgcgg 360 gtggcctgtc ctacacgatg accggtacgc cgtcgcgggc actggtgacg caggcctccg 420 acggcctgca ccggctcacc gcggaattca tggcgcggat gggcgtgacg aacgcgccgc 480 acctcgacgt gatcctggac ggcgcgatcc cgcacggccg gggtctcggct ccagcgcgg 540 ccggctcacg cgcgatcgcc ttggccctcg ccgacctctt cggccacgaa ctggccgagc 600 acacggcgta cgaactggtg cagacggccg agaacatggc gcacggccgg gccagcggcg 660 tggacgcgat gacggtcggc gcgtcccggc cgctgctgtt ccagcagggc cgcaccgagc 720 gactggccat cggctgcgac agcctgttca tcgtcgccga cagcggcgtc ccgggcagca 780 ccaaggaagc ggtcg agatg ctgcgggagg gattcacccg cagcgccgga acacaggagc 840 ggttcgtcgg ccgggcgacg gaactgaccg aggccgcccg gcaggccctc gccgacggcc 900 ggcccgagga gctgggctcg cagctgacgt actaccacga gctgctccat gaggcccgcc 960 tgagcaccga cggcatcgat gcgctggtcg aggccgcgct gaaggcaggc agcctcggag 1020 ccaagatcac cggcggtggt ctgggcggct gcatgatcgc acaggcccgg cccgaacagg 1080 cccgggaggt cacccggcag ctccacgagg ccggtgccgt acagacctgg gtcgtaccgc 1140 tgaaagggct cgacaaccat gcgcagtgaa cacccgacca cgaccgtgct ccagtcgcgg 1200 gagcagggca gcgcggccgg cgccaccgcg gtcgcgcacc caaacatcgc gctgatcaag 1260 tactggggca agcgcgacga gcggctgatc ctgccctgca ccaccagcct gtcgatgacg 1320 ctggacgtct tccccacgac caccgaggtc cggctcgacc ccgccgccga gcacgacacg 1380 gccgccctca acggcgaggt ggccacgggc gagacgctgc gccgcatcag cgccttcctc 1440 tccctggtgc gggaggtggc gggcagcgac cagcgggccg tggtggacac ccgcaacacc 1500 gtgcccaccg gggcgggcct ggcgtcctcc gccagcgggt tcgccgccct cgccgtcgcg 1560 gccgcggccg cctacgggct cgaactcgac gaccgcgggc tgtcccggct ggcccgacgt 1620 ggatccggct ccgcctcgcg gtc gatcttc ggcggcttcg ccgtctggca cgccggcccc 1680 gacggcacgg ccacggaagc ggacctcggc tcctacgccg agccggtgcc cgcggccgac 1740 ctcgacccgg cgctggtcat cgccgtggtc aacgccggcc ccaagcccgt ctccagccgc 1800 gaggccatgc gccgcaccgt cgacacctcg ccgctgtacc ggccgtgggc cgactccagt 1860 aaggacgacc tggacgagat gcgctcggcg ctgctgcgcg gcgacctcga ggccgtgggc 1920 gagatcgcgg agcgcaacgc gctcggcatg cacgccacca tgctggccgc ccgccccgcg 1980 gtgcggtacc tgtcgccggc cacggtcacc gtgctcgaca gcgtgctcca gctccgcaag 2040 gacggtgtcc tggcctacgc gaccatggac gccggtccca acgtgaaggt gctgtgccgg 2100 cgggcggacg ccgagcgggt ggccgacgtc gtacgcgccg ccgcgtccgg cggtcaggtc 2160 ctcgtcgccg ggccgggaga cggtgcccgc ctgctgagcg agggcgcatg acgacaggtc 2220 agcgcacgat cgtccggcac gcgccgggca agctgttcgt cgcgggcgag tacgcggtcg 2280 tggatccggg caacccggcg atcctggtag cggtcgaccg gcacatcagc gtcaccgtgt 2340 ccgacgccga cgcggacacc ggggccgccg acgtcgtgat ctcctccgac ctcggtccgc 2400 aggcggtcgg ctggcgctgg cacgacggcc ggctcgtcgt ccgcgacccg gacgacgggc 2460 agcaggcgcg cagcgccctg gcccacgtg g tgtcggcgat cgagaccgtg ggccggctgc 2520 tgggcgaacg cggacagaag gtccccgctc tcaccctctc cgtcagcagc cgcctgcacg 2580 aggacggccg gaagttcggc ctgggctcca gcggcgcggt gaccgtggcg accgtagccg 2640 ccgtcgccgc gttctgcgga ctcgaactgt ccaccgacga acggttccgg ctggccatgc 2700 tcgccaccgc ggaactcgac cccaagggct ccggcgggga cctcgccgcc agcacctggg 2760 gcggctggat cgcctaccag gcgcccgacc gggcctttgt gctcgacctg gcccggcgcg 2820 tgggagtcga ccggacactg aaggcgccct ggccggggca ctcggtgcgc cgactgccgg 2880 cgcccaaggg cctcaccctg gaggtcggct ggaccggaga gcccgcctcc accgcgtccc 2940 tggtgtccga tctgcaccgc cgcacctggc ggggcagcgc ctcccaccag aggttcgtcg 3000 agaccacgac cgactgtgtc cgctccgcgg tcaccgccct ggagtccggc gacgacacga 3060 gcctgctgca cgagatccgc cgggcccgcc aggagctggc ccgcctggac gacgaggtcg 3120 gcctcggcat cttcacaccc aagctgacgg cgctgtgcga cgccgccgaa gccgtcggcg 3180 gcgcggccaa gccctccggg gcaggcggcg gcgactgcgg catcgccctg ctggacgccg 3240 aggcgtcgcg ggacatcaca catgtacggc aacggtggga gacagccggg gtgctgcccc 3300 tgcccctgac tcctgccctg gaagggatct aaga atgacc agcgcccaac gcaaggacga 3360 ccacgtacgg ctcgccatcg agcagcacaa cgcccacagc ggacgcaacc agttcgacga 3420 cgtgtcgttc gtccaccacg ccctggccgg catcgaccgg ccggacgtgt ccctggccac 3480 gtccttcgcc gggatctcct ggcaggtgcc gatctacatc aacgcgatga ccggcggcag 3540 cgagaagacc ggcctcatca accgggacct ggccaccgcc gcccgcgaga ccggcgtccc 3600 catcgcgtcc gggtccatga acgcgtacat caaggacccc tcctgcgccg acacgttccg 3660 tgtgctgcgc gacgagaacc ccaacgggtt cgtcatcgcg aacatcaacg ccaccacgac 3720 ggtcgacaac gcgcagcgcg cgatcgacct gatcgaggcg aacgccctgc agatccacat 3780 caacacggcg caggagacgc cgatgccgga gggcgaccgg tcgttcgcgt cctgggtccc 3840 gcagatcgag aagatcgcgg cggccgtcga catccccgtg atcgtcaagg aggtcggcaa 3900 cggcctgagc cggcagacca tcctgctgct cgccgacctc ggcgtgcagg cggcggacgt 3960 cagcggccgc ggcggcacgg acttcgcccg catcgagaac ggccgccggg agctcggcga 4020 ctacgcgttc ctgcacggct gggggcagtc caccgccgcc tgcctgctgg acgcccagga 4080 catctccctg cccgtcctcg cctccggcgg tgtgcgtcac ccgctcgacg tggtccgcgc 4140 cctcgcgctc ggcgcccgcg ccgtcggctc ctccgccggc ttcctgcgca ccctgatgga 4200 cgacggcgtc gacgcgctga tcacgaagct cacgacctgg ctggaccagc tggcggcgct 4260 gcagaccatg ctcggcgcgc gcaccccggc cgacctcacc cgctgcgacg tgctgctcca 4320 cggcgagctg cgtgacttct gcgccgaccg gggcatcgac acgcgccgcc tcgcccagcg 4380 ctccagctcc atcgaggccc tccagacgac gggaagcaca cgatgacgga aacgcacgcc 4440 atagccgggg tcccgatgag gtgggtggga ccccttcgta tttccgggaa cgtcgccgag 4500 accgagaccc aggtcccgct cgccacgtac gagtcgccgc tgtggccgtc ggtgggccgc 4560 ggggcgaagg tctcccggct gacggagaag ggcatcgtcg ccaccctcgt cgacgagcgg 4620 atgacccgct cggtgatcgt cgaggcgacg gacgcgcaga ccgcgtacat ggccgcgcag 4680 accatccacg cccgcatcga cgagctgcgc gaggtggtgc gcggctgcag ccggttcgcc 4740 cagctgatca acatcaagca cgagatcaac gcgaacctgc tgttcatccg gttcgagttc 4800 accaccggtg acgcctccgg ccacaacatg gccacgctcg cctccgatgt gctcctgggg 4860 cacctgctgg agacgatccc tggcatctcc tacggctcga tctccggcaa ctactgcacg 4920 gacaagaagg ccaccgcgat caacggcatc ctcggccgcg gcaagaacgt gatcaccgag 4980 ctgctggtgc cgcgggacgt cgtcgagaac aacctgcaca ccacg gctgc caagatcgtc 5040 gagctgaaca tccgcaagaa cctgctcggc accctgctcg ccggcggcat ccgctcggcc 5100 aacgcccact tcgcgaacat gctgctcggc ttctacctgg ccaccggcca ggacgccgcc 5160 aacatcgtcg agggctcgca gggcgtcgtc atggccgagg accgcgacgg cgacctctac 5220 ttcgcctgca ccctgccgaa cctgatcgtc ggcacggtcg gcaacggcaa gggtctcggc 5280 ttcgtggaga cgaacctcgc ccggctcggc tgccgagccg accgcgaacc cggggagaac 5340 gcccgccgcc tcgccgtcat cgcggcagcg accgtgctgt gcggtgaact ctcgctgctc 5400 gcggcacaga cgaacccggg cgaactcatg cgcgcgcacg tccagctgga acgcgacaac 5460 aagaccgcaa aggttggtgc atagggcatg tccatctcca taggcattca cgacctgtcg 5520 ttcgccacaa ccgagttcgt cctgccgcac acggcgctcg ccgagtacaa cggcaccgag 5580 atcggcaagt accacgtcgg catcggccag cagtcgatga gcgtgccggc cgccgacgag 5640 gacatcgtga ccatggccgc gaccgcggcg cggcccatca tcgagcgcaa cggcaagagc 5700 cggatccgca cggtcgtgtt cgccacggag tcgtcgatcg accaggcgaa ggcgggcggc 5760 gtgtacgtgc actccctgct ggggctggag tcggcctgcc gggtcgtcga gctgaagcag 5820 gcctgctacg gggccaccgc cgcccttcag ttcgccatcg gcctggtgcg gcgcgacccc 5880 gcccagcagg tcctggtcat cgccagtgac gtctccaagt acgagctgga cagccccggc 5940 gaggcgaccc agggcgcggc cgcggtggcc atgctggtcg gcgccgaccc ggccctgctg 6000 cgtatcgagg agccgtcggg cctgttcacc gccgacgtca tggacttctg gcggcccaac 6060 tacctcacca ccgctctggt cgacggccag gagtccatca acgcctacct gcaggccgtc 6120 gagggcgcct ggaaggacta cgcggagcag gacggccggt cgctggagga gttcgcggcg 6180 ttcgtctacc accagccgtt cacgaagatg gcctacaagg cgcaccgcca cctgctgaac 6240 ttcaacggct acgacaccga caaggacgcc atcgagggcg ccctcggcca gacgacggcg 6300 tacaacaacg tcatcggcaa cagctacacc gcgtcggtgt acctgggcct ggccgccctg 6360 ctcgaccagg cggacgacct gacgggccgt tccatcggct tcctgagcta cggctcgggc 6420 agcgtcgccg agttcttctc gggcaccgtc gtcgccgggt accgcgagcg tctgcgcacc 6480 gaggcgaacc aggaggcgat cgcccggcgc aagagcgtcg actacgccac ctaccgcgag 6540 ctgcacgagt acacgctccc gtccgacggc ggcgaccacg ccaccccggt gcagaccacc 6600 ggccccttcc ggctggccgg gatcaacgac cacaagcgca tctacgaggc gcgctagcga 6660 cacccctcgg caacggggtg cgccactgtt cggcgcaccc cgtgccgggc tttcgc acag 6720 ctattcacga ccatttgagg ggcgggcagc cgcatgaccg acgtccgatt ccgcattatc 6780 ggtacgggtg cctacgta 6798

<210> 2 <211> 1038 <212> DNA <213> Streptomyces <400> 2 atg cag aaa aga caa agg gag ctg agt gcg ttg aca cta 39 Met Gln Lys Arg Gln Arg Glu Leu Ser Ala Leu Thr Leu 1 5 10 ccg acc tcg gct gag ggg gta tca gaa agc cac cgg gcc cgc tcg gtc 87 Pro Thr Ser Ala Glu Gly Val Ser Glu Ser His Arg Ala Arg Ser Val 15 20 25 ggc atc ggt cgc gcc cac gcc aag gcc atc ctg ctg gga gag cat gcg 135 Gly Ile Gly Arg Ala His Ala Lys Ala Ile Leu Leu Gly Glu His Ala 30 35 40 45 gtc gtc tac gga gcg ccg gca ctc gct ctg ccg att ccg cag ctc acg 183 Val Val Tyr Gly Ala Leu Ala Leu Pro Ile Pro Gln Leu Thr 50 55 60 gtc acg gcc agc gtc ggc tgg tcg tcc gag gcc tcc gac agt gcg ggt 231 Val Thr Ala Ser Val Gly Trp Ser Ser Glu Ala Ser Asp Ser Ala Gly 65 70 75 ggc ctg tcc tac acg atg acc ggt acg ccg tcg cgg gca ctg gtg acg 279 Gly Leu Ser Tyr Thr Met Thr Gly Thr Pro Ser Arg Ala Leu Val Thr 80 85 90 cag gcc tcc gac ggc ctg cac cgg ctc acc gcg gaa ttc atg gcg 327 Gln Ala Ser Asp Gly Leu His Arg Leu Thr Ala Glu Phe Met Ala Arg 95 100 105 atg ggc gtg acg aac gcg ccg cac ctc gac gtg atc ctg gac ggc gcg 375 Met Gly Val Thr Asn Ala Pro His Leu Asp Val Ile Leu Asp Gly Ala 110 115 120 125 atc ccg cac ggc cgg ggt ctc ggc tcc agc gcg gcc ggc tca cgc gcg 423 Ile Pro His Gly Arg Gly Leu Gly Ser Ser Ala Ala Gly Ser Arg Ala 130 135 140 atc gcc ttg gcc ctc gcc gac ctc ttc ggc cac gag cc gac gag cc gac gag Ile Ala Leu Ala Leu Ala Asp Leu Phe Gly His Glu Leu Ala Glu His 145 150 155 acg gcg tac gaa ctg gtg cag acg gcc gag aac atg gcg cac ggc cgg 519 Thr Ala Tyr Glu Leu Val Gln Thr Ala Glu Asn Met Ala Gly Arg 160 165 170 gcc agc ggc gtg gac gcg atg acg gtc ggc gcg tcc cgg ccg ctg ctg 567 Ala Ser Gly Val Asp Ala Met Thr Val Gly Ala Ser Arg Pro Leu Leu 175 180 185 ttc cag cag ggc cgc acc gcc atc ggc tgc gac agc ctg 615 Phe Gln Gln Gly Arg Thr Glu Arg Leu Ala Ile Gly Cys Asp Ser Leu 190 195 200 205 ttc atc gtc gcc gac agc ggc gtc ccg ggc agc acc aag gaa Ascg gtc 66 S er Gly Val Pro Gly Ser Thr Lys Glu Ala Val 210 215 220 gag atg ctg cgg gag gga ttc acc cgc agc gcc gga aca cag gag cgg 711 Glu Met Leu Arg Glu Gly Phe Thr Arg Ser Ala Gly Thr Gln Glu Arg 225 230 235 ttc gtc ggc cgg gcg acg gaa ctg acc gag gcc gcc cgg cag gcc ctc 759 Phe Val Gly Arg Ala Thr Glu Leu Thr Glu Ala Ala Ala Arg Gln Ala Leu 240 245 250 gcc gac ggc cgg ccc gag gag ctg gc tg ca tac cac 807 Ala Asp Gly Arg Pro Glu Glu Leu Gly Ser Gln Leu Thr Tyr Tyr His 255 260 265 gag ctg ctc cat gag gcc cgc ctg agc acc gac ggc atc gat gcg ctg 855 Glu Leu Leu His Glu Ala Arg Leu Ser Thrp Gly Ile Asp Ala Leu 270 275 280 285 gtc gag gcc gcg ctg aag gca ggc agc ctc gga gcc aag atc acc ggc 903 Val Glu Ala Ala Leu Lys Ala Gly Ser Leu Gly Ala Lys Ile Thr Gly 290 295 ggg ggt tgc atg atc gca cag gcc cgg ccc gaa cag gcc 951 Gly Gly Leu Gly Gly Cys Met Ile Ala Gln Ala Arg Pro Glu Gln Ala 305 310 315 cgg gag gtc acc cgg cag ctc cac gag gcc ggt gcc gta cag acc tgg 999 Arg V al Thr Arg Gln Leu His Glu Ala Gly Ala Val Gln Thr Trp 320 325 330 gtc gta ccg ctg aaa ggg ctc gac aac cat gcg cag tga 1038 Val Val Pro Leu Lys Gly Leu Asp Asn His Ala Gln 335 340

<210> 3 <211> 1053 <212> DNA <213> Streptomyces <400> 3 atg cgc agt gaa cac ccg acc acg acc gtg ctc 33 Met Arg Ser Glu His Pro Thr Thr Thr Val Leu 15 10 cag tcg cgg gag cag ggc agc gcg gcc ggc gcc acc gcg gtc gcg cac 81 Gln Ser Arg Glu Gln Gly Ser Ala Ala Gly Ala Thr Ala Val Ala His 15 20 25 cca aac atc gcg ctg atc aag tac tgg ggc ag cgg gg ctg 129 Pro Asn Ile Ala Leu Ile Lys Tyr Trp Gly Lys Arg Asp Glu Arg Leu 30 35 40 atc ctg ccc tgc acc acc agc ctg tcg atg acg ctg gac gtc ttc ccc 177 Ile Leu Pro Cys Thr Thr Ser Leu Ser Met Thr Leu Asp Val Phe Pro 45 50 55 acg acc acc gag gtc cgg ctc gac ccc gcc gcc gag cac gac acg gcc 225 Thr Thr Thr Glu Val Arg Leu Asp Pro Ala Ala Glu His Asp Thr Ala 60 65 70 75 gcc ctc aac ggc gag gtg gcc acg ggc gag acg ctg cgc cgc atc agc 273 Ala Leu Asn Gly Glu Val Ala Thr Gly Glu Thr Leu Arg Arg Ile Ser 80 85 90 gcc ttc ctc tcc ctg gtg cgg gag gtg gcg ggc agc Gac cag cgg Pg Ser Leu Val Arg Glu Val Ala Gly Ser Asp Gln Arg Ala 95 100 105 gtg gtg gac acc cgc aac acc gtg ccc acc ggg gcg ggc ctg gcg tcc 369 Val Val Asp Thr Arg Asn Thr Val Pro Thr Gly Ala Gly Leu Ala Ser 110 115 120 tcc gcc agc ggg ttc gcc gcc ctc gcc gtc gcg gcc gcg gcc gcc tac 417 Ser Ala Ser Gly Phe Ala Ala Leu Ala Val Ala Ala Ala Ala Ala Tyr 125 130 135 ggg ctc gaa ctc gac gac cgc ggg ctg tcc cgg ctg gcc cga cgt gga Le Gly Leu Asp Arg Gly Leu Ser Arg Leu Ala Arg Arg Gly 140 145 150 155 tcc ggc tcc gcc tcg cgg tcg atc ttc ggc ggc ttc gcc gtc tgg cac 513 Ser Gly Ser Ala Ser Arg Ser Ile Phe Gly Gly Phe Ala Val Trp His 160 165 170 gcc ggc ccc gac ggc acg gcc acg gaa gcg gac ctc ggc tcc tac gcc 561 Ala Gly Pro Asp Gly Thr Ala Thr Glu Ala Asp Leu Gly Ser Tyr Ala 175 180 185 gag ccg gtg ccc gcg gcc gac ctc gac cc atc gcc gtg 609 Glu Pro Val Pro Ala Ala Asp Leu Asp Pro Ala Leu Val Ile Ala Val 190 195 200 gtc aac gcc ggc ccc aag ccc gtc tcc agc cgc gag gcc atg cgc cgc 657 Val Asn Ala Gly Pro Lys Pro Val Ser Ser A rg Glu Ala Met Arg Arg 205 210 215 acc gtc gac acc tcg ccg ctg tac cgg ccg tgg gcc gac tcc agt aag 705 Thr Val Asp Thr Ser Pro Leu Tyr Arg Pro Trp Ala Asp Ser Ser Lys 220 225 230 235 gac gac ctg gac gag atg cgc tcg gcg ctg ctg cgc ggc gac ctc gag 753 Asp Asp Leu Asp Glu Met Arg Ser Ala Leu Leu Arg Gly Asp Leu Glu 240 245 250 gcc gtg ggc gag atc gcg gag cgc aac gcg cc gac gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gcc Val Gly Glu Ile Ala Glu Arg Asn Ala Leu Gly Met His Ala Thr 255 260 265 atg ctg gcc gcc cgc ccc gcg gtg cgg tac ctg tcg ccg gcc acg gtc 849 Met Leu Ala Ala Arg Pro Ala Val Arg Tyr Leu Ser Ala Thr Val 270 275 280 acc gtg ctc gac agc gtg ctc cag ctc cgc aag gac ggt gtc ctg gcc 897 Thr Val Leu Asp Ser Val Leu Gln Leu Arg Lys Asp Gly Val Leu Ala 285 290 295 tac gcg acc atg gac gcc ggt ccc aag gtg ctg tgc cgg cgg 945 Tyr Ala Thr Met Asp Ala Gly Pro Asn Val Lys Val Leu Cys Arg Arg 300 305 310 315 gcg gac gcc gag cgg gtg gcc gac gtc gta cgc gcc gcc gcg tcc ggc 993 Ala Asp Ala Gla A la Asp Val Val Arg Ala Ala Ala Ser Gly 320 325 330 ggt cag gtc ctc gtc gcc ggg ccg gga gac ggt gcc cgc ctg ctg agc 1041 Gly Gln Val Leu Val Ala Gly Pro Gly Asp Gly Ala Arg Leu Leu Ser 335 340 340 g ggc gca tga 1053 Glu Gly Ala 350

<210> 4 <211> 1125 <212> DNA <213> Streptomyces <400> 4 atg acg aca 9 Met Thr Thr 1 ggt cag cgc acg atc gtc cgg cac gcg ccg ggc aag ctg ttc gtc gcg 57 Gly Gln Arg Thr Ile Val Arg His Ala Pro Gly Lys Leu Phe Val Ala 5 10 15 ggc gag tac gcg gtc gtg gat ccg ggc aac ccg gcg atc ctg gta gcg 105 Gly Glu Tyr Ala Val Val Asp Pro Gly Asn Pro Ala Ile Leu Val Ala 20 25 30 35 gtc gac cgg cac atc agc gtc acc gtg tcc gac gcc gac gcg gac acc 153 Val Asp Arg His Ile Ser Val Thr Val Ser Asp Ala Asp Ala Asp Thr 40 45 50 ggg gcc gcc gac gtc gtg atc tcc tcc gac ctc ggt ccg cag gcg gtc 201 Gly Ala Ala Asp Val Val Ile Ser Ser Asp Leu Gly Pro Gln Ala Val 55 60 65 ggc tgg cgc tgg cac gac ggc cgg ctc gtc gtc cgc gac ccg gac gac 249 Gly Trp Arg Trp Hisp Arg Leu Val Val Arg Asp Pro Asp Asp 70 75 80 ggg cag cag gcg cgc agc gcc ctg gcc cac gtg gtg tcg gcg atc gag 297 Gly Gln Gln Ala Arg Ser Ala Leu Ala His Val Val Ser Ala Ile Glu 85 90 95 acc gtg ggc cgg ctg ctg ggc gaa cgc gga cag aag gt c ccc gct ctc 345 Thr Val Gly Arg Leu Leu Gly Glu Arg Gly Gln Lys Val Pro Ala Leu 100 105 110 115 acc ctc tcc gtc agc agc cgc ctg cac gag gac ggc cgg aag ttc ggc 393 Thr Leu Ser Val Ser Ser Arg Leu His Glu Asp Gly Arg Lys Phe Gly 120 125 130 ctg ggc tcc agc ggc gcg gtg acc gtg gcg acc gta gcc gcc gtc gcc 441 Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val Ala Ala Val Ala 135 140 145 gcg ttc tgc gga ctc gaa ctg tcc acc gac gaa cgg ttc cgg ctg gcc 489 Ala Phe Cys Gly Leu Glu Leu Ser Thr Asp Glu Arg Phe Arg Leu Ala 150 155 160 atg ctc gcc acc gcg gaa ctc gac ccc aag ggc gcc ccg Met Leu Ala Thr Ala Glu Leu Asp Pro Lys Gly Ser Gly Gly Asp Leu 165 170 175 gcc gcc agc acc tgg ggc ggc tgg atc gcc tac cag gcg ccc gac cgg 585 Ala Ala Ser Thr Trp Gly Gly Trp Ile Ala Tyr Gln Ala Pro Asp Arg 180 185 190 195 gcc ttt gtg ctc gac ctg gcc cgg cgc gtg gga gtc gac cgg aca ctg 633 Ala Phe Val Leu Asp Leu Ala Arg Arg Val Gly Val Asp Arg Thr Leu 200 205 210 aag gcg ccc tgg tcc ggc gt g cgc cga ctg ccg gcg ccc aag 681 Lys Ala Pro Trp Pro Gly His Ser Val Arg Arg Leu Pro Ala Pro Lys 215 220 225 ggc ctc acc ctg gag gtc ggc tgg acc gga gag ccc gcc tcc acc gcg 729 Gly Leu Thr Leu Glu Val Gly Trp Thr Gly Glu Pro Ala Ser Thr Ala 230 235 240 tcc ctg gtg tcc gat ctg cac cgc cgc acc tgg cgg ggc agc gcc tcc 777 Ser Leu Val Ser Asp Leu His Arg Arg Thr Trp Arg Gly Ser Ala Ser 245 250 255 cac cag agg ttc gtc gag acc acg acc gac tgt gtc cgc tcc gcg gtc 825 His Gln Arg Phe Val Glu Thr Thr Thr Asp Cys Val Arg Ser Ala Val 260 265 270 270 275 acc gcc ctg gag tcc ggc gac gac acg agc ctg ctg cg gag atc cgc 873 Thr Ala Leu Glu Ser Gly Asp Asp Thr Ser Leu Leu His Glu Ile Arg 280 285 290 cgg gcc cgc cag gag ctg gcc cgc ctg gac gac gag gtc ggc ctc ggc 921 Arg Ala Arg Gln Glu Leu Ala Asp Glu Val Gly Leu Gly 295 300 305 atc ttc aca ccc aag ctg acg gcg ctg tgc gac gcc gcc gaa gcc gtc 969 Ile Phe Thr Pro Lys Leu Thr Ala Leu Cys Asp Ala Ala Glu Ala Val 310 315 320 ggc ggcggc cc c tcc ggg gca ggc ggc ggc gac tgc ggc atc 1017 Gly Gly Ala Ala Lys Pro Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile 325 330 335 gcc ctg ctg gac gcc gag gcg tcg cgg gac atc aca cat gta cag caca Leu Asp Ala Glu Ala Ser Arg Asp Ile Thr His Val Arg Gln 340 345 350 350 355 cgg tgg gag aca gcc ggg gtg ctg ccc ctg ccc ctg act cct gcc ctg 1113 Arg Trp Glu Thr Ala Gly Val Leu Pro Leu Pro Leu Thr Pro Ala Leu 360 365 370 gaa ggg atc taa 1125 Glu Gly Ile

<210> 5 <211> 1092 <212> DNA <213> Streptomyces <400> 5 atg acc agc gcc caa cgc aag 21 Met Thr Ser Ala Gln Arg Lys 15 gac gac cac gta cgg ctc gcc atc gag cag cac aac gcc cac agc gga 69 Asp Asp His Val Arg Leu Ala Ile Glu Gln His Asn Ala His Ser Gly 10 15 20 cgc aac cag ttc gac gac gtg tcg ttc gtc cac cac gcc ctg gcc ggc 117 Arg Asn Gln Phe Asp Val Ser Phe Val His His Ala Leu Ala Gly 25 30 35 atc gac cgg ccg gac gtg tcc ctg gcc acg tcc ttc gcc ggg atc tcc 165 Ile Asp Arg Pro Asp Val Ser Leu Ala Thr Ser Phe Ala Gly Ile Ser 40 45 50 55 tgg cag gtg ccg atc tac atc aac gcg atg acc ggc ggc agc gag aag 213 Trp Gln Val Pro Ile Tyr Ile Asn Ala Met Thr Gly Gly Ser Glu Lys 60 65 70 acc ggc ctc atc aac cgg gac ctg gcc acc gcc ggc gag accg ggc 261 Thr Gly Leu Ile Asn Arg Asp Leu Ala Thr Ala Ala Arg Glu Thr Gly 75 80 85 gtc ccc atc gcg tcc ggg tcc atg aac gcg tac atc aag gac ccc tcc 309 Val Pro Ile Ala Ser Gly Ser Met Asn Ala Tyr Ile Lys Asp Pro Ser 90 95 100 tgc gcc gac a cg ttc cgt gtg ctg cgc gac gag aac ccc aac ggg ttc 357 Cys Ala Asp Thr Phe Arg Val Leu Arg Asp Glu Asn Pro Asn Gly Phe 105 110 115 gtc atc gcg aac atc aac gcc acc acg acg gtc gac aac gcg ca Val Ile Ala Asn Ile Asn Ala Thr Thr Thr Val Asp Asn Ala Gln Arg 120 125 130 135 gcg atc gac ctg atc gag gcg aac gcc ctg cag atc cac atc aac acg 453 Ala Ile Asp Leu Ile Glu Ala Asn Ala Leu Gln Ile His Ile Asn Thr 140 145 150 gcg cag gag acg ccg atg ccg gag ggc gac cgg tcg ttc gcg tcc tgg 501 Ala Gln Glu Thr Pro Met Pro Glu Gly Asp Arg Ser Phe Ala Ser Trp 155 160 165 gtc ccg cag atc gag aag atg gcg gcc gtc gac atc ccc gtg atc 549 Val Pro Gln Ile Glu Lys Ile Ala Ala Ala Val Asp Ile Pro Val Ile 170 175 180 gtc aag gag gtc ggc aac ggc ctg agc cgg cag acc atc ctg ctg ctc lug Lys Asn Gly Leu Ser Arg Gln Thr Ile Leu Leu Leu 185 190 195 gcc gac ctc ggc gtg cag gcg gcg gac gtc agc ggc cgc ggc ggc acg 645 Ala Asp Leu Gly Val Gln Ala Ala Asp Val Ser Gly Arg Gly Gthl Two 15 gac ttc gcc cgc atc gag aac ggc cgc cgg gag ctc ggc gac tac gcg 693 Asp Phe Ala Arg Ile Glu Asn Gly Arg Arg Glu Leu Gly Asp Tyr Ala 220 225 230 ttc ctg cac ggc tgg ggg gcc accg ctg gac gcc 741 Phe Leu His Gly Trp Gly Gln Ser Thr Ala Ala Cys Leu Leu Asp Ala 235 240 245 cag gac atc tcc ctg ccc gtc ctc gcc tcc ggc ggt gtg cgt cac ccg 789 Gln Asp Ile Ser Leu Pro Val Leu A Gly Gly Val Arg His Pro 250 255 260 ctc gac gtg gtc cgc gcc ctc gcg ctc ggc gcc cgc gcc gtc ggc tcc 837 Leu Asp Val Val Arg Ala Leu Ala Leu Gly Ala Arg Ala Val Gly Ser 265 270 270 275 tcc gcc gg cgc acc ctg atg gac gac ggc gtc gac gcg ctg 885 Ser Ala Gly Phe Leu Arg Thr Leu Met Asp Asp Gly Val Asp Ala Leu 280 285 290 295 atc acg aag ctc acg acc tgg ctg gac cag ctg gcg gcg ctg cag acc 933 Ile Thr Lys Leu Thr Thr Trp Leu Asp Gln Leu Ala Ala Leu Gln Thr 300 305 310 atg ctc ggc gcg cgc acc ccg gcc gac ctc acc cgc tgc gac gtg ctg 981 Met Leu Gly Ala Arg Thr Pro Ala Asp Leu Thr Arg Cys Val L eu 315 320 325 ctc cac ggc gag ctg cgt gac ttc tgc gcc gac cgg ggc atc gac acg 1029 Leu His Gly Glu Leu Arg Asp Phe Cys Ala Asp Arg Gly Ile Asp Thr 330 335 340 cgc cgc ctc gcc cagcgccgc gag gcc ctc cag acg acg 1077 Arg Arg Leu Ala Gln Arg Ser Ser Ser Ile Glu Ala Leu Gln Thr Thr 345 350 355 gga agc aca cga tga 1092 Gly Ser Thr Arg 360

<210> 6 <211> 1062 <212> DNA <213> Streptomyces <400> 6 atg acg gaa acg 12 Met Thr Glu Thr 1 cac gcc ata gcc ggg gtc ccg atg agg tgg gtg gga ccc ctt cgt att 60 His Ala Ile Ala Gly Val Pro Met Arg Trp Val Gly Pro Leu Arg Ile 5 10 15 20 tcc ggg aac gtc gcc gag acc gag acc cag gtc ccg ctc gcc acg tac 108 Ser Gly Asn Val Ala Glu Thr Glu Thr Gln Val Pro Leu Ala Thr Tyr 25 30 35 gag tcg ccg ctg tgg ccg tcg gtg ggc cgc ggg gcg aag gtc tcc cgg 156 Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala Lys Val Ser Arg 40 45 50 ctg acg gag aag ggc atc gc acc ctc gtc gac gag cgg atg acc 204 Leu Thr Glu Lys Gly Ile Val Ala Thr Leu Val Asp Glu Arg Met Thr 55 60 65 cgc tcg gtg atc gtc gag gcg acg gac gcg cag acc gcg tac atg gcc 252 Arg Ser Val Ile Val Glu Ala Thr Asp Ala Gln Thr Ala Tyr Met Ala 70 75 80 gcg cag acc atc cac gcc cgc atc gac gag ctg cgc gag gtg gtg cgc 300 Ala Gln Thr Ile His Ala Arg Ile Asp Glu Leu Arg Glu Val Val Arg 85 90 95 100 ggc tgc agc cgg ttc gcc cag ctg atc a ac atc aag cac gag atc aac 348 Gly Cys Ser Arg Phe Ala Gln Leu Ile Asn Ile Lys His Glu Ile Asn 105 110 115 gcg aac ctg ctg ttc atc cgg ttc gag ttc acc acc ggt gac gcc tcc 396 Ala Asn Leu Leu Phe Arg Phe Glu Phe Thr Thr Gly Asp Ala Ser 120 125 130 ggc cac aac atg gcc acg ctc gcc tcc gat gtg ctc ctg ggg cac ctg 444 Gly His Asn Met Ala Thr Leu Ala Ser Asp Val Leu Leu Gly His Leu 135 140 145 ctg gag acg atc cct ggc atc tcc tac ggc tcg atc tcc ggc aac tac 492 Leu Glu Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile Ser Gly Asn Tyr 150 155 160 tgc acg gac aag aag gcc acc gcg atc aac ggc atc ccc ggc 540 Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile Leu Gly Arg Gly 165 170 175 180 aag aac gtg atc acc gag ctg ctg gtg ccg cgg gac gtc gtc gag aac 588 Lys Asn Val Ile Thr Glu Leu Leu Val Ar Asp Val Val Glu Asn 185 190 195 aac ctg cac acc acg gct gcc aag atc gtc gag ctg aac atc cgc aag 636 Asn Leu His Thr Thr Ala Ala Lys Ile Val Glu Leu Asn Ile Arg Lys 200 205 210 aac ctg ctc ggc acc ctg c tc gcc ggc ggc atc cgc tcg gcc aac gcc 684 Asn Leu Leu Gly Thr Leu Leu Ala Gly Gly Ile Arg Ser Ala Asn Ala 215 220 225 cac ttc gcg aac atg ctg ctc ggc ttc tac ctg gcc acc ggc ca ggg ca Asn Met Leu Leu Gly Phe Tyr Leu Ala Thr Gly Gln Asp 230 235 240 gcc gcc aac atc gtc gag ggc tcg cag ggc gtc gtc atg gcc gag gac 780 Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Val Met Ala Glu Asp 250 255 260 cgc gac ggc gac ctc tac ttc gcc tgc acc ctg ccg aac ctg atc gtc 828 Arg Asp Gly Asp Leu Tyr Phe Ala Cys Thr Leu Pro Asn Leu Ile Val 265 270 275 ggc acg gtc ggc agg gc ggc ag gc cag c gtg gag acg aac ctc 876 Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val Glu Thr Asn Leu 280 285 290 gcc cgg ctc ggc tgc cga gcc gac cgc gaa ccc ggg gag aac gcc cgc 924 Ala Arg Leu Acy Cyg Arg Glu Pro Gly Glu Asn Ala Arg 295 300 305 cgc ctc gcc gtc atc gcg gca gcg acc gtg ctg tgc ggt gaa ctc tcg 972 Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys Gly Glu Leu Serg 310 ct 320 ct 320 g ca cag acg aac ccg ggc gaa ctc atg cgc gcg cac gtc 1020 Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met Arg Ala His Val 325 330 335 340 cag ctg gaa cgc gac aac aag acc gca aag gtt ggt tag Leu Glu Arg Asp Asn Lys Thr Ala Lys Val Gly Ala 345 350

<210> 7 <211> 1170 <212> DNA <213> Streptomyces <400> 7 atg tcc atc tcc ata ggc att cac gac 27 Met Ser Ile Ser Ile Gly Ile His Asp 15 ctg tcg ttc gcc aca acc gag ttc gtc ctg ccg cac acg gcg ctc gcc 75 Leu Ser Phe Ala Thr Thr Glu Phe Val Leu Pro His Thr Ala Leu Ala 10 15 20 25 gag tac aac ggc acc gag atc ggc aag tac cac gtc ggc atc ggc cag 123 Glu Tyr Asn Gly Thr Glu Ile Gly Lys Tyr His Val Gly Ile Gly Gln 30 35 40 cag tcg atg agc gtg ccg gcc gcc gac gag gac atc gtg acc atg gcc 171 Gln Ser Met Ser Val Pro Ala Ala Asp Glu Asp Ile Val Thr Met Ala 45 50 55 gcg acc gcg gcg cgg ccc atc atc gag cgc aac ggc aag agc cgg atc 219 Ala Thr Ala Ala Arg Pro Ile Ile Glu Arg Asn Gly Lys Ser Arg Ile 60 65 70 cgc acg gtc gtg ttc gcc acg gag tc gac cag gcg aag gcg 267 Arg Thr Val Val Phe Ala Thr Glu Ser Ser Ile Asp Gln Ala Lys Ala 75 80 85 ggc ggc gtg tac gtg cac tcc ctg ctg ggg ctg gag tcg gcc tgc cgg 315 Gly Gly Val Tyr Val His Seru Leu Gly Leu Glu Ser Ala Cys Arg 90 95 1 00 105 gtc gtc gag ctg aag cag gcc tgc tac ggg gcc acc gcc gcc ctt cag 363 Val Val Glu Leu Lys Gln Ala Cys Tyr Gly Ala Thr Ala Ala Leu Gln 110 115 120 ttc gcc atc ggc ctg gtg cgg cgc gcc gcc gcc cag gtc ctg gtc 411 Phe Ala Ile Gly Leu Val Arg Arg Asp Pro Ala Gln Gln Val Leu Val 125 130 135 atc gcc agt gac gtc tcc aag tac gag ctg gac agc ccc ggc gag gcg 459 Ile Ala Ser Asp Val Ser Lys Tyr Glu Leu Asp Ser Pro Gly Glu Ala 140 145 150 acc cag ggc gcg gcc gcg gtg gcc atg ctg gtc ggc gcc gac ccg gcc 507 Thr Gln Gly Ala Ala Ala Val Ala Met Leu Val Gly Ala Asp Pro Ala 155 160 165 ctg ctg cg gag gag ccg tcg ggc ctg ttc acc gcc gac gtc atg 555 Leu Leu Arg Ile Glu Glu Pro Ser Gly Leu Phe Thr Ala Asp Val Met 170 175 180 185 gac ttc tgg cgg ccc aac tac ctc acc acc gct ctg gtc gacgg Asp Phe Trp Arg Pro Asn Tyr Leu Thr Thr Ala Leu Val Asp Gly Gln 190 195 200 gag tcc atc aac gcc tac ctg cag gcc gtc gag ggc gcc tgg aag gac 651 Glu Ser Ile Asn Ala Tyr Leu Gln Ala Val Glu Gly Ala Trp L ys Asp 205 210 215 tac gcg gag cag gac ggc cgg tcg ctg gag gag ttc gcg gcg ttc gtc 699 Tyr Ala Glu Gln Asp Gly Arg Ser Leu Glu Glu Phe Ala Ala Phe Val 220 225 230 tac cac cag ccg ttc acg tac aag gcg cac cgc cac ctg 747 Tyr His Gln Pro Phe Thr Lys Met Ala Tyr Lys Ala His Arg His Leu 235 240 245 ctg aac ttc aac ggc tac gac acc gac aag gac gcc atc gag ggc gcc 795 Leu Asn Phe Asn Gyr Asp Thr Asp Lys Asp Ala Ile Glu Gly Ala 250 255 260 265 ctc ggc cag acg acg gcg tac aac aac gtc atc ggc aac agc tac acc 843 Leu Gly Gln Thr Thr Ala Tyr Asn Asn Val Ile Gly Asn Ser Tyr Thr 270 275 280 gcg tcg gtg tac ctg ggc ctg gcc gcc ctg ctc gac cag gcg gac gac 891 Ala Ser Val Tyr Leu Gly Leu Ala Ala Leu Leu Asp Gln Ala Asp Asp 285 290 295 ctg acg ggc cgt tc atc ggcc cc gc cc gc cc gc cc gc cc gg cc gc cc gc cc gg cc gc cc gg cc gc cc gg cc gc cc gc cc gg cc gg cc gc gc agc gtc 939 Leu Thr Gly Arg Ser Ile Gly Phe Leu Ser Tyr Gly Ser Gly Ser Val 300 305 310 gcc gag ttc ttc tcg ggc acc gtc gtc gcc ggg tac cgc gag cgt ctg 987 Ala Glu Phe Phe Ser Gly Thr Val Val Ala Gly T yr Arg Glu Arg Leu 315 320 325 cgc acc gag gcg aac cag gag gcg atc gcc cgg cgc aag agc gtc gac 1035 Arg Thr Glu Ala Asn Gln Glu Ala Ile Ala Arg Arg Lys Ser Val Asp 330 335 340 345 345 tacgcc acc tac gag ctg cac gag tac acg ctc ccg tcc gac ggc 1083 Tyr Ala Thr Tyr Arg Glu Leu His Glu Tyr Thr Leu Pro Ser Asp Gly 350 355 360 ggc gac cac gcc acc ccg gtg cag acc acc ggc ccc ttc cgg ctg gcc 1131 G His Ala Thr Pro Val Gln Thr Thr Gly Pro Phe Arg Leu Ala 365 370 375 ggg atc aac gac cac aag cgc atc tac gag gcg cgc tag 1170 Gly Ile Asn Asp His Lys Arg Ile Tyr Glu Ala Arg 380 385

<210> 8 <211> 345 <212> PRT <213> Streptomyces <400> 8 Met Gln Lys Arg Gln Arg Glu Leu Ser Ala Leu Thr Leu 1 5 10 Pro Thr Ser Ala Glu Gly Val Ser Glu Ser His Arg Ala Arg Ser Val 15 20 25 Gly Ile Gly Arg Ala His Ala Lys Ala Ile Leu Leu Gly Glu His Ala 30 35 40 45 Val Val Tyr Gly Ala Pro Ala Leu Ala Leu Pro Ile Pro Gln Leu Thr 50 55 60 Val Thr Ala Ser Val Gly Trp Ser Ser Glu Ala Ser Asp Ser Ala Gly 65 70 75 Gly Leu Ser Tyr Thr Met Thr Gly Thr Pro Ser Arg Ala Leu Val Thr 80 85 90 Gln Ala Ser Asp Gly Leu His Arg Leu Thr Ala Glu Phe Met Ala Arg 95 100 105 Met Gly Val Thr Asn Ala Pro His Leu Asp Val Ile Leu Asp Gly Ala 110 115 120 125 Ile Pro His Gly Arg Gly Leu Gly Ser Ser Ala Ala Gly Ser Arg Ala 130 135 140 Ile Ala Leu Ala Leu Ala Asp Leu Phe Gly His Glu Leu Ala Glu His 145 150 155 Thr Ala Tyr Glu Leu Val Gln Thr Ala Glu Asn Met Ala His Gly Arg 160 165 170 Ala Ser Gly Val Asp Ala Met Thr Val Gly Ala Ser Arg Pro Leu Leu 175 180 185 Phe Gln Gln Gly Arg Thr Glu Arg Leu Ala Ile Gly Cys Asp Ser Leu 190 195 200 205 Phe Ile Val Ala Asp Ser Gly Val Pro Gly Ser Thr Lys Glu Ala Val 210 215 220 Glu Met Leu Arg Glu Gly Phe Thr Arg Ser Ala Gly Thr Gln Glu Arg 225 230 235 Phe Val Gly Arg Ala Thr Glu Leu Thr Glu Ala Ala Arg Gln Ala Leu 240 245 250 Ala Asp Gly Arg Pro Glu Glu Leu Gly Ser Gln Leu Thr Tyr Tyr His 255 260 265 Glu Leu Leu His Glu Ala Arg Leu Ser Thr Asp Gly Ile Asp Ala Leu 270 275 280 285 Val Glu Ala Ala Leu Lys Ala Gly Ser Leu Gly Ala Lys Ile Thr Gly 290 295 300 Gly Gly Leu Gly Gly Cys Met Ile Ala Gln Ala Arg Pro Glu Gln Ala 305 310 315 Arg Glu Val Thr Arg Gln Leu His Glu Ala Gly Ala Val Gln Thr Trp 320 325 330 Val Val Pro Leu Lys Gly Leu Asp Asn His Ala Gln 335 340

<210> 9 <211> 350 <212> PRT <213> Streptomyces <400> 9 Met Arg Ser Glu His Pro Thr Thr Thr Val Leu 1 5 10 Gln Ser Arg Glu Gln Gly Ser Ala Ala Gly Ala Thr Ala Val Ala His 15 20 25 Pro Asn Ile Ala Leu Ile Lys Tyr Trp Gly Lys Arg Asp Glu Arg Leu 30 35 40 Ile Leu Pro Cys Thr Thr Ser Leu Ser Met Thr Leu Asp Val Phe Pro 45 50 55 Thr Thr Thr Glu Val Arg Leu Asp Pro Ala Ala Glu His Asp Thr Ala 60 65 70 75 Ala Leu Asn Gly Glu Val Ala Thr Gly Glu Thr Leu Arg Arg Ile Ser 80 85 90 Ala Phe Leu Ser Leu Val Arg Glu Val Ala Gly Ser Asp Gln Arg Ala 95 100 105 Val Val Asp Thr Arg Asn Thr Val Pro Thr Gly Ala Gly Leu Ala Ser 110 115 120 Ser Ala Ser Gly Phe Ala Ala Leu Ala Val Ala Ala Ala Ala Ala Tyr 125 130 135 Gly Leu Glu Leu Asp Asp Arg Gly Leu Ser Arg Leu Ala Arg Arg Gly 140 145 150 155 Ser Gly Ser Ala Ser Arg Ser Ile Phe Gly Gly Phe Ala Val Trp His 160 165 170 Ala Gly Pro Asp Gly Thr Ala Thr Glu Ala Asp Leu Gly Ser Tyr Ala 175 180 185 Glu Pro Val Pro Ala Ala Asp Leu Asp Pro Ala Leu Val Ile Ala Val 190 195 200 Val Asn Ala Gly Pro Lys Pro Val Ser Ser Arg Glu Ala Met Arg Arg 205 210 215 Thr Val Asp Thr Ser Pro Leu Tyr Arg Pro Trp Ala Asp Ser Ser Lys 220 225 230 235 Asp Asp Leu Asp Glu Met Arg Ser Ala Leu Leu Arg Gly Asp Leu Glu 240 245 250 Ala Val Gly Glu Ile Ala Glu Arg Asn Ala Leu Gly Met His Ala Thr 255 260 265 Met Leu Ala Ala Arg Pro Ala Val Arg Tyr Leu Ser Pro Ala Thr Val 270 275 280 Thr Val Leu Asp Ser Val Leu Gln Leu Arg Lys Asp Gly Val Leu Ala 285 290 295 Tyr Ala Thr Met Asp Ala Gly Pro Asn Val Lys Val Leu Cys Arg Arg 300 305 310 315 Ala Asp Ala Glu Arg Val Ala Asp Val Val Arg Ala Ala Ala Ser Gly 320 325 330 Gly Gln Val Leu Val Ala Gly Pro Gly Asp Gly Ala Arg Leu Leu Ser 335 340 345 Glu Gly Ala 350

<210> 10 <211> 374 <212> PRT <213> Streptomyces <400> 10 Met Thr Thr 1 Gly Gln Arg Thr Ile Val Arg His Ala Pro Gly Lys Leu Phe Val Ala 5 10 15 Gly Glu Tyr Ala Val Val Asp Pro Gly Asn Pro Ala Ile Leu Val Ala 20 25 30 35 Val Asp Arg His Ile Ser Val Thr Val Ser Asp Ala Asp Ala Asp Thr 40 45 50 Gly Ala Ala Asp Val Val Ile Ser Ser Asp Leu Gly Pro Gln Ala Val 55 60 65 Gly Trp Arg Trp His Asp Gly Arg Leu Val Val Arg Asp Pro Asp Asp 70 75 80 Gly Gln Gln Ala Arg Ser Ala Leu Ala His Val Val Ser Ala Ile Glu 85 90 95 Thr Val Gly Arg Leu Leu Gly Glu Arg Gly Gln Lys Val Pro Ala Leu 100 105 110 115 Thr Leu Ser Val Ser Ser Arg Leu His Glu Asp Gly Arg Lys Phe Gly 120 125 130 Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val Ala Ala Val Ala 135 140 145 Ala Phe Cys Gly Leu Glu Leu Ser Thr Asp Glu Arg Phe Arg Leu Ala 150 155 160 Met Leu Ala Thr Ala Glu Leu Asp Pro Lys Gly Ser Gly Gly Asp Leu 165 170 175 Ala Ala Ser Thr Trp Gly Gly Trp Ile Ala Tyr Gln Ala Pro Asp Arg 180 185 190 195 Ala Ph e Val Leu Asp Leu Ala Arg Arg Val Gly Val Asp Arg Thr Leu 200 205 210 Lys Ala Pro Trp Pro Gly His Ser Val Arg Arg Leu Pro Ala Pro Lys 215 220 225 Gly Leu Thr Leu Glu Val Gly Trp Thr Gly Glu Pro Ala Ser Thr Ala 230 235 240 Ser Leu Val Ser Asp Leu His Arg Arg Thr Trp Arg Gly Ser Ala Ser 245 250 255 His Gln Arg Phe Val Glu Thr Thr Thr Asp Cys Val Arg Ser Ala Val 260 265 270 275 275 Thr Ala Leu Glu Ser Gly Asp Asp Thr Ser Leu Leu His Glu Ile Arg 280 285 290 Arg Ala Arg Gln Glu Leu Ala Arg Leu Asp Asp Glu Val Gly Leu Gly 295 300 305 Ile Phe Thr Pro Lys Leu Thr Ala Leu Cys Asp Ala Ala Glu Ala Val 310 315 320 Gly Gly Ala Ala Lys Pro Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile 325 330 335 Ala Leu Leu Asp Ala Glu Ala Ser Arg Asp Ile Thr His Val Arg Gln 340 345 350 355 Arg Trp Glu Thr Ala Gly Val Leu Pro Leu Pro Leu Thr Pro Ala Leu 360 365 370 Glu Gly Ile

<210> 11 <211> 363 <212> PRT <213> Streptomyces <400> 11 Met Thr Ser Ala Gln Arg Lys 15 Asp Asp His Val Arg Leu Ala Ile Glu Gln His Asn Ala His Ser Gly 10 15 20 Arg Asn Gln Phe Asp Asp Val Ser Phe Val His His Ala Leu Ala Gly 25 30 35 Ile Asp Arg Pro Asp Val Ser Leu Ala Thr Ser Phe Ala Gly Ile Ser 40 45 50 55 Trp Gln Val Pro Ile Tyr Ile Asn Ala Met Thr Gly Gly Ser Glu Lys 60 65 70 Thr Gly Leu Ile Asn Arg Asp Leu Ala Thr Ala Ala Arg Glu Thr Gly 75 80 85 Val Pro Ile Ala Ser Gly Ser Met Asn Ala Tyr Ile Lys Asp Pro Ser 90 95 100 Cys Ala Asp Thr Phe Arg Val Leu Arg Asp Glu Asn Pro Asn Gly Phe 105 110 115 Val Ile Ala Asn Ile Asn Ala Thr Thr Thr Val Asp Asn Ala Gln Arg 120 125 130 135 Ala Ile Asp Leu Ile Glu Ala Asn Ala Leu Gln Ile His Ile Asn Thr 140 145 150 Ala Gln Glu Thr Pro Met Pro Glu Gly Asp Arg Ser Phe Ala Ser Trp 155 160 165 Val Pro Gln Ile Glu Lys Ile Ala Ala Ala Val Asp Ile Pro Val Ile 170 175 180 Val Lys Glu Val Gly Asn Gly Leu Ser Arg Gln Thr Ile Leu Leu Leu 18 5 190 195 Ala Asp Leu Gly Val Gln Ala Ala Asp Val Ser Gly Arg Gly Gly Thr 200 205 210 215 Asp Phe Ala Arg Ile Glu Asn Gly Arg Arg Glu Leu Gly Asp Tyr Ala 220 225 230 Phe Leu His Gly Trp Gly Gln Ser Thr Ala Ala Cys Leu Leu Asp Ala 235 240 245 Gln Asp Ile Ser Leu Pro Val Leu Ala Ser Gly Gly Val Arg His Pro 250 255 260 Leu Asp Val Val Arg Ala Leu Ala Leu Gly Ala Arg Ala Val Gly Ser 265 270 275 Ser Ala Gly Phe Leu Arg Thr Leu Met Asp Asp Gly Val Asp Ala Leu 280 285 290 295 Ile Thr Lys Leu Thr Thr Trp Leu Asp Gln Leu Ala Ala Leu Gln Thr 300 305 310 Met Leu Gly Ala Arg Thr Pro Ala Asp Leu Thr Arg Cys Asp Val Leu 315 320 325 Leu His Gly Glu Leu Arg Asp Phe Cys Ala Asp Arg Gly Ile Asp Thr 330 335 340 Arg Arg Leu Ala Gln Arg Ser Ser Ser Ile Glu Ala Leu Gln Thr Thr 345 350 355 Gly Ser Thr Arg 360

<210> 12 <211> 353 <212> PRT <213> Streptomyces <400> 12 Met Thr Glu Thr 1 His Ala Ile Ala Gly Val Pro Met Arg Trp Val Gly Pro Leu Arg Ile 5 10 15 20 Ser Gly Asn Val Ala Glu Thr Glu Thr Gln Val Pro Leu Ala Thr Tyr 25 30 35 Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala Lys Val Ser Arg 40 45 50 Leu Thr Glu Lys Gly Ile Val Ala Thr Leu Val Asp Glu Arg Met Thr 55 60 65 Arg Ser Val Ile Val Glu Ala Thr Asp Ala Gln Thr Ala Tyr Met Ala 70 75 80 Ala Gln Thr Ile His Ala Arg Ile Asp Glu Leu Arg Glu Val Val Arg 85 90 95 100 Gly Cys Ser Arg Phe Ala Gln Leu Ile Asn Ile Lys His Glu Ile Asn 105 110 115 Ala Asn Leu Leu Phe Ile Arg Phe Glu Phe Thr Thr Gly Asp Ala Ser 120 125 130 Gly His Asn Met Ala Thr Leu Ala Ser Asp Val Leu Leu Gly His Leu 135 140 145 Leu Glu Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile Ser Gly Asn Tyr 150 155 160 Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile Leu Gly Arg Gly 165 170 175 180 Lys Asn Val Ile Thr Glu Leu Leu Val Pro Arg Asp Val Val Glu Asn 185 190 195 As n Leu His Thr Thr Ala Ala Lys Ile Val Glu Leu Asn Ile Arg Lys 200 205 210 Asn Leu Leu Gly Thr Leu Leu Ala Gly Gly Ile Arg Ser Ala Asn Ala 215 220 225 His Phe Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala Thr Gly Gln Asp 230 235 240 Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Val Met Ala Glu Asp 245 250 255 260 Arg Asp Gly Asp Leu Tyr Phe Ala Cys Thr Leu Pro Asn Leu Ile Val 265 270 275 Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val Glu Thr Asn Leu 280 285 290 Ala Arg Leu Gly Cys Arg Ala Asp Arg Glu Pro Gly Glu Asn Ala Arg 295 300 305 Arg Leu Ala Val Ile Ala Ala Ala Thr Val Val Leu Cys Gly Glu Glu Leu Ser 310 315 320 Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met Arg Ala His Val 325 330 335 340 Gln Leu Glu Arg Asp Asn Lys Thr Ala Lys Val Gly Ala 345 350

<210> 13 <211> 389 <212> PRT <213> Streptomyces <400> 13 Met Ser Ile Ser Ile Gly Ile His Asp 15 Leu Ser Phe Ala Thr Thr Glu Phe Val Leu Pro His Thr Ala Leu Ala 10 15 20 25 Glu Tyr Asn Gly Thr Glu Ile Gly Lys Tyr His Val Gly Ile Gly Gln 30 35 40 Gln Ser Met Ser Val Pro Ala Ala Asp Glu Asp Ile Val Thr Met Ala 45 50 55 Ala Thr Ala Ala Arg Pro Ile Ile Glu Arg Asn Gly Lys Ser Arg Ile 60 65 70 Arg Thr Val Val Phe Ala Thr Glu Ser Ser Ile Asp Gln Ala Lys Ala 75 80 85 Gly Gly Val Tyr Val His Ser Leu Leu Gly Leu Glu Ser Ala Cys Arg 90 95 100 105 Val Val Glu Leu Lys Gln Ala Cys Tyr Gly Ala Thr Ala Ala Leu Gln 110 115 120 Phe Ala Ile Gly Leu Val Arg Arg Asp Pro Ala Gln Gln Val Leu Val 125 130 135 Ile Ala Ser Asp Val Ser Lys Tyr Glu Leu Asp Ser Pro Gly Glu Ala 140 145 150 Thr Gln Gly Ala Ala Ala Val Ala Met Leu Val Gly Ala Asp Pro Ala 155 160 165 Leu Leu Arg Ile Glu Glu Glu Pro Ser Gly Leu Phe Thr Ala Asp Val Met 170 175 180 185 185 Asp Phe Trp Arg Pro Asn Tyr Leu Thr Thr Ala Leu Val As p Gly Gln 190 195 200 Glu Ser Ile Asn Ala Tyr Leu Gln Ala Val Glu Gly Ala Trp Lys Asp 205 210 215 Tyr Ala Glu Gln Asp Gly Arg Ser Leu Glu Glu Phe Ala Ala Phe Val 220 225 230 Tyr His Gln Pro Phe Thr Lys Met Ala Tyr Lys Ala His Arg His Leu 235 240 245 Leu Asn Phe Asn Gly Tyr Asp Thr Asp Lys Asp Ala Ile Glu Gly Ala 250 255 260 265 Leu Gly Gln Thr Thr Ala Tyr Asn Asn Val Ile Gly Asn Ser Tyr Thr 270 275 280 Ala Ser Val Tyr Leu Gly Leu Ala Ala Leu Leu Asp Gln Ala Asp Asp 285 290 295 Leu Thr Gly Arg Ser Ile Gly Phe Leu Ser Tyr Gly Ser Gly Ser Val 300 305 310 Ala Glu Phe Phe Ser Gly Thr Val Val Ala Gly Tyr Arg Glu Arg Leu 315 320 325 Arg Thr Glu Ala Asn Gln Glu Ala Ile Ala Arg Arg Lys Ser Val Asp 330 335 340 345 Tyr Ala Thr Tyr Arg Glu Leu His Glu Tyr Thr Leu Pro Ser Asp Gly 350 355 360 Gly Asp His Ala Thr Pro Val Gln Thr Thr Gly Pro Phe Arg Leu Ala 365 370 375 Gly Ile Asn Asp His Lys Arg Ile Tyr Glu Ala Arg 380 385

[Brief description of the drawings]

FIG. 1 shows the structure of the gene of the present invention, the structure of a deletion mutant, and the results of the presence or absence of growth of each transformant under various culture conditions.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI theme coat ゛ (Reference) C12R 1:19) C12N 15/00 ZNAA (72) Inventor Shunji Takahashi 641 Yagicho, Chuo-ku, Chiba-shi, Chiba Green Heights B-102 (72) Inventor Motoki Takagi 5-18-12 Hongo, Bunkyo-ku, Tokyo Futaki Heights 306 F-term (reference) 4B024 AA01 AA03 AA11 BA07 BA08 BA10 BA80 CA03 CA09 DA06 EA04 FA02 GA11 GA19 GA27 HA13 HA14 4B050 CC01 CC03 DD02 EE01 LL01 LL03 LL05 4B065 AA26X AA50Y AB01 AC14 AC15 AC16 BA02 BA25 BB01 BC03 BC26 BD15 BD16 BD28 CA27 CA28 CA29 CA44 CA46

Claims (11)

[Claims] 1. DNA having any of the following: (1) a nucleotide sequence of SEQ ID NO: 1; (2) one to several nucleotides are deleted, substituted, added and / or inserted in SEQ ID NO: 1 A nucleotide sequence that encodes all enzymes necessary for the function of the mevalonate pathway; or (3) a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions. A nucleotide sequence encoding all enzymes necessary for the function of the mevalonate pathway. 2. The method according to claim 1, wherein the enzymes required for functioning the mevalonate pathway are at least phosphomevalonate kinase, diphosphomevalonate decarboxylase, mevalonate kinase, HMG-CoA reductase and HMG-CoA synthase. DNA. 3. A protein encoded by the DNA according to claim 1 or 2. 4. A DNA having any of the following: (1) a base sequence of SEQ ID NO: 2, a base sequence in which one to several bases are deleted, substituted, added and / or inserted in SEQ ID NO: 2, and a base sequence encoding phosphomevalonate kinase; or A nucleotide sequence capable of hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, wherein the nucleotide sequence encodes phosphomevalonate kinase; (2) the nucleotide sequence of SEQ ID NO: 3; A base sequence in which several bases are deleted, substituted, added and / or inserted, wherein the base sequence encodes diphosphomevalonate decarboxylase;
A base sequence capable of hybridizing under stringent conditions with the base sequence of SEQ ID NO: 4; a base sequence encoding diphosphomevalonate decarboxylase; (3) a base sequence of SEQ ID NO: 4; Is a nucleotide sequence having deletion, substitution, addition and / or insertion of a base, which is capable of hybridizing under stringent conditions to the nucleotide sequence encoding mevalonate kinase or the nucleotide sequence of SEQ ID NO: 4. A nucleotide sequence encoding mevalonate kinase; (4) a nucleotide sequence of SEQ ID NO: 5; or (5) a nucleotide sequence of SEQ ID NO: 7; , A base sequence that has been substituted, added and / or inserted and that codes for HMG-CoA synthase, A nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions of SEQ ID NO: 7, the nucleotide sequence encoding HMG-CoA synthase. 5. A protein encoded by the DNA according to claim 4. 6. A protein having any one 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, which has phosphomevalonate kinase activity; (2) the amino acid sequence of SEQ ID NO: 9, and one to several amino acids in SEQ ID NO: 9 Is an amino acid sequence having a diphosphomevalonate decarboxylase activity or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 9 An amino acid sequence having diphosphomevalonate decarboxylase activity; (3 An amino acid sequence having SEQ ID NO: 10, an amino acid sequence in which one to several amino acids have been deleted, substituted, added and / or inserted in SEQ ID NO: 10, which has a mevalonate kinase activity; (4) an amino acid sequence of SEQ ID NO: 11; or (5) an amino acid sequence of SEQ ID NO: 13, which has 60% or more homology with the amino acid sequence of 13. An amino acid sequence in which one to several amino acids have been deleted, substituted, added and / or inserted in HMG-C
An amino acid sequence having oA synthase activity or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 13, which has HMG-CoA 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 isoprenoid compound in a culture, and an isoprenoid compound from the culture. A method for producing an isoprenoid compound, comprising a step of collecting a compound. 11. The method for producing an isoprenoid compound according to claim 10, wherein the isoprenoid compound is an isoprenoid compound selected from ubiquinone, vitamin K ₂ , or carotenoid.