WO2001057223A1 - Enzyme in non-mevalonate pathway and gene encoding the same - Google Patents

Abstract

An enzyme which catalyzes an unknown reaction step in the non-mevalonate pathway, in particular, the reaction step with the use of 2-phospho-4-(cytidine-5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME2P) as the substrate. Namely, an enzyme protein having a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity and showing the following physicochemical properties is provided. (I) Action: in the presence of Mg2+, forming 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) and cytidine 5'-monophosphate (CMP) from CDP-ME2P serving as the substrate. (1) Molecular weight: about 22 kDa when measured by SDS-PAGE.

Description

 Specification

 TECHNICAL FIELD The present invention relates to non-mevalonate pathway enzymes and genes encoding the same.

 The present invention relates to novel enzymes in the non-mevalonate pathway, genes encoding them, and uses thereof. Background art

 Isoprenide is a generic term for compounds having a basic skeleton of isoprene units having 5 carbon atoms, and is biosynthesized by polymerization of isopentenyl pyrophosphate (IPP). There are a wide variety of isoprenide compounds in nature, many of which are useful to humankind.

 For example, ubiquinone plays an important role in the body as an essential component of the electron transport system, and is used as a drug effective for heart disease.In Europe and the United States, demand for health foods is increasing. ing.

 Biluminmin K is an important bimin which is involved in the blood coagulation system, is used as a hemostatic agent, and has recently been suggested to be involved in bone metabolism, and is expected to be applied to the treatment of osteoporosis. And menaquinone are licensed as medicines.

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

 In addition, compounds based on the isoprene skeleton of 40 carbon atoms, called carotenoids, have an antioxidant effect, and have anti-cancer and immunostimulatory activities such as 力 rotilin, astaxanthin, and cryptoxanthin. Some are expected to have. As described above, isoprenoid compounds contain many useful substances, and if these inexpensive production methods are established, they will have great social and medical benefits.

The production of isoprenide compounds by fermentation has been studied for a long time. Investigations have been made and strain breeding by mutation treatment has been attempted, and further attempts have been made to improve the production volume by genetic engineering techniques. However, its effect is limited to individual compound species, and no effective method is known for isoprenide compounds in general.

 Isopentenyl biphosphoric acid (hereinafter also referred to as IPP), which is the basic skeleton unit of isoprenoid compounds, is used in eukaryotes such as animals and yeasts from acetyl CoA via mevalonic acid. It has been proven to be biosynthesized (mevalonate pathway: see Figure 1A).

 In the mevalonate pathway, 3-hydroxy-3-methylglyuryl-CoA (HM6-CoA) reductase is considered to be the rate-limiting factor (Mol. Biol. Cell, 5, 655 (1994)), and in yeast. Attempts have been made to increase the expression of HMG-CoA reductase and to increase carotenoid productivity (Mizawa et al., Proceedings of the Carotenoid Research Symposium (1997)).

In E. coli, IPP is synthesized through a primary metabolic pathway called the non-mevalonate pathway (see Figure 1B). This non-mevalonic acid pathway will be described. The first step in this pathway is the production of 1-deoxy-D-xylulose 5-phosphate (DXP) by the condensation of pyruvate and glyceraldehyde triphosphate, and the enzyme that catalyzes this reaction is DXP synthesis. It is an enzyme. In the second step, DXP is converted to 2-C-methyl-D-erythritol tetraphosphate (MEP) by DXP reductoisomerase. In the third step, MEP is treated with MEP cytidylyltransferase to give 4- (cytidine 5 '? Diphospho) -1-C-methyl? It is converted to D-erythritol (CD P-ME) (Proc. Natl. Acad. Sci. USA, 96, 11758, (1999), Tetrahedron Lett, in press). More recently, the following enzymatic reaction using CDP-ME as a substrate has been clarified, and the hydroxyl group at the position of CDP-ME has been phosphorylated to give 2-phospho-4- (cytidine 5'-diphospho). ) -2-C-methyl-D-erythritol (CDP—ME2P) was found to be produced (Proc. Natl. Acad. Sci. USA, 97, 1062 (2000), Tetrahedron Lett, in press) PNAS, February 1, 1062-1067, Vol. 97, No. 3, 2000). However, the biosynthetic intermediates after CDP-ME2P, and the enzymes and genes involved in their synthesis remain unclear. Disclosure of the invention

 The present invention catalyzes an unknown reaction step in the non-mevalonate pathway, particularly a reaction step using 2-phospho-4- (cytidine-5, -diphospho) -2-C-methyl-D-erythritol as a substrate. It is an object of the present invention to provide an enzyme capable of producing the enzyme and a gene encoding the enzyme.

 Another object of the present invention is to provide a method for improving the productivity of isoprenide using the above enzyme or gene.

 Another object of the present invention is to provide a method for screening a non-mevalonate pathway inhibitor useful as an antibacterial agent or a herbicide using the above enzyme or gene.

 In order to solve the above problems, the present inventors first isolated a mutant strain deficient in a gene encoding an enzyme that catalyzes an unknown reaction step of the non-mevalonate pathway, and complemented the mutation. To obtain the gene. Next, as a result of analyzing the functions of those genes, the reaction using 2-phospho-4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol (CDP-ME2P) as a substrate was catalyzed. We succeeded in isolating a gene that encodes an enzyme that functions. Furthermore, they have found that by expressing this gene in large amounts in E. coli, the productivity of isoprenoid is improved. The present invention has been completed based on these findings.

 That is, according to the present invention, the following (1) to (15) are provided.

 (1) An enzyme having the following physicochemical properties and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity.

 (I) Action:

 2-phospho-4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol acts on 2-C-methyl-D-erythritol 2,4-cyclodiphosphoric acid and cytidine 5,1-monolith Produces acid (CMP).

(II) Substrate specificity: 2-phospho-4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol is used as a substrate.

 (III) Molecular weight:

 When measured by SDS-PAGE, it shows a molecular weight of about 22 kDa.

 (IV) Reaction conditions:

Mg ²⁺ is required for the reaction catalyzed by this enzyme.

 (2) An enzyme protein having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity having any one of the following amino acid sequences:

 (A) the amino acid sequence of SEQ ID NO: 1;

 (B) an amino acid sequence in which one to several amino acids have been deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1, wherein 2-C-methyl-D-erythritol 2, An amino acid sequence having 4-cyclodiphosphate synthase activity; or

(C) an amino acid sequence having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity Amino acid sequence.

 (3) A DNA encoding the protein of (1) or (2).

 (4) DNA encoding a protein having a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity having any one of the following nucleotide sequences.

 (A) the nucleotide sequence of SEQ ID NO: 2;

 (B) a base sequence in which one to several bases have been deleted, substituted, added and / or inserted in SEQ ID NO: 2, and comprising 2-C-methyl-D-erythritol 2,4-cyclodirin A nucleotide sequence encoding a protein having acid synthase activity; or

 (C) a base sequence capable of hybridizing with the base sequence of SEQ ID NO: 2 under stringent conditions, wherein the protein has 2-C-methyl-D-erythritol 2,4-cyclodylinate synthase activity A nucleotide sequence encoding

 (5) A recombinant vector containing the DNA according to (3) or (4).

(6) A transformant having the recombinant vector according to (5). (7) The transformant according to (6), which is Escherichia coli.

 (8) a step of culturing a transformant produced by transforming a vector containing the DNA according to (3) or (4) into a host to produce an isoprenoid compound in the culture; and A method for producing an isoprenide compound, comprising a step of collecting an isoprenide compound.

(9) The method for producing an isoprenoid compound according to (8), wherein the isoprenoid compound is an isoprenoid compound selected from ubiquinone, vitamin K ₂ , or carotenide.

 (10) A method for screening a non-mevalonate pathway inhibitor, comprising searching for a substance that inhibits a reaction in the non-mevalonate pathway catalyzed by the enzyme according to (1) or (2).

 (11) The screening method according to (10), wherein the non-mevalonate pathway inhibitor is an antibacterial active substance.

 (12) The screening method according to (10), wherein the non-mevalonate pathway inhibitor is a herbicidally active substance.

 (13) An inhibitor of a non-mevalonic acid pathway obtained by the screening method according to (10).

 (14) An antibacterial substance obtained by the screening method according to (11).

(15) A herbicidally active substance obtained by the screening method according to (12). BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 illustrates the mevalonate and non-mevalonate pathways for synthesizing isopentenyl pyrophosphate (II).

 Figure 2 shows that the ygbB gene product catalyzes a reaction using 2-phospho-4- (cytidine 5, -diphospho) -2-C-methyl-D-erythritol (CDP-ME2P) as a substrate. It is a figure showing that it produces methyl-D-erythritol 2,4-cyclodiphosphate (MECDP).

Figure 3 shows the chemical structure of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP). It is a figure showing structure. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, embodiments and a method of implementing the present invention will be described in detail.

 (1) Description of an embodiment of the present invention

 As used herein, the phrase "one to several bases are deleted, substituted, added and / or inserted" means, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 0. , More preferably 1 to 5 arbitrary numbers of bases are 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, it means that any number of 1 to 5 amino acids have been deleted, substituted, added and / or inserted.

As used herein, the phrase "can be hybridized under stringent conditions" means that the DNA is used as a probe, and the colony hybridization method, the Braak hybridization method, or the Southern blot method is used. DNA obtained by using the hybridization method or the like.Specifically, DNA obtained from colonies or plaque-derived DNA or a DNA on which a fragment of the DNA is immobilized, After performing hybridization at 65 ° C in the presence of 0.7 to 1.0 M NaCl, 0.1 to 2 times the SSC solution (The composition of the 1 times concentrated SSC solution is 15 OmM sodium chloride, DNA that can be identified by washing the filter using 65 mM (15 mM sodium citrate) at 65 ° C. Hypride Daisylation is described in Molecular Cloning: A laboratory Manual, 2 ^nD ED., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989. It can be done according to the method.

DNA that can hybridize under stringent conditions include: DNA having a certain degree of homology with the nucleotide sequence of the DNA to be used as the probe may be mentioned.Homology is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more. Above, particularly preferably at least 95%, most preferably at least 98%.

 One embodiment of the present invention relates to an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity. The present invention relates to an enzyme protein having an amino acid sequence. The homology with the amino acid sequence of SEQ ID NO: 1 is not particularly limited as long as it is 60% or more, for example, 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly Preferably it is at least 95%, most preferably at least 98%.

 One aspect of the present invention relates to a novel enzyme protein having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity. As used herein, “2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity” refers to 2-phospho-4- (cytidine 5′-diphospho) -2- substrate as a substrate. It acts on C-methyl-D-erythritol to break the bond between phosphoric acid and phosphoric acid, resulting in 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) and CMP. It broadly means the activity of catalyzing the resulting reaction. The present invention also provides (A) the nucleotide sequence of SEQ ID NO: 2;

 (B) a base sequence in which one to several bases have been deleted, substituted, added and / or inserted in SEQ ID NO: 2, comprising 2-C-methyl-D-erythritol 2,4-cyclodylline A nucleotide sequence encoding a protein having acid synthase activity; or

 (C) a nucleotide sequence capable of hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, wherein the nucleotide sequence is 2-C-methyl-D-erythritol 2,4-cyclodylinic acid synthase activity A nucleotide sequence encoding a protein having

Using DNA having any of the following nucleotide sequences as a DNA encoding a protein having 2-C-methyl-D-erythritol 2,4-cyclophosphoric acid synthase activity: For example, the present invention relates to production of a protein having 2,4-cyclodiphosphate synthase activity using the above DNA. (2) Method for obtaining 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and DNA encoding the same

 (2-A) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and cloning of DNA encoding it

 The present inventors have constructed a recombinant DNA encoding an enzyme of the mevalonate pathway and a recombinant DNA obtained by mutating the recombinant DNA. One such recombinant DNA, pUMV19AS, is a plasmid in which DNA encoding mevalonate kinase, phosphomevalonate kinase, and phosphomevalonate decarboxylase has been introduced into an E. coli vector, PUC118. 1 1—3 4 8 3 7 5 See the detailed description: Japanese Patent Application No. 11-34 837 75, the entire contents of which are incorporated herein by reference. Escherichia coli transformed with a plasmid containing DNA encoding mevalonate kinase, phosphomevalonate kinase, and phosphomevalonate decarboxylase is able to biosynthesize IPP via the mevalonate pathway in the presence of mevalonate.

 Next, an E. coli strain having a mevalonate pathway introduced by such transformation

(This Escherichia coli also originally has a non-mevalonate pathway) to obtain a mutant strain deficient in a non-mevalonate pathway enzyme. That is, Escherichia coli having both the mevalonate pathway and the non-mevalonate pathway are treated with a mutagen, cultured on an agar plate, and the grown colonies are grown on an LB agar plate and LB agar containing IPTG and mevalonic acid. Replicate to media plate. After selecting those that show the requirement of IPTG and mevalonic acid, strains that cannot grow on LB agar medium containing methylerythritol (ME) can be selected as the mutant strain of interest. By using this Escherichia coli non-mevalonate pathway-deficient mutant to obtain a gene that complements the mutant as described below, a gene encoding the enzyme of the present invention can be isolated.

First, the chromosomal DNA of E. coli is treated with an appropriate restriction enzyme, and the obtained DNA fragment is obtained. Is ligated, and a DNA fragment having a suitable size (for example, 1 to 3 kb) is ligated into a vector which has been treated with a restriction enzyme to prepare a chromosome genome library of Escherichia coli. Using this chromosomal library, Escherichia coli non-mevalonate pathway-deficient mutants were transformed by a conventional method, and the resulting transformants were spread on an agar medium, cultured, and plasmids were extracted from the obtained colonies. By determining the nucleotide sequence of the plasmid, DNA (ygbB gene) (also referred to as the DNA of the present invention) encoding the enzyme of the present invention can be isolated.

 The DNA (ygbB gene) of the present invention has been isolated by the method described above. Given the nucleotide sequence of SEQ ID NO: 2 in the present specification, those skilled in the art can appropriately design primers for PCR and perform PCR using Escherichia coli chromosomal DNA as a DNA (ygbB gene) can be isolated. Specifically, the ygbB gene is amplified by performing PCR using the chromosomal DNA of E. coli W3110 strain as type I and primers having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 and Taq DNA polymerase. be able to.

 The DNA of the present invention can be cloned into a suitable vector that can be amplified in a suitable host (eg, E. coli). Cloning is carried out in a conventional manner, for example, as described in Molecular Protocols, Second Edition, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter, `` Current Protocols in Molecular Biology ''). '' Biology), DNA Clonin 1: Method described in CoreTechniques, A Practical Approach, Second Edition, Oxford University Press (1995), or a commercially available kit, for example, Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning t This can be performed by using Life Techno Kuchijizu) or ZAP-cDNA Synthesis Kit (Stratagene).

As the cloning vector, any phage vector, plasmid vector, or the like may be used as long as it can replicate autonomously in the host. Escherichia coli expression vector may be used as the cloning vector. Specifically, ZAP Express Strategies, 5, 58 (1992)], pBluescrlpt II SK (+) CNuclelc Acids Research, 17, 9494 (1989)], Lambda ZAP11 (Stratagene), gtlO, Agtll CDNA Cloning, A Practical Approach, 1, 49 (1985)], person TriplEx (manufactured by Clonetech), person ExCell (manufactured by Pharmacia), PT7T318U (manufactured by Pharmacia), pcD2 CMol. Cen. Biol., 3, 280 (1983)], pM218 (manufactured by Wako Pure Chemical Industries), pUC118 (manufactured by Takara Shuzo), pEG400 CJ.Bac, 172, 2392 (1990)], pQE-30 (manufactured by QIAGEN), etc. Can be.

 From the resulting transformant, a plasmid containing the target DNA can be prepared by a conventional method, for example, Molecular Cloning, 2nd edition, Current 'Protocols', 'Molecularity, Bio-mouth, DNA Clonin'. 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), etc.

An example of the amino acid sequence of the enzyme of the present invention is shown in SEQ ID NO: 1, and an example of the nucleotide sequence of the DNA of the present invention encoding the enzyme is shown in SEQ ID NO: 2. However, these are merely examples, and mutant enzymes and mutant DNAs having mutations in these sequences are also included in the present invention as long as they retain 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity. Included in the range. Such mutant enzymes and mutant DNAs can also be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, a mutated DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 2 and introducing a mutation into these DNAs. For example, the method can be carried out using a method in which DNA having the nucleotide sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like. Site-directed mutagenesis, one of the genetic techniques, is useful because it allows the introduction of specific mutations at specific positions, and is useful in Molecular Cloning, 2nd edition, Current-Protocol. Izuru'in 'Molecular' biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Pro Natl. Acad. Sci. USA, 79, 6409, 1982, Pro Natl. Acad. Sci. USA, 82, 488, 1985, Gene, 34, 315, 1985, Gene, 102, 67, 1991, etc. Can be. (2-B) Preparation of transformant having DNA encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and expression of the above enzyme protein

 In order to express the DNA obtained as described above in a host cell, first, the desired DNA fragment is digested with a restriction enzyme or a DNA degrading enzyme to a suitable length containing the gene. After the DNA fragment is inserted into the expression vector, the DNA fragment is inserted downstream of the promoter in the expression vector, and then the expression vector into which the DNA has been inserted is introduced into a host cell suitable for the expression vector.

 Any host cell that can express the gene of interest can be used. For example, bacteria belonging to the genera Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc .; And yeasts, animal cells, insect cells and the like belonging to the genus Schiziomyces and the like.

 As the expression vector, a vector which is capable of autonomous replication in the host cell or capable of integration into a chromosome and which contains a promoter at a position capable of transcribing the desired DNA is used.

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

Examples of the expression vector include pBTrP2, pBTac and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-Promega), pQE-8 (QIAGEN), pQE-30 (QIAGEN), pKYPIO (58-110600), pKYP200 [Agrc. Biol. Chem., 48, 669 (1984)], PLSA1 [Agrc, Biol. Chem. , 53, 277 (1989)), pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescrlptl l SK +, pBluescriptl l SK (-) (Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191) 39094, US5160735), pSupex pUBllO pTP5, pC194, pl) C18 (Gene, 33, 103 (1985)), pUC19 (Gene, 33, 103 (1985)), pSTV28 (Takara Shuzo), pSTV29 (Manufactured by Takara Shuzo), pUC118 (manufactured by Takara Shuzo), pPAl (JP-A-63-233798), PEG400 [J. Bacteriol., 172, 2392 (1990)], pQE-30 (manufactured by QIAGEN) and the like are exemplified. be able to.

Any promoter may be used as long as it can be expressed in the host cell. For example, trp promoter evening one (P trp), lac promoter (P lac), P _L promoter - evening one, promoters from P _R promoter evening one, P _SE promoter Isseki one such as Escherichia coli, phage and the like, SP01 Promo One, SP02 Promoter, penP Promo One, etc. It is also possible to use promoters designed and modified artificially, such as Promoter (Ptrp x 2), Tac Promoter, Letl Promoter, and lacT7 promoter in which two P trps are connected in series. .

 The ribosome binding sequence may be any as long as it can be expressed in the host cell, but the distance between the Shine-Dalgamo sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). It is preferable to use a purified plasmid. Although a transcription termination sequence is not necessarily required for expression of a desired DNA, it is desirable to arrange a transcription termination sequence immediately below a structural gene.

Host cells include Escherichia, Corynebacterium, Brevibacterium, Bacillus, Microbacterium, Serratia, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter j¾, Azobacter, Chromatium, win微生物, microorganisms belonging to the genera Methyl bacterium, Phormidium, Rhodobacter, Rhodopseudomonas, Rhodospirilium, Scenedesmun, Streptomyces, Synnecoccus, Zymomonas, etc., preferably Escherichia, Corynebacterium Genus, Brevibacterium, Bacil lus, Pseudomonas, Agrobacterium, Alicyclobacil lus, Anabaena, Anacystis Genus, Arthrobacter, Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas, Rhodospiril lum 厲, Scenedesmun, Streptomyces exhibition, Syrmecoccus, Zymomonas .

Specific examples of the microorganism include, for example, Escherichia coli XL1-Blue ゝ Escherichia col i XL2-Blue, Escherichia col i DH1, Escherichia col i DH5, Escherichia coli MClOOOs Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, coli HB10 Escherichia col i No49, Escherichia col i W3110, Escherichia coli NY49, Escherichia col i MP347, Escherichia coli 522, Bacil lus subti lis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes N Brevibacterium immariophi lum ATCC14068, Brevibacterium saccharolyticum ATCC14066 S Brevibacterium f lavum ATCC14067 S Brevibacterium lactofermentum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14297, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphi lum ATCC15354, Serratia f icaria _N Serratia fonticola, Serratia sicoma ceres opera spermia ceractia serocerium spercier serocerium spercier serocerium spercier serocerium serocerium sera ceres ceremonial serocerium sera ceres gersacti s ercerium serocerium sera ceres ceres gersa cer a. ogenes, Agrobacterium rubi, Anabaena cyl indrica, Anabaenadol iolum _s Anabaena f losaquae ^ Arthrobacter aurescens _s Arthrobacter citreus, Arthrobacter globformis, Arthrobacter hydrocarboglutamicus Arthrobacter mysorens, Arthrobacter nicotianae, Arthrobacter nicotianae, Arthrobacter nicotianae, Arthrobacter paraff inhr, protohrs, Arthrobacter nicotianae, Arthrobacter paraffhrhr Chromatium buderi, Chromatium tepidum, Chromatium vinosum , Chromatium warmingi i Chromatium f luviatile s Erwinia uredovora, Erwinia carotovora, Erwinia ananas, Erwnia herbicola Erwinia punctata N Erwinia terreusヽMethylobacterium rhodesianum, Methylobacterium extorquens, Phormidium sp . ATCC29409, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodopseudomonas blastica, Rhodopseudomonas marina, Rhodopseudomonas palustris, Rhodospiri llum rubrum, Rhodospiri llum salexigens Rhodospirillum sal inarum Streptomyces ambofaciens N Streptomyces aureofaciens, Streptomyces aureus, Streptomyces fimgicidicus, Streptomyces griseochromogenes Streptomyces griseus, Streptomyces l ividans, Streptomyces olivogriseus, Streptomyces rameus, Streptomyces tanashiensis s Streptomyces vinaceus Zymomonas mobil is and the like.

 Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells. For example, a method using calcium ions! Acad. SCI. 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 YEpl3 (ATCC37115), YEp24 (ATCC37051), Ycp50 (ATCC37419), pHS19, and pHS15.

 Any promoter can be used as long as it can be expressed in yeast, for example, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gall promoter, Promoters such as gallO promoter, heat shock protein Promoter, MF HI Promoter, and CUP1 Promoter can be listed.

Examples of host cells include Saccharomyces cerevisae, Schizosaccharomyces pombe _N Kluyveromyces lactis, Trichosporon pullulans schwans, cho uv ).

As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, an electroporation method (Methods. Enzymol, 194, 182 (1990)), spheroblast method [Proc. Natl. Acad. Sci. USA, 75, 1929 (I)], lithium acetate method [J. Bacteriol., 153, 163 (1983)], or Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). When animal cells are used as host cells, expression vectors include, for example, pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 (Japanese Unexamined Patent Publication No. 3-22979; Cytotechnology, 3, 133, (1990)), pAS3- 3 (Japanese Patent Laid-Open No. 2-227075), p-band [; Nature, 329, 840, (1987)], pcDNAI / AmPUnvitrogen, pREP4 (Invitrogen), pAGE103 [J. Blochem., 101, 1307 (1987) )], PAGE210 and the like.

 Any promoter can be used as long as it can be expressed in animal cells. For example, the promoter of the IE (i ediate early) gene of cytomegalovirus (human CMV) and the promoter of SV40 can be used. Early Promo One Night, Retrovirus Promo One-Meta Mouth Choonein Promo One One, Heat Shock Promo One One One, SR Hypro One-One Night, etc. In addition, the human CMV IE gene enhancer may be used together with the promoter.

 Examples of the host cell include Namalba cell, HBT5637 (JP-A-63-299), C0S1 cell, C0S7 cell, CH0 cell and the like.

 As a method for introducing the recombinant vector into animal cells, any method that can introduce DNA into animal cells can be used.For example, the electoral poration method CCytotechnology, 3, 133 (1990)], calcium phosphate Natl. Acad. Sci., USA, 84, 7413 (1987)], virology, 52, 456 (1973), and the like. The transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.

When insect cells are used as hosts, for example, baculovirus 'Expression Vectors', 'A' Laboratory, Baculovirus Expression Vectors (A Laboratory Manual), current 'Protocols''Molekiura-by the method described in Biology, Bio / Technology, 6, 47 (1988), etc. The protein can be expressed.

 That is, the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to insect cells to express the protein. Can be done.

Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacI II (all manufactured by Invitrogen) and the like. The baculovirus, e.g., burglar Gaka insects infection the virus is a § © Togurafa californica force-Nuclear poly to Doroshisu virus (Autographa californica nuclear polyhedrosis virus), etc. The Yore, ₀ insect cell capable Rukoto Examples include Spodoptera frugiperda ovarian cells Sf9, Sf21 (baculovirus 'expression' vectors, a 'laboratory 1' manual, doubling 'h-freeman' and Company.H. Freeman and Company), New York (New York), (1992)], and High5 (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.

 Examples of a method for co-introducing the above-mentioned recombinant gene introduction vector and the above baculovirus into insect cells to prepare a recombinant virus include, for example, a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

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

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

A transformant having the recombinant DNA incorporating the above DNA is cultured in a medium, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase is produced and accumulated in the culture. By collecting the enzyme protein from the culture, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase can be isolated. The method for culturing the transformant carrying the DNA of the present invention in a medium can be performed according to a usual method used for culturing a host.

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

 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, propionic acid And organic acids such as ethanol and alcohols such as propanol.

 Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn starch, etc. Plyka, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.

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

 The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably up to 40 ° C, and the culture time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.

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

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an Indian user may be added to the medium, if necessary. For example, a microorganism transformed with an expression vector using the lac promoter When culturing a microorganism that has been transformed with an expression vector using the trp promoter, for example, indole acrylic acid (IM), etc., when culturing a microorganism transformed with isopropropyl-1 /?-D-thiogalactobyranoside (IPTG). It may be added.

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

Culture is carried out usually pH6~8, 30~40 ° C, 5% C0 2 present 1 to 7 days under conditions such as lower. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

 As a medium for culturing the transformant obtained by using insect cells as host cells, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400, ExCell405 [all manufactured by JRH Biosciences], Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used. Cultivation is usually carried out under conditions of pH 6 to 7, 25 to 30 ° C, etc. for 1 to 5 days.

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

 To isolate and purify 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (the protein of the present invention) from the culture of the transformant of the present invention, the usual enzyme Isolation and purification methods may be used.

For example, when the protein of the present invention is expressed in a lysed state in cells, the cells are recovered by centrifugation after cell culture, suspended in an aqueous buffer, and then sonicated with a homogenizer, french press, and Mentongaulin homogen. The cells are disrupted using a Nyzer, Dynomill, etc. 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, etc. Salt method, precipitation method using organic solvent, anion exchange chromatography using resin such as getylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), S-Sepharose FF (manufactured by Pharmacia), etc. Chromatography using cation-exchange resin, hydrophobic chromatography using resin such as Ni-NTA agarose, butyl sepharose, phenylsepharose, gel filtration using molecular sieve, A purified sample can be obtained by using techniques such as electrophoresis, such as the method of mouth-to-mouth chromatography, chromatofocusing, and isoelectric focusing, alone or in combination.

 When the protein is expressed in an insoluble form in the cells, the cells are similarly recovered, crushed, and the protein is recovered by a usual method from the precipitate fraction obtained by centrifugation. Thereafter, the insoluble form of the protein is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution to a solution containing no protein denaturing agent or a diluting concentration of the protein denaturing agent such that the protein is not denatured, the protein is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method.

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

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

 (3) Production of isoprenide compounds using 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase or DNA encoding it

The transformant obtained in the above (2) is cultured according to the method described in the above (2), an isoprenoid compound is produced and accumulated in the culture, and the isoprenide compound is collected from the culture. By this culture, isoprenoid compounds such as ubiquinone, vitamin K ₂ , and carotenoid can be produced. Specific examples include 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, microorganisms belonging to the genus Arthrobacter Production of bismuth 1 < _{2 using} E. coli as a transformant, production of astaxanthin using a microorganism belonging to the genus Agrobacterium as a transformant, use of a microorganism belonging to the genus Erwinia as a transformant, copen, ^ Ichirotin, Production of zeaxanthin can be mentioned.

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

 (4) A method for screening non-mevalonate pathway inhibitors using 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase.

 (4-A) Screening method for inhibitors of non-mevalonate pathway

 The present invention relates to a non-mevalonate pathway, which comprises searching for a substance that inhibits a reaction in the non-mevalonate pathway catalyzed by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. It also relates to methods for screening for inhibitors.

The above “reaction in the non-mevalonate pathway catalyzed by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase” refers to the substrate 2-phospho-4- (cytidine 5′- This is a reaction that acts on diphospho) -2-C-methyl-D-erythritol to produce 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) and CMP. This reaction Can inhibit the non-mevalonate pathway.

 Specific examples of “substances that inhibit a reaction in the non-mevalonate pathway catalyzed by 2-C_methyl-D-erythritol 2,4-cyclodiphosphate synthase” include 2-C-methyl- Substances that inhibit D-erythritol 2,4-cyclodiphosphate synthase activity.

 A substance that inhibits the reaction in the non-mevalonate pathway catalyzed by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase can inhibit the growth of microorganisms and plants having the non-mevalonate pathway. Inhibit.

 Since the non-mevalonate pathway is present in microorganisms and plants, but not in animals or humans, an inhibitor of the non-mevalonate pathway that does not affect humans or animals is obtained by the screening method described above. be able to. Such substances are useful as antibacterial agents and herbicides.

 (4-B) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase assay

 The measurement of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity can be carried out according to the usual enzyme activity measurement method.

 That is, the pH of the buffer used in the reaction solution for measuring the activity may be within the pH range that does not inhibit the target enzyme activity, and is preferably in the range including the optimum pH.

 For example, in the case of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, pH 5 to: 10, preferably? ~ 9.

 Any buffer can be used as long as it does not inhibit the enzyme activity and can achieve the above pH. As the buffer, a Tris-HCl buffer, a phosphate buffer, a borate buffer, a HEPS buffer, a MOPS buffer, a bicarbonate buffer, and the like can be used, and a Tris-HCl buffer is preferred.

 The buffer may be used at any concentration as long as the enzyme activity is not inhibited, but is preferably 1 mM to 1 M.

Also, Mg ^{2 +} is added to the reaction solution. These metal ions are added as metal salts And can be added as chlorides, sulfates, carbonates, phosphates and the like. The concentration of the metal ion may be any concentration as long as it does not inhibit the reaction, but is generally 0.01 mM to 100 mM and 0.11111 ^ to 101111 ^.

 Add 2-phospho-4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol as a substrate for the enzyme to the reaction mixture. The substrate may be used at any concentration as long as it does not interfere with the reaction, but is preferably 0.01 mM to 0.2M. The concentration of the enzyme used in the reaction is not particularly limited, but the reaction is usually carried out at a concentration in the range of 0.01 mg / ml to 10 Omg / ml. The enzyme used does not necessarily have to be purified to a single level, and it is sufficient that the enzyme has a purity that does not inhibit the reaction. Cell extracts containing 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity or cells having the enzyme activity can also be used.

 The reaction temperature may be within a temperature range that does not inhibit the activity of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, and is preferably a temperature range including the optimum temperature. The reaction temperature is usually 10 ° C to 60 ° C, preferably 30 to 40 ° C.

 The activity can be detected by a method capable of measuring the substrate or the reaction product by reducing the substrate or increasing the reaction product accompanying the reaction.

 The method includes, for example, a method of separating and quantifying a target substance by high performance liquid chromatography (HPLC), if necessary.

 (4-C) 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase catalyzes substances that inhibit the reaction in the non-mevalonate pathway

 To screen for a substance that inhibits 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity, a test substance is added to the enzyme activity measurement system described in (4-B) above. The enzymatic reaction is performed in the same manner as above, and a substance that suppresses the amount of reduction of the substrate or a substance that suppresses the production of the reaction product than when no test substance is added is screened.

Screening methods include the method of temporarily tracking the amount of decrease in the substrate or the increase in the amount of the reaction product, the amount of decrease in the substrate after a certain period of reaction, or the reaction. Methods for measuring the amount of increase in the product and the like can be mentioned.

 In a method that tracks the amount of decrease in substrate or increase in reaction product over time, measure the amount of decrease in substrate or increase in reaction product at intervals of about 15 seconds to 20 minutes during the reaction. It is preferable to measure at intervals of 1 to 3 minutes.

 In a method for measuring the amount of decrease in the substrate or the amount of increase in the reaction product after reacting for a certain period of time, the reaction time is preferably 15 minutes to 1 day, more preferably 30 minutes to 2 hours.

 The present invention is specifically illustrated by the following examples, but the present invention is not limited by these examples. Example

Example 1: Construction of Escherichia coli mutant having mevalonate pathway

 pUMV19AS is a plasmid in which DNA encoding mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, HMG-CoA reductase, etc. has been introduced into an Escherichia coli vector, PUC118 (Japanese Patent Application No. Hei 10-284,197). 1 1—3 4 8 3 7 5). E. coli JM109 strain transformed with this plasmid will be able to biosynthesize IPP via the mevalonate pathway in the presence of IPTG and mevalonic acid.

 PUMV19AS cannot be introduced into the wild-type E. coli W3110 strain due to too many copies. This time, in order to obtain the target mutant from E. coli W3110, only the vector part of pUMV19AS was replaced with the vector PTTQ18, which can be introduced into E. coli W3110, by the following procedure.

pUMV19AS was digested with two restriction enzymes EcoRI (manufactured by Takara Shuzo) and Hindlll (manufactured by Takara Shuzo) and incorporated into plasmid pTTQ18 (manufactured by Amersham) treated with the same two restriction enzymes. It was named. The E. coli W3110 strain was transformed with pTTQMV19ZS according to a standard method. This E. coli W3110 (pTTQMV19AS) strain is expected to induce gene expression with IPTG and to be able to biosynthesize IPP via the mevalonate pathway by adding mevalonic acid. This was demonstrated by the following experiment. Fosdomycin specifically inhibits the non-mevalonate pathway (Figure IB, Tetrahedron Lett. 39: 7913, 1998.). Therefore, E. coli W3110, which has only the non-mevalonic acid pathway, cannot grow in a medium containing 20 g / ml of fosmidomycin because it cannot synthesize IPP essential for growth. In fact, the E. coli W3110 strain was cultured for 14 hours at 37 ° C in LB medium containing 20 µg / ml fosmidomycin, but no growth was confirmed (Table 1). On the other hand, the above-mentioned transformed E. coli strain, E. coli W3110 (PTTQMV19AS) strain, which we have produced, can be used only in the presence of 0.1 mM IPTG and 0.02 ° mevalonic acid in the presence of 20 μg / ml fosmidomycin. It was viable (Table 1). In addition, growth was displayed in the 660 nm turbidity (0. D. _{6 6 0).} Table 1. E. coli W3110 strain in the presence of fosmidomycin

E. coli W3110 (pTTQMV19AS) stock growth of (0. D. _{6 6 0)}

 Medium components

 LB medium only LB medium +0.1 mM IPTG + 0.02¾; mevalonic acid

W3110 shares 0 0

 W3110 (pTTQMV19AS) strain 0 350 The results in Table 1 demonstrate that in this transformed E. coli strain, the mevalonate pathway is activated, and that IPP is also biosynthesized in the mevalonate pathway (Figure 1A). . Therefore, the E. coli W3110 (pTTQMV19AS) strain was found to be E. coli expressing the mevalonate pathway by adding IPTG and mevalonic acid. In other words, it is clear that E. coli W3110 (pTTQMV19AS) can biosynthesize IPP not only in the non-mevalonate pathway but also in the mevalonate pathway when 0.1 mM IPTG and 0.02% mevalonic acid are added. Was. Example 2: Obtaining a gene that complements a non-mevalonate pathway-deficient mutant

(1) Acquisition of Escherichia coli non-mevalonate pathway defective mutant The E. coli W3110 (pTTQMV19AS) strain was inoculated into an LB liquid medium containing 50 g / ml of ampicillin, and cultured at 37 ° C until the exponential growth phase.

After the culture, cells were obtained from the resulting culture by centrifugation. The fungus body was washed with 0.05 M Tris / maleate buffer (pH 7.0), cell concentration was suspended in the same buffer cormorants by to 10 ⁹ cells / ml. To the suspension was added N-methyl-N'-nitro-N-nitrosguanidine (hereinafter abbreviated as NTG; NTG is a mutagen) to a final concentration of 2 mg / ml, and the mixture was added at room temperature. Mutation treatment was performed by holding for 40 minutes.

 The obtained mutant-treated cells were spread on an LB agar plate containing O.lmM IPTG and 0.02% mevalonic acid, and cultured at 37 ° C.

 The colonies that grew on the agar medium were replicated on an LB agar plate and an LB agar plate containing 0.1 mM IPTG and mevalonic acid 0.02, which showed the requirement of IPTG and mevalonic acid, that is, O. After selecting a strain that can grow on LB agar medium containing lmM IPTG and 0.02% mevalonic acid but cannot grow on LB agar medium, it is also intended to be a strain that cannot grow on LB agar medium containing methyl erythritol (ME) Mutant, ie, the third step of the non-mevalonate pathway (ie, MEP is 4 4 (cytidine 5 ′? Diphospho) -12-C-methyl? D-erythritol (MEP) by MEP cytidylyltransferase. It was selected as a mutant strain of a gene encoding an enzyme that catalyzes the subsequent reactions).

This screening shows that the first step of the non-mevalonate pathway (ie, the condensation of pyruvate with glyceraldehyde triphosphate to form 1-doxy-D-xylulose 5-phosphate (DXP), The enzyme that catalyzes is DXP synthase) and the second stage (ie, the reaction by which DXP is converted to 2-C-methyl-D-erythritol tetraphosphate (MEP) by DXP reductoisomerase). Mutants of the gene to be coded were eliminated, and the efficiency of obtaining the desired mutant was improved (Fig. 1B). By selecting a strain that cannot grow on an LB agar medium containing CDP-ME, a mutant strain of the gene encoding the enzyme in the third step can be eliminated. Not used when selecting mutant strains Did not. Therefore, in the above-mentioned method for obtaining a non-mevalonate pathway-deficient mutant, a mutant having a mutation in the gene encoding the enzyme of the third stage of the non-mevalonate pathway may be selected.

 By this selection, four types of Escherichia coli non-mevalonate pathway-deficient mutants were obtained.

 (2) Obtaining a gene that complements a non-mevalonate pathway-deficient mutant of Escherichia coli

 The E. coli W3110 strain was inoculated into an LB liquid medium, cultured at 37 ° C until the logarithmic growth phase, and then centrifuged to collect the cells. Chromosomal DNA was isolated and purified from the obtained cells according to a conventional method. 200 µg of the chromosomal DNA was partially digested with a restriction enzyme Sau3AI (Takara Shuzo), and the resulting digested DNA fragment was subjected to sucrose density gradient ultracentrifugation (2600 rpm, 20 ° C, 20 hours). , Size fractionation.

 The DNA fragment having a size of 1 to 3 kb obtained by the fractionation was ligated to a vector PMW118 (manufactured by Futatsu Gene) digested with a restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.). A chromosomal genome library of E. coli W3110 strain was prepared.

 Using the chromosome library prepared above, the four types of Escherichia coli non-mevalonate pathway-deficient mutants isolated in (1) above were transformed according to a conventional method. The resulting transformant was spread on an LB agar medium containing 50 zg / ml of ampicillin, and cultured at 37 ° C at room temperature. In the culture, a plasmid was extracted from a colony of a transformant obtained from the mutant strain, and the nucleotide sequence of the plasmid was determined according to a standard method (Molecular Cloning: A laboratory Manual, 2nd ED.). In the determination of the nucleotide sequence, a thermosequenase cycle sequencing kit (manufactured by Amersham Pharmacia Biotech) was used as a sequence reaction reagent, and a DNA sequencer model 4000L (manufactured by Licor) was used as a sequence analyzer.

The plasmid retained by the transformant obtained from one of the above four mutants was named PMEW243. The DNA fragment inserted into PMEW243 was found to contain the full length of the ygbB gene of unknown function based on the chromosomal nucleotide sequence information of Escherichia coli based on the database of the National Institute of Genetics. The nucleotide sequence of the ygbB gene is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence encoded by the ygbB gene is shown in SEQ ID NO: 1.

 In order to determine whether the protein encoded by the ygbB gene is an enzyme that uses CDP-E2P as a substrate, PCR was performed using a recombinant vector that sufficiently expressed each of these genes. Science, 230, 1350 (1985)).

 Sense primer (GGGGATCCCGMTTGGACACGGTTTTGACG: SEQ ID NO: 3) and antisense primer (GGGGATCCTTTTGTTGCCTTMTGAGTAGC: SEQ ID NO: 4) were purchased (Amersham Pharmacia Biotech). BamHI restriction enzyme sites were added to the 5 and 5 ends of the sense primer and the antisense primer, respectively.

 Using the chromosome DNA of E. coli W3110 strain as type III, the primer and Taq DNA polymerase (manufactured by Boehriger) were used to perform PCR with a DNA Thermal Cycler (manufactured by MJ Reserch) to amplify the ygbB gene. The PCR was performed under the conditions that the reaction process consisted of 1 cycle and 25 cycles of a reaction process consisting of 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 2 minutes, followed by a reaction at 72 ° C for 10 minutes. .

 After the amplified DNA fragment and pUC118 (Takara Shuzo) were digested with the restriction enzyme BamHI, each DNA fragment was purified by agarose gel electrophoresis.

 After mixing these purified fragments, ethanol precipitation was carried out, and the obtained DNA precipitate was dissolved in 51 distilled water, and a recombinant DNA was obtained by performing a Ligation reaction.

After confirming that the recombinant DNA is the ygbB gene by determining the DNA sequence, plasmid was extracted from the recombinant, digested with the restriction enzyme BajnHI, and then subjected to agarose gel electrophoresis and treated with BamHI. _c obtained DNA fragment containing ygbB gene

After digesting PQE30 (manufactured by QIAGEN) with the restriction enzyme {εΒη}, agarose gel electrophoresis was performed to obtain a BamHI-treated pQE30 fragment.

After mixing the BamHI-treated ygbB gene-containing DNA fragment obtained above with the BamHI-digested pQE30 fragment, ethanol precipitation is performed, and the resulting DNA precipitate is subjected to a 51 Recombinant DNA was obtained by dissolving in distilled water and performing a Reigeshon reaction and named pQEYGBB. Example 3: Activity measurement of ygbB gene product and reaction product

 (1) Purification of ygbB gene product

 The pQEYGBB constructed in Example 2 was introduced into an E. coli Ml 5 strain (manufactured by QIAGEN) having pREP4 by a conventional method, and was resistant to ampicillin 200 μg / ml and kanamycin 25 μg / ml. col i M15 (pREP4, pQEYGBB) strain was obtained.

 E. col i M15 (pREP4, pQEYGBB) was cultured at 37 ° C in 1 ^ liquid medium 100 1111 containing 200 μg / ml ampicillin and kanamycin 25 at the time when the turbidity at 660 nm reached 0.6. IPTG was added to a final concentration of 0.1 mM. After further culturing at 37 ° C for 5 hours, the culture supernatant was removed by centrifugation (3000 rpm, 10 minutes). The cells were suspended in 6 ml of 100 mM Tris-HCl buffer (pH 8.0), and crushed with an ultrasonic crusher (BRANSON) while cooling on ice. The obtained cell lysate was centrifuged (10000 rpm, 20 minutes, 4 ° C), and the supernatant was recovered. The cell extract centrifuged supernatant is passed through a Ni-NTA agaro-resin column (manufactured by QIAGEN), and 20 ml of a washing buffer [100 mM Tris-HCl

(pH 8.0), 50 mM imidazole, 0.5% Tween 20]. Then, 10 mM of elution buffer [100 mM Tris-HCl (pH 8.0), 200 mM imidazole] was passed through the column, and the eluate was fractionated by 1 ml.

 The protein amount of each fraction was measured using a protein amount quantification kit (manufactured by BioRad), and the protein-containing fraction was used as a purified protein fraction.

 (2) Preparation of substrate CDP-ME2P

 The reaction substrate, CDP-ME2P, was prepared according to the literature (Proc. Natl. Acad. Sci. USA, 97, 1062 (2000), Tetrahedron Lett, in press).

 (3) Measurement of enzyme activity of ygbB gene product

To 50 ml of a reaction solution containing 10 mM Tris-HCl (pH 8.0), 1 mM MgCl ₂ and 2 m of the above ygbB gene product, add 50 mg of the CDP-ME2P synthesized as above, and add Time ink I did it. The amount of CDP-ME2P during the incubation was monitored by measuring the absorption at 280 nm by HPLC (manufactured by JASCO Corporation) using an Asahipak GS-320 HQ column (manufactured by Shodex). -It was found that the peak at 11.6 minutes derived from ME2P decreased, and a peak derived from cytidine 5'-monophosphate occurred at 9.6 minutes. The HPLC was performed at 30 C with a 10 mM potassium phosphate buffer (pH 2.5) flowing at a flow rate of 1.0 ml / min.

 In order to confirm the structure of the reaction product in the above reaction, the reaction product was isolated by the following method. After diluting the entire amount of the above reaction solution to 100 ml with water, the solution was passed through a Dowex 1-X8 (C1-type, 2 × 6 cm) column.

 The column was eluted with 100 ml of 1% saline, then passed through Sephadex G-10 (1.8 × 100 cm), and eluted with water. The eluted fraction was lyophilized to isolate 2.8 m of the reaction product. In order to track the reaction products, Ή-NMR was used to collect fractions showing 1.43 ppm of methyl sig- nates specific to the reaction products.

 (4) Structural analysis of reaction products

For the molecular formula of the reaction product, a value of m / z = 344.9481 (M + Na) ⁺ , Δ -1.2μu was obtained in a high-resolution FABMS spectrum using a mass spectrometer (HX-110, manufactured by JEOL). since the obtained was determined to _{C 5 H 10 0 9 P 2} Na 2. In this measurement, glycerol was used for the matrix.

The chemical shift values of ¹ H-NMR, ¹³ C-NMR, and ³¹ P-NMR spectra of the reaction product in heavy water are shown below. For the measurement, a nuclear magnetic resonance analyzer (A500, manufactured by JEOL) was used.

 Awake (500 MHz): 54.21 (m, H-4a), 4.13 (m, H-4b), 4.13 (m, H-3), 3.77 (d, J = 12.5 Hz, H-la), 3.62 ( d, J2 12.5 Hz, H-lb), 1.43 (s, 2-Me);

^{1 C NMR (125 MHz):} 584.3 (d, J = 8.5 Hz, C- 2), 68.9 (d, J rather 3.0 Hz, C- 3), 67.4 (J = 4.5 Hz, C - 1), 66.2 ( d, J = 6.5, C-4), 16.8 (J = 5.5 Hz, 2-Me);

³¹ P NMR (202 MHz): (5-10.9 (d, J = 23.2 Hz), -14.9 (d, J = 23.2 Hz) From these spectra, the structure of the reaction product -Methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) (Fig. 3). The ygbB gene product was named 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECDP synthase). This 2-C-methyl-D-erythritol 2,4-cyclophosphate synthase (MECDP synthase) is a novel enzyme that has not been reported before.

 The ygbB gene product catalyzes a reaction using 2-phospho-4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol (CDP-ME2P) as a substrate to produce 2-C-methyl-D -Produces erythritol 2,4-cyclodiphosphate (MECDP). This reaction is shown in FIG.

 (5) Properties of 2-C-methyl-D-erythritol 2,4-cyclohexyl diphosphate synthase (MECDP synthase)

 Analysis of the purified 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase by SDS-PAGE showed a molecular weight of about 22 kDa.

2-C-methyl - D-erythritol tall 2,4 Shikurojirin acid Shin evening The reaction Ichize, Mg ² + was essential. Example 4: Carotenoid production by recombinant Escherichia coli

 Brassmid pACCAR25AcrtX is a kind of isoprenoid and contains a gene encoding an enzyme for synthesizing carotene. E. coli JM109 (pACCAR25Z crtX) transformed with this plasmid is Carotene can be synthesized (J. Bacteriol. 172, 6704, (1990)).

 The method for quantifying Ichirotin is described below.

 Plasmid pQEYGBB or pQE30 as a control was introduced into E. coli JM109 (pACCAR25AcrtX), respectively, and transformed E. coli resistant to 200 μg / ml ampicillin and 34 jg / ml chloramphenicol JM109 (pACCAR25AcrtX, pQEYGBB) ヽ and E. coli JM109 (pACCAR25AcrtX, pQE30) were obtained.

E. coli JM109 (pACCAR25AcrtX, pQEYGBB) and E. coli JM109 (pACCAR25AcrtX, pQE30) were converted to 200 μg / ml ampicillin and 34 μg / ml, respectively. 42 hours at 30 ° C in 100 ml of 2 x YT medium (Tryptone Pepton (16 g / 1), Bacto Yeast Extract (10 g / l), NaCl (5 g / l), pH 7.0) containing chloramphenicol After culturing, the cells were collected by centrifugation. The collected cells were freeze-dried, the dried cells were weighed, 60 ml of a solution of black form: methanol = 2: 1 was added, and the mixture was allowed to stand for 1 hour to extract carotene. Then, the chloroform / methanol solution was evaporated in the evaporator overnight, and 1 ml of acetone was added to the remaining solid to dissolve it. The amount of /?-Carotene in this acetone solution was determined by high-performance liquid chromatography (manufactured by JASCO Corporation) using a Sensi-Pack PEGASIL 0DS column (manufactured by Sensyu Kagaku) using isopropyl alcohol: methanol = 1: 2. It was eluted at 1 ml / min in a solvent system and quantified. Under these conditions, 一 rotin is eluted at 8.0 minutes. In addition, the amount of? -Carotene was represented by the relative value of the area of a peak corresponding to 5-force rotin in the above chromatogram. The results obtained are shown in Table 2 below. Table 2

 Plasmid bacterial mass retained by JM109 strain? —Carotene——Carotene / microbial mass pACCAR25AcrtX, pQE30 0.12g 171855 1400000

pACCARZ5AcrtX, pQEYGBB 0.12g 2222749 19QQQ00

E. col i JM109 (pACCAR25AcrtX ₅ pQEYGBB) produced about 1.3 times the amount of 力 rotorin per culture volume (100 ml) compared to that of E. col i JM109 (pACCAR25AcrtX, pQE30) ( See table above; 222749/171855 = about 1.3). In addition, the production amount of per-cell rotin per bacterial mass in E. col i JM109 (pACCAR25AcrtX, pQEYGBB) was about 1.35 times that of E. col i JM109 (pACCAR25AcrtX, pQE30) ( See table above; 1900000/1400000 = about 1.35).

These results indicated that the non-mevalonate pathway gene ygbB derived from Escherichia coli was effective in improving the productivity of isoprenide. Industrial applicability

 According to the present invention, an unknown reaction step in the non-mevalonate pathway, specifically, a reaction step using 2-phospho-4- (cytidine 5′-diphospho) -2-C-methyl-D-erythritol as a substrate is performed. An enzyme that catalyzes, as well as a gene that encodes it, has been provided. Further, it has become possible to provide a method for improving productivity of isoprenoid using the above enzyme or gene, and a method for screening an antibacterial agent using the above enzyme or gene.

Claims

The scope of the claims

1. An enzyme protein having the following physicochemical properties and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity.

 (I) Action:

 2-phospho-4- (cytidine 5, -diphospho) -2-C-methyl-D-erythritol acts on 2-C-methyl-D-erythritol 2,4-cyclodiphosphate and cytidine 5'-monolyl Generates acid (CMP).

 (II) Substrate specificity:

 Use 2-phospho-4- (cytidine 5, -diphospho) -2-C-methyl-D-erythritol as a substrate.

 (III) molecular weight:

 When measured by SDS-PAGE, it shows a molecular weight of about 22 kDa.

 (IV) Reaction conditions:

Mg ²⁺ is required for the reaction catalyzed by this enzyme.

2. 2-C-methyl having any of the following amino acid sequences - D-erythritol Thor _{2, 4} - Shikurojirin acid Shin evening enzyme protein having Ichize activity.

(A) the amino acid sequence of SEQ ID NO: 1;

 (B) an amino acid sequence in which one to several amino acids have been deleted, substituted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, comprising 2-C-methyl-D-erythritol An amino acid sequence having 2,4-cyclodiphosphate synthase activity; or

(C) an amino acid sequence having at least 60% homology with the amino acid sequence of SEQ ID NO: 1 and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity .

 3. A DNA encoding the protein of claim 1 or 2.

4. DNA encoding a protein having any of the following base sequences and having 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity. (A) the nucleotide sequence of SEQ ID NO: 2;

 (B) a base sequence in which one to several bases have been deleted, substituted, added and / or inserted in SEQ ID NO: 2, and comprising 2-C-methyl-D-erythritol 2,4-cyclodylline A nucleotide sequence encoding a protein having acid synthase activity; or

 (C) a nucleotide sequence capable of hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, wherein the protein has 2-C-methyl-D-erythritol 2,4-cyclodylinate synthase activity. The base sequence to be coded.

 5. A recombinant vector containing the DNA according to claim 3 or 4.

 6. A transformant having the recombinant vector according to claim 5.

 7. The transformant according to claim 6, which is Escherichia coli.

 8. A step of culturing a transformant produced by transforming the vector containing the DNA of claim 3 or 4 into a host to produce an isoprenide compound in the culture, and from the culture. A method for producing an isoprenide compound, comprising a step of collecting an isoprenoid compound.

9. Isopurenoi de compound, Yubikinon a Isopurenoi de compound selected from vitamin K ₂ or Karochino I de, method of Isopureno I de A compound according to claim 8.

 10. A method for screening a non-mevalonate pathway inhibitor, comprising searching for a substance that inhibits a reaction in the non-mevalonate pathway catalyzed by the enzyme according to claim 1 or 2.

 11. The screening method according to claim 10, wherein the non-mevalonate pathway inhibitor is an antibacterial active substance.

 12. The screening method of claim 10, wherein the non-mevalonate pathway inhibitor is a herbicidally active substance.

 1 3. An inhibitor of the non-mevalonate pathway obtained by the screening method according to claim 10.

14. An antibacterial active substance obtained by the screening method according to claim 11.

15. Herbicidally active substance c obtained by the screening method according to claim 12.