JPH11178590A - Gene for enzyme for synthesizing decaprenyl diphosphate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new recombinant protein having a specific amino acid sequence, having the activity of an enzyme for synthesizing decaprenyl diphoshate, and useful for producing the decaprenyl diphosphate and ubiquinone-10 which is an electronic transmission system component, etc. SOLUTION: This recombinant protein comprises a protein comprising an amino acid sequence of the formula or an amino acid sequence of the formula wherein at least one amino acid is deleted, replaced or added, and having the activity of an enzyme for synthesizing decaprenyl diphosphate. The new recombinant protein is useful for producing decaprenyl diphosphate metabolized into the prenyl side chain of ubiquinone-10 which is a component of an electronic transmission system produced by bacteria, etc. The new recombinant protein is obtained by screening a genome DNA library originating from a soil bacterium: Paracoccus denitrificans with a probe comprising a fragment amplified by a PCR using the genome DNA as a template, and subsequently expressing the obtained gene in an expression vector.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to decaprenyl diphosphate synthase, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and decaprenyl diphosphate synthase. And a method for producing ubiquinone-10.

[0002]

BACKGROUND OF THE INVENTION Isoprenoids are 23,000 in nature
It is the most diverse group of compounds present in more than one species, including sterols, carotenoids, sugar carrier lipids, prenylquinones, prenylated proteins and the like (FIG. 1). Formation of a basic carbon skeleton in the biosynthesis process of these isoprenoid compounds, that is, the heap of isopentenyl diphosphate (IPP) which is an isoprene unit having 5 carbon atoms
Enzymes that catalyze d-to-tail type polymerization condensation reactions are collectively referred to as prenyl diphosphate synthases. Each prenyl diphosphate synthase is roughly classified into four types according to differences in chain length, steric structure and the like of the prenyl diphosphate produced (Table 1).

[0003]

[Table 1]

Geranyl diphosphate belongs to the short chain prenyl diphosphate synthase (prenyltransferase I)
(GPP, C10) Synthase, farnesyl diphosphate (FPP, C15)
Synthase (Eberhardt, NL et al., (1975) J. Biol. Chem.
250,863-866), geranylgeranyl diphosphate (GGPP, C2
0) Synthase (Sagami, H. et al., (1994) J. Biol. Chem. 26
9, 20561-20566). The short-chain prenyl diphosphate biosynthesized by these enzymes is water-soluble and is also supplied as an allylic primer substrate for other groups of polyprenyl diphosphate synthases.

[0005] Medium-chain prenyl diphosphate synthase (prenyltransferase II) includes hexaprenyl diphosphate (H
exPP, C30) Synthase (Fujii, H. et al., (1982) J. Biol. C
hem.257,14610), heptaprenyl diphosphate (HepPP, C3
5) Synthase (Takahashi, I. etal., (1980) J. Biol. Chem. 25
5,4539) and so on. The major difference between these enzymes and the short-chain prenyl diphosphate synthase described above is that they are heterodimeric enzymes formed from two types of proteins that do not have a catalytic function by themselves. These are usually dissociated, but associate with the presence of a substrate to express the function as an enzyme. The products of these enzymes are highly hydrophobic and have the property of forming micelles, but do not require lipids or surfactants for the expression of enzyme activity. This is thought to be because medium-chain prenyl diphosphate synthase is a special enzyme system that repeats such dynamic dissociation and association.

The E-type long-chain prenyl diphosphate synthase (prenyltransferase III) includes octaprenyl diphosphate (OctPP, C40) synthase and decaprenyl diphosphate (DP
P, C50) Synthetic enzymes and the like belong. These enzymes, unlike prenyltransferase II, are non-dissociating homodimers and are activated by a polyprenyl diphosphate carrier protein (Ohnuma, S. et al., (1991) J. Biol. C.
hem. 266, 23706-23713). This activation is believed to maintain catalyst turnover by removing hydrophobic reaction products from the active site of the enzyme.

[0007] Nonaprenyl diphosphate is a type Z long chain prenyl diphosphate synthase (prenyltransferase IV).
(E, E-farnesyl-all-Z-hexaprenyl diphosphate, C4
5) Synthetic enzymes, such as undecaprenyl diphosphate (E, E-farnesyl-all-Z-octaprenyl diphosphate, C55) synthase. The products of these enzyme reactions act as sugar carrier lipids during bacterial cell wall biosynthesis. Activity expression requires the addition of phospholipids and surfactants. In addition, D which is classified as prenyltransferase III
It is known that a surfactant is required for PP synthase to exhibit its activity.

By the way, the soil bacterium Paracoccus denitrificans is a bacterium which is considered to be the source of human mitochondria. The respiratory chain and the oxidative phosphorylation mechanism are more efficient than other bacteria, and have characteristics closer to mitochondria in that they are organized as a single tissue (John, P. et. al., (1975) Nature 254,
495-498). From Paracoccus denitrificans,
So far, three types of prenyl diphosphate synthase activities have been confirmed (FIG. 2). That is, dimethylallyl diphosphate
(DMAPP) and two molecules of IPP are condensed to form E to form FPP
PP synthase, NPP synthase (Ishii, 6), in which six molecules of IPP are condensed to Z-type from FPP to produce nonaprenyl diphosphate (NPP)
K. et al., (1986) Biochem. J. 233, 773-777), and D from which 7 molecules of IPP are condensed to form E from FPP to form DPP.
PP synthase (Ishii, K. et al., (1983) Biochem. Biophy
s.Res. Commun. 116, 500-506).

[0009] NPP produced by the NPP synthase is a sugar carrier lipid essential for the cell wall biosynthesis of the bacterium. However, unlike several E-type prenyl diphosphate synthases that have already been cloned and analyzed, prenyl diphosphates that catalyze Z-type condensation reactions such as NPP synthase and undecaprenyl diphosphate synthase. For phosphate synthase,
The relationship between its structure and catalytic function has not yet been clarified.

[0010] DPP produced by DPP synthase is
It is metabolized to the prenyl side chain of ubiquinone-10, a component of the electron transport system produced by this bacterium. C30 to C50 polyprenyl diphosphate biosynthesized in enzymes classified as bacterial prenyltransferases II and III are all supplied as precursors of the side chains of menaquinone and ubiquinone, respectively. Therefore, the chain length of the product of each enzyme directly reflects on the side chain length of the prenylquinone of the bacterium from which the enzyme originated. Among prenylquinones, ubiquinone-10 is industrially extracted from Paracoccus denitrificans because it has the same side chain length as human coenzyme Q (CoQ), and is used as a pharmaceutical.
Ubiquinone has been known to be effective for chronic heart disease for some time, and is also effective as an antiarrhythmic agent, so it is also used for prevention of arrhythmia by administration of an anthracycline anticancer agent (Fujioka, T. et al.). et al., Tohoku J.
Exp. Med. (1983) 141 suppl. 453-463).

[0011]

The present invention relates to decaprenyl diphosphate synthase, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and decaprenyl diphosphate. An object of the present invention is to provide a method for producing synthase and ubiquinone-10.

[0012]

Means for Solving the Problems As a result of intensive studies based on the above problems, the present inventors have succeeded in cloning a long-chain decaprenyl diphosphate synthase gene from Paracoccus denitrificans. Thus, the present invention has been completed. That is, the present invention is the following recombinant protein (a) or (b). (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2; (b) a protein consisting of an amino acid sequence in which at least one amino acid has been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Protein having phosphate synthase activity

Further, the present invention provides the above (a) or (b)
Is a gene that encodes a recombinant protein. Examples of the gene include a gene containing the base sequence represented by SEQ ID NO: 1. Further, the present invention is a recombinant vector containing the gene. Furthermore, the present invention is a transformant transformed by the above-mentioned recombinant vector.

Further, the present invention provides a method for producing decaprenyl diphosphate synthase, which comprises culturing the transformant in a medium and collecting decaprenyl diphosphate synthase from the resulting culture. Furthermore, the present invention is a method for producing ubiquinone-10, comprising extracting ubiquinone-10 from the transformant.

Hereinafter, the present invention will be described in detail. Heretofore, it is known that prenyl diphosphate synthase (hereinafter also referred to as prenyltransferase) has seven highly conserved regions across species (Koyama,
T. et al. (1993) J. Biochem. 113, 355). In the present invention, degenerate oligonucleotides for primers are designed based on amino acid sequences highly conserved among various prenyltransferases, and these primers are variously combined to obtain soil bacteria Paracoccus denitrificans (Paracoccus denitrificans; hereinafter, "P.denitrific
ans ") as a template
The gene of the present invention can be obtained by screening using the amplified partial sequence as a probe.

1. Cloning of Gene Encoding Prenyl Diphosphate Synthase (1) Preparation of Genomic DNA First, genomic DNA is prepared from cultured cells of a prenyl diphosphate synthase-producing bacterium, for example, a soil bacterium P. denitrificans. For preparation of genomic DNA, any known technique can be used. For example, medium (80 containing yeast extract 2g, polypeptone _{10g, MgSO 4 · 7H 2 O} 1g, distilled water 1L
2), and cultured at 30 ° C. for one to several days (until saturation). Subsequently, the microbial cells are treated with lysozyme, and further treated with a surfactant such as sodium lauryl sulfate.Then, the protein is removed with an organic solvent such as phenol, chloroform, or ether, and the genomic DNA is easily precipitated by precipitation with ethanol. Can be prepared (Sait
o, H. et al., (1963) Biochim. Biophys Acta 72, 619-
629).

Next, the genomic DNA obtained is ligated to a vector plasmid to prepare a genomic DNA library. The preparation can be performed according to a known method.
For example, suitable restriction enzymes (eg, EcoRI, BamHI, Hind
III, Sau3AI, MboI, PstI)
After the strand and the DNA strand of the plasmid have been cleaved, they are treated with DNA ligase (eg, T4 DNA ligase) or, depending on the state of the cleaved ends, with terminal transferase or DNA polymerase.
Bind DNA strands with NA ligase (Molecu
lar cloning, Cold Spring Harbor Laboratory, 269, 1
982; Nelson T. et al., Method in Enzymol., 68, 41,
(1979)). The vectors used here include λ phage vectors (λgt10, Charon 4A, EMBL-3, etc.), plasmid vectors (pBR322, pSC101, pUC19, pUC119, pA
CYC117). After incorporating the DNA fragment into these vectors, E. coli (DH1, HB101, JM109
, C600, MV1184, TH2), etc.
A library can be obtained.

(2) Preparation of screening probe First, a probe for screening the genomic DNA by hybridization is prepared. In order to prepare a probe having higher specificity for the target DNA, it is considered appropriate to prepare an oligonucleotide that encodes a region where amino acid residues are highly conserved among the respective species. The probe can be obtained by ordinary chemical synthesis. Fig. 3
The following conserved amino acid sequence is selected based on

Region I "(Gly or Glu) Gly Lys Arg Ile
Arg Pro ”sequence (SEQ ID NO: 3)“ (Thr or Met) Ala (Ser or Thr) Leu (Val,
Ile or Leu) His Asp ”sequence (SEQ ID NO: 4),“ Ala Se
r Leu Leu His Asp Asp ”sequence (SEQ ID NO: 5) and“ Al
“A Asp Leu Arg Arg Gly” sequence (SEQ ID NO: 6) “Leu Ala Gly Asp Phe Leu Leu” sequence in region III (SEQ ID NO: 7) “Gly Glu Leu Leu Gln Leu” sequence in region IV (SEQ ID NO: 8) Region V "Lys Thr Ala Leu Leu Ile" sequence of SEQ ID NO: 9 "Phe Gln Leu Ile Asp Asp" sequence of region VI (SEQ ID NO: 9)
10), “Asp Asp IleLeu Asp Phe” sequence (SEQ ID NO: 1)
1), “Gly Lys Asn Val Gly Asp Asp” sequence (SEQ ID NO: 12) and “Asp Asp (Leu, Ile or Met) Leu Asp (Tyr
Or Phe) (Asn or Thr) "sequence (SEQ ID NO: 13)

Here, regions I, II, III, IV, V and VI
Is based on Koike-Takeshita, A. et al., (1995) J. Biol. Che.
m. 270, 18396-18400, the amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophylus described in 43-53, 74-95, 110 to 11
9th, 145th to 150th, 170th to 175th, 204th
This is the 230th region.

The examination of conserved amino acid sequences among species is carried out by examining Bacillus stearothermophilus, Escherichia coli, Saccharomyces cerevisiae, rat and human FPS, Erwinia herbicola ( Erwinia
herbicola) and Erwinia Uredbora (Erwini)
auredovora) and known prenyl diphosphate synthases such as Saccharomyces cerevisiae HexPS. The following oligonucleotide probes are prepared based on these amino acid sequences.

That is, according to the present invention, the amino acid sequence highly conserved among various prenyltransferases, especially hexaprenyl diphosphate (HexPP, C30),
Heptaprenyl diphosphate (HepPP, C35) (Koike-Takeshita,
A. et al. (1995) J. Biol. Chem. 270, 18396-18400)
And octaprenyl diphosphate (OctPP, C40) (Jeong, J.
H., et al. DNA Seq. 4, 59-67 (1993)), based on sequences characteristic of medium- and long-chain prenyltransferases.
Design 12 types.

Sense primer S1 (designed based on the sequence of SEQ ID NO: 3): 5 ′-(CG) (AG) CGG (AT) AA (AG) C (AG) (CGT) AT (CGT) CGTCC-3 '(SEQ ID NO: 14) S2 (designed based on the sequence of SEQ ID NO: 4): 5'-A (CT) (ACGT) GC (GT) (AT) C (ACGT) CT (ACGT) (CGT) T ( ACGT) CACGA-3 '(SEQ ID NO: 15) S3 (designed based on the sequence of SEQ ID NO: 3): 5'-GG (ACGT) GG (ACGT) AA (AG) CG (ACGT) AT (ACT) CG ( ACGT) CC-3 '(SEQ ID NO: 16) S4 (designed based on the sequence of SEQ ID NO: 5): 5'-GC (ACGT) TC (ACGT) CT (ACGT) CT (ACGT) CA (CT) GACGA- 3 '(SEQ ID NO: 17) S5 (designed based on the sequence of SEQ ID NO: 6): 5'-GC (ACGT) GA (CT) TT (AG) (AC) G (ACGT) (AC) G (ACGT) GG-3 '(SEQ ID NO: 18) S6 (designed based on the sequence of SEQ ID NO: 7): 5'-(CT) T (ACGT) GC (ACGT) GG (ACGT) GA (CT) TT (CT) TT (AG) TT-3 '(SEQ ID NO: 19)

A1 (designed based on the sequence of SEQ ID NO: 13): 5 ′-(GT) T (AG) (AT) AATCGAG (TA) A (ACT) (AG) TC (AG) TC -3 '(SEQ ID NO: 20) A2 (designed based on the sequence of SEQ ID NO: 8): 5'-(ACGT) A (AG) (CT) TG (CT) AA (ACGT) A (AG) (CT) TC (ACGT) CC-3 '(SEQ ID NO: 21) A3 (designed based on the sequence of SEQ ID NO: 9): 5'-(AGT) AT (ACGT) AG (ACGT) AG (ACGT) GC (ACGT) GT (TC) TT-3 '(SEQ ID NO: 22) A4 (designed based on the sequence of SEQ ID NO: 10): 5'-(AG) TC (AG) TC (AGT) AT (CT) AA (CT) TG ( AG) AA-3 '(SEQ ID NO: 23) A5 (designed based on the sequence of SEQ ID NO: 11): 5'-(AG) AA (AG) TC (ACGT) A (AG) (AGT) AT (AG) TC (AG) TC-3 '(SEQ ID NO: 24) A6 (designed based on the sequence of SEQ ID NO: 12): 5'-(AG) TC (AG) TC (ACGT) CC (ACGT) AC (AG) TT (CT) TT (ACGT) CC-3 '(SEQ ID NO: 25)

(3) Cloning of Part of the Prenyl Diphosphate Synthase Gene The screening of the gene of the present invention from the genomic DNA of P. denitrificans can be performed by a known method, for example, Southern hybridization, colony hybridization, or the like.
It can be performed by PCR and a combination thereof. For example, PCR is performed on the genomic DNA of P. denitrificans in combination with the above primers to amplify DNA containing the prenyl diphosphate synthase gene.
A DNA fragment suspected of containing the target gene is separated and recovered by electrophoresis, and after ligation reaction with the vector, Escherichia coli is transformed and cloned. The DN thus obtained
The A fragment (pCR14) is suitable as a probe for obtaining the full length prenyl diphosphate synthase gene.

(4) Cloning of full length prenyl diphosphate synthase gene Probe pCR14 is a DNA fragment containing a part of the prenyl diphosphate synthase gene of P. denitrificans as described above. Therefore, to screen for a gene encoding a prenyl diphosphate synthase peptide of the present invention, pCR14 is used, for example, as follows. A 10-5 kbp portion of the genomic DNA partially digested with Sau3AI was extracted from an agarose gel and inserted into the BamHI site of pUC119,
E. coli JM109 strain is transformed to prepare a DNA library. Then, colony hybridization is performed using a probe.

(5) Determination of Nucleotide Sequence The clone obtained as described above is digested with an appropriate restriction enzyme, electrophoresed in an agarose gel, and a restriction map is prepared from the migration pattern and migration distance. DNA fragmentation (small fragmentation) based on this restriction map
To obtain the smallest clone showing the activity,
The base sequence is analyzed for NA. The nucleotide sequence may be determined using deletion clones deleted from one side of the insert and prepared in both forward and reverse directions.

The clones thus screened are digested with appropriate restriction enzymes such as EcoRI, PstI, etc., and then pUC119,
It is cloned into a plasmid such as pUC19 and used in a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sangar et al.
anger, F. et al., Proc. Natl. Acad. Sci. USA, 74,
5463 (1977)) and the like. The determination of the base sequence can be performed using a base sequence automatic analyzer such as T7 Sequencing Kit (Pharmacia).

(6) Identification of Gene A region predicted to be a prenyl diphosphate synthase gene is inserted into an expression vector, and Escherichia coli is transformed. By measuring the activity of the crude enzyme extract obtained by crushing the cultured cells, the prenyl diphosphate synthase of the present invention, in particular, decaprenyl diphosphate synthase can be identified.
Further, the gene can be identified by measuring the ubiquinone side chain length of the transformant.

SEQ ID NO: 1 shows the nucleotide sequence of the gene encoding prenyltransferase, and SEQ ID NO: 2 shows the amino acid sequence of the prenyltransferase of the present invention. The amino acid sequence represented by SEQ ID NO: 2 has the prenyltransferase activity. Mutations such as deletion, substitution, and addition may occur in at least one (preferably one or several). In addition to the base sequence represented by SEQ ID NO: 1, a gene encoding the same polypeptide that differs only in degenerate codons is also included in the gene of the present invention.

The above mutation can be introduced by a known method, for example, the Kunkel method (Kunkel, TA Proc. Natl. Acad. Sc).
i. (1985) 82, 488).
After the base sequence is determined in this way, the target gene can be obtained by chemical synthesis or by hybridization using a DNA fragment obtained by PCR or the like.

2. Preparation of Recombinant Vector and Transformant The recombinant vector of the present invention can be obtained by incorporating the gene of the present invention into an appropriate vector. Also,
The transformant of the present invention comprises the recombinant vector of the present invention,
It can be obtained by introducing into a host compatible with the vector used in producing the recombinant vector. The purified gene is inserted into a restriction enzyme site or a multiple cloning site of an appropriate vector DNA to prepare a recombinant vector, and a host cell is transformed with the recombinant vector.

Vector DNA for inserting DNA fragment
Is not particularly limited as long as it can be replicated in a host cell, and examples thereof include plasmid DNA and phage DNA. As plasmid DNA, for example, plasmid pUC11
8 (Takara Shuzo), pUC119 (Takara Shuzo), pBluescript S
K + (Stratagene), pGEM-T (Promega) and the like.Phage DNAs include, for example, M13mp18, M13m
p19 and the like. The host is not particularly limited as long as it can express the target gene, and any of eukaryotic cells and prokaryotic cells can be used. For example,
Escherichia coli, Bacillus subtilis
(Bacillus subtilis), yeasts such as Saccharomyces cerevisiae, and animal cells such as COS cells and CHO cells.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention may be capable of autonomous replication in the host and may contain a promoter, a gene of the present invention, and a transcription termination sequence. preferable. For example, Escherichia coli includes XL1-Blue (Stratagene), JM109
(Manufactured by Takara Shuzo Co., Ltd.).
For example, pTrc99A, pET expression system and the like can be mentioned. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli, phage, etc., such as trp promoter, lac promoter, PL promoter, and PR promoter are used. In the present invention, transformation of E. coli
Hanahan's method (Hanahan DJ, Mol. Biol. (1983) 16
6, 557).

When yeast is used as a host, examples of expression vectors include YEp13 and YCp50. As the promoter, for example, gal 1 promoter, gal
10 promoters and the like. As a method for introducing a recombinant vector into yeast, for example, an electroporation method (Becker DM, Methods. Enzymol., 194, 182-187)
(1991)), spheroplast method (Hinnen A., Proc. Nat.
l. Acad. Sci. USA, 75, 1929-1933 (1978)), lithium acetate method (Ito H., J. Bacteriol., 153, 163-168 (198
3)) and the like.

When an animal cell is used as a host, for example, pSG5, pREP4, pZeoSV or the like is used as an expression vector. Methods for introducing recombinant DNA into animal cells include:
For example, an electroporation method, a calcium phosphate precipitation method and the like can be mentioned. When a plasmid DNA is used as the vector DNA, for example, when inserting an EcoRI DNA fragment, the plasmid DNA is digested with a restriction enzyme EcoRI. Next, the DNA fragment and the cleaved vector DNA
Are mixed, and the mixture is reacted with, for example, T4 DNA ligase (Takara Shuzo) to obtain a recombinant vector.

3. Production of prenyltransferase The prenyltransferase of the present invention can be produced by culturing the transformant containing the recombinant vector obtained as described above. The culture method may be an ordinary solid culture method, but preferably a liquid culture method. As a medium for culturing the transformant, for example, one or more nitrogen sources selected from yeast extract, peptone, meat extract, etc., and one kind of inorganic salts such as dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, etc. What added the above, and what is necessary added the saccharide raw material, the antibiotic, the vitamin, etc. suitably is used. If necessary, add IPTG to the medium.
May be added to induce gene expression. The pH of the medium at the start of cultivation is adjusted to 6.8 to 7.5, and
The culture is carried out at -42 ° C, preferably around 37 ° C for 5 hours to overnight by aeration, stirring, shaking, and the like.

After completion of the cultivation, ordinary protein purification means can be used to collect the prenyltransferase of the present invention from the culture. That is, the cells are disrupted by a lysis treatment using an enzyme such as lysozyme, an ultrasonic crushing treatment, a grinding treatment, or the like, and the prenyltransferase is discharged outside the cells. Next, insolubles are removed by filtration or centrifugation to obtain a crude polypeptide solution.

In order to further purify the polypeptide from the crude polypeptide solution, an ordinary protein purification method can be used. For example, an ammonium sulfate salting-out method, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis, etc. are used alone or in an appropriate combination.

4. Production of Ubiquinone-10 Ubiquinone is known as a component of the electron transport system in many organisms, and the length of its isoprenoid side chain differs depending on the species. In Escherichia coli, the side chain of isoprene of isoprene unit 8 supplied by OPP synthase, the budding yeast Saccharomyces cerevisiae (Saccha
romyces cerevisiae) has 6 isoprene side chains, and human has 10 isoprene side chains.

In general, ubiquinone possessed by Escherichia coli does not have 10 isoprene side chains, but ubiquinone (ubiquinone-10) having 10 isoprene side chains is obtained from a transformant having the gene of the present invention introduced into Escherichia coli. be able to. That is, ubiquinone-10 is obtained by disrupting Escherichia coli by ultrasonic treatment or the like, extracting the cell components with hexane, and finally subjecting the cells to HPLC.

[0042]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. [Example 1] Cloning of prenyltransferase gene So far, it has been known that prenyltransferase has seven highly conserved regions across species. Therefore, in the present invention, a genomic DNA of P. denitrificans is designed by designing a degenerate oligonucleotide based on an amino acid sequence highly conserved among various prenyltransferases and combining these primers in various ways.
Was used as a template, and cloning was performed by screening using the partial sequence amplified by the PCR as a probe. In cloning, Takara Shuzo, Toyobo and New England BioLabs used restriction enzymes and other DNA modifying enzymes.

[0043] (1) Preparation P.denitrificans Preparation and genomic libraries P.denitrificans of genomic DNA, 802 medium (polypeptone 10 g, yeast extract _{2g, MgSO 4 · 7H 2 O} 1g, distilled water 1L, pH 7.0)
The cells were inoculated into 100 ml and cultured at 30 ° C. until saturation. Thereafter, the cells were collected, and genomic DNA was prepared according to the method of Frederick et al. P. denitrificans is American Type Cu
lture Collection (ATCC14907). P.denitrifi
The genomic DNA of cans is partially digested with a restriction enzyme, and a DNA fragment of a certain length is recovered to prepare a library. Thereby, the screening efficiency is improved as compared with the case where screening is performed from the entire genomic library.

First, 1 U of Sau3AI was added to 50 μg of genomic DNA.
Was added and incubation was started at 37 ° C. After the incubation was started, a fixed amount was sampled every 5 minutes up to 90 minutes to stop the reaction. After electrophoresis of the sample on a 0.8% agarose gel, a 10 to 5 kbp fragment was recovered, each DNA fragment was ligated to a pUC119-BamHI vector, and E. coli DH5
α was transformed. The transformed E. coli was cultured in LB medium to prepare a glycerol stock at a glycerol concentration of 30%. In this way, ten libraries containing about 2000 clones were prepared, and plasmid DNA was recovered from these libraries.

(2) Design of PCR primers In the present invention, amino acid sequences highly conserved among various prenyltransferases, and in particular, hexaprenyl diphosphate (HexPP, C30), heptaprenyl diphosphate (He
pPP, C35) (Koike-Takeshita A. et al. (1995) J. Biol.
Chem. 270, 18396-18400) and octaprenyl diphosphate
(OctPP, C40) (Jeong, JH, et al. DNASeq. 4, 59-67
(1993)), for example, 12 types of degenerate primers shown below were designed based on the sequences characteristic of medium- and long-chain prenyltransferases.

Sense primer: S1: 5 '-(CG) (AG) CGG (AT) AA (AG) C (AG) (CGT) AT (CGT) CGTCC-
3 '(SEQ ID NO: 14) S2: 5'-A (CT) (ACGT) GC (GT) (AT) C (ACGT) CT (ACGT) (CGT) T (A
CGT) CACGA-3 '(SEQ ID NO: 15) S3: 5'-GG (ACGT) GG (ACGT) AA (AG) CG (ACGT) AT (ACT) CG (ACG
T) CC-3 '(SEQ ID NO: 16) S4: 5'-GC (ACGT) TC (ACGT) CT (ACGT) CT (ACGT) CA (CT) GACGA-
3 '(SEQ ID NO: 17) S5: 5'-GC (ACGT) GA (CT) TT (AG) (AC) G (ACGT) (AC) G (ACGT) GG
-3 '(SEQ ID NO: 18) S6: 5'-(CT) T (ACGT) GC (ACGT) GG (ACGT) GA (CT) TT (CT) TT (A
G) TT-3 '(SEQ ID NO: 19)

Antisense primer: A1: 5 '-(GT) T (AG) (AT) AATCGAG (TA) A (ACT) (AG) TC (AG) TC-
3 '(SEQ ID NO: 20) A2: 5'-(ACGT) A (AG) (CT) TG (CT) AA (ACGT) A (AG) (CT) TC (ACG
T) CC-3 '(SEQ ID NO: 21) A3: 5'-(AGT) AT (ACGT) AG (ACGT) AG (ACGT) GC (ACGT) GT (TC) T
T-3 '(SEQ ID NO: 22) A4: 5'-(AG) TC (AG) TC (AGT) AT (CT) AA (CT) TG (AG) AA-3 '(SEQ ID NO: 23) A5: 5' -(AG) AA (AG) TC (ACGT) A (AG) (AGT) AT (AG) TC (AG) TC-
3 '(SEQ ID NO: 24) A6: 5'-(AG) TC (AG) TC (ACGT) CC (ACGT) AC (AG) TT (CT) TT (ACG
T) CC-3 '(SEQ ID NO: 25)

(3) Amplification of Prenyl Transferase Gene Fragment by PCR PCR was performed using TaKaRa Taq from Takara Shuzo Co., Ltd. The composition of a typical reaction is as follows. The template is P.denitrif
icans genomic DNA was used.

PCR was performed using DNA Thermal Cycler PJ from Takara Shuzo.
The following cycle was performed using 2000. That is,
One cycle at 97 ° C for 30 seconds, 40 ° C for 30 seconds and 70 ° C for 1 minute
This is done for 5 cycles, followed by 30 minutes at 97 ° C.
This cycle was repeated 30 times at a cycle of 30 seconds at 55 ° C. and 1 minute at 70 ° C. After PCR, 1 × TBE
Performed electrophoresis on 5% acrylamide gel and amplified
The DNA fragment was confirmed (FIG. 4), and the DNA fragment was recovered by a gel recovery method. Primers S4 (SEQ ID NO: 17) and A6 (SEQ ID NO: 2
The DNA clone obtained by the reaction using 5) is designated as pCR14. Thereafter, pCR14 was purified and the pT7BlueT- vector (No.
vagen), the nucleotide sequence was determined by an automatic nucleotide sequencer (ABIPRISM ^™ 310 Genetic Analyzer), and analyzed with the gene analysis software GENETIX, and compared with the amino acid sequences of other prenyltransferases.

As a result, the amino acid sequence encoded by pCR14 differs from the amino acid sequence of OctPP synthase of E. coli by 45.7%.
%, The amino acid sequence of B. stearothermophilus HepPP synthase and 35.5%, the amino acid sequence of E. coli FPP synthase
It showed 31.8% homology (FIG. 5).

(4) Analysis by Southern Blot The PCR product having the base sequence determined by subcloning and determining the base sequence was newly prepared based on the sequence inside the above-mentioned degenerated oligo primers (S4 and A6). And BA; FIG. 6) was designed, and a DNA fragment to be used as a probe for hybridization was amplified by PCR and collected. Sense primer BS: 5'-CCGGCCGACGCAAACCTT-3 '(SEQ ID NO: 26) Antisense primer BA: 5'-CTGCTGCACCGCCGGGTC-3' (SEQ ID NO: 27) Amplification: 30 seconds at 97 ° C, 30 seconds at 55 ° C and 72 ° C This was performed for 30 cycles with 1 minute as one cycle.

Using the amplified 300 bp fragment as a probe, P. denitrificans genomic DNA was subjected to Southern blot analysis. For PCR products, use a commercially available kit (R
eady To Go DNA Labeling Beads (Pharmacia)) and labeled with [α- ³⁵ S] dCTP (Amersham). The labeling method followed the kit protocol. The filter is P.deni
Transfer 10 μg of chromosomal DNA of trificans to ApaI, EcoRI and Ba.
The whole digested with mHI was electrophoresed on 0.5 × TBE 0.7% agarose gel, denatured with alkali, and then nitrocellulose membrane filter (BIORAD zeta probe.
The sample was transferred to a blotting membrane (Zeta Probe Brotting Membranes) and Amersham's Hybond-N +) to prepare. The composition of the hybridization solution was changed as follows depending on the homology with the probe, and the filter was incubated at a constant temperature of 42 ° C. to perform pre-hybridization and hybridization.

[0053]

[0054]

The washing conditions after hybridization were also changed as follows. Severe conditions (corresponding to 100% homology) 0.1% SDS ・ 0.1 × SSC 68 ℃ Medium conditions (corresponding to 50% homology) 0.1% SDS ・ 2 × SSC 55 ℃ 55 ° C And analyzed using a Fuji BAS-2000 Bioimage Analyzer System.

As a result, under severe hybridization conditions (the homology between the detected band and the primer is equivalent to 100%), the band of 16.5 kbp when the genomic DNA was cut with ApaI and the band of 18.5 kbp when cut with EcoRI were used. But also B
When digested with amHI, a 11.2 kbp band and a slightly weakly hybridized 4.2 kbp band were respectively confirmed (FIG. 7A). 4.2 kb slightly weaker when cut with BamHI
The p band contains a sequence highly homologous to the sequence considered to be the prenyltransferase gene obtained this time, that is, another prenyltransferase gene (FPP synthase gene) of P. denitrificans. is expected.

On the other hand, under moderate hybridization conditions (the homology between the detected band and the primer is equivalent to 50%),
When the genomic DNA was cut with ApaI, a band of 7.4 kbp was further confirmed, when cut with EcoRI, a band of 5.3 kbp, and when cut with BamHI, a band of 5.2 kbp was confirmed (FIG. 7B). It is expected that these are likely to be other prenyltransferase genes.

(5) Recovery of full-length gene by colony hybridization In order to recover a full-length gene including a gene fragment amplified by PCR, colony hybridization was performed using the probe used for Southern hybridization. First, genomic DNA of P. denitrificans was converted to Sau3AI.
After limited digestion, the 10-5 kbp fragment was recovered and pUC
By subcloning into 119 BamHI vectors, a library containing approximately 2000 clones was
This was produced. Plasmids were recovered from these libraries, cut with the restriction enzyme EcoRI, and then subjected to Southern hybridization to reliably select libraries containing the target gene sequence.

As a result, 10 libraries (No. 1 to N
o.10), bands strongly hybridized to the No. 9 and No. 10 libraries were detected (FIG. 8). When colony hybridization was performed on No. 10 of the library in which the band was most strongly detected in the Southern hybridization, three positive clones were obtained. Plasmids were collected for them and designated as p11A1, p11A2, p11C1. Since these clones had an insert of about 7 kbp, it was confirmed by PCR whether or not the target gene was contained.
PCR was performed using the PCR primers BS and BA used when preparing the hybridization primer, and pCR1
Judgment was made based on whether the same band as in Example 4 was amplified (FIG. 9). PCR at 98 ° C for 30 seconds, 67 ° C for 30 seconds and 74 ° C
This was performed for 25 cycles with the reaction of 30 seconds as one cycle.

As a result, only p11A1 (lane 1) amplified about 300 bp of DNA similar to pCR14 (lane 4) (FIG. 9). p
Since 11A2 and p11C1 were not amplified by PCR under these conditions, it is considered that they do not contain the full length or are other prenyltransferase genes.

Next, where the same sequence as pCR14 is contained in the insert of about 7 kbp for p11A1 and whether the full length of the prenyltransferase gene is contained, it is confirmed by preparing a restriction enzyme map. (Figure 10). As a result, it was found that the same sequence as pCR14 was located at about 1.1 to 1.5 kbp from the end of the insert of p11A1. The average gene length of prenyltransferase is about 1 kbp, and pCR14 contains the conserved region II.
It was predicted that the insert of p11A1 would contain the full-length prenyltransferase gene sequence from the inclusion of p11A1 to pVI.

(6) Deletion of p11A1 and Determination of Nucleotide Sequence First, the entire nucleotide sequence of the prenyltransferase gene contained in p11A1 was determined. The deletion was allowed to proceed from the BglII site located 4 kbp downstream of the same sequence as pCR14, and finally cut out at the BamHI site located 430 bp upstream of pCR14, followed by SmaI-BamHI of pUC119.
Ligated into vector. Screened by colony hybridization and recovered p11A1 for BglII
After digestion with, the DNA was digested from the end with Exo nuclease III, and the reaction was stopped at an appropriate time. Thereafter, the DNA ends were made blunt by treating with Mungbean nuclease and Klenow fragment, and finally Dam was digested with BamHI.
The NA fragment was cut out.

After electrophoresis on a 3.5% acrylamide gel, the DNA fragment was recovered by gel and ligated to the BamHI-SmaI vector of pUC119. Using this to transform Escherichia coli DH5α,
Several single transformants were selected and the plasmid was recovered.
Plasmids containing these deletion products were applied to an ABI sequencer to determine the nucleotide sequence of the full-length gene. As a result, an ORF containing the same nucleotide sequence as pCR14 and having seven conserved regions characteristic of prenyltransferase in its amino acid primary sequence was found (FIG. 11; SEQ ID NO: 28). In the ORF, there were four ATG codons that could be translation initiation sites. this house,
The third methionine, which is close to the Shine-Dalgarno consensus sequence and at a reasonable distance, is considered to be the translation initiation site.

When the primary sequence of the amino acid of this ORF was compared with the primary sequence of the main prenyltransferase cloned so far, 34.9% of the FPP synthase of E. coli, and 3% of the FPP synthase of B. stearothermophilus.
1.1%, 31.8% of GGPP synthase from Erwinia uredovora,
HexPP synthase of M.luteusBP-26 is 26.3%, B.stearoth
ermophilus HepPP synthase 34.4%, E. coli OctPP
There was 44.2% homology with the synthase (FIG. 12). In addition,
In the course of the deletion, the nucleotide sequence of about 1 kbp downstream of the ORF of prenyltransferase contained in p11A1 was analyzed.

As a result, 25 bases downstream of the termination codon TGA of the ORF (11th base in the base sequence shown in FIG. 11 and SEQ ID NO: 28)
A typical terminator sequence consisting of a repeating sequence and a T sequence was found at positions 74 to 1201). Therefore, it was found that there was no ORF constituting the operon downstream of this prenyltransferase gene. In addition, Ba
The nucleotide sequence of about 1 kbp upstream of the mHI site was also determined, and a 3.3 kbp operon of the β-ketothiolase gene and acetyl-CoA reductase gene of P. denitrificans, which had already been cloned and analyzed, was 3.3 kbp upstream of the ORF of the prenyltransferase gene. (Yabutani, T. et al., (1995) FEMS Micro
bial. Lett. 133, 85-90). These two genes form an operon, but are terminated with a terminator, and thus do not form an operon with the ORF of the gene of the present invention (FIG. 13).

[Example 2] Construction of prenyltransferase large-scale expression system In the present invention, an NcoI site was introduced at the position of ATG at the start codon of ORF, and subcloned into the NcoI site of pTrc99A, which is a vector for large-scale expression, to obtain pTrc99A. We constructed a system for forcible expression with the strong trc promoter and SD sequence. Based on the seven conserved regions of prenyltransferase known so far, an expression plasmid was introduced into which an ORF was introduced in which the methionine codon of ATG, which is present at an approximate position of the expected start codon, was used as the start codon.

(1) Preparation of Plasmid for Mass Expression Of the ORFs considered to be prenyltransferases identified as a result of confirming the nucleotide sequence, the ORF having the third methionine as the start codon was replaced with the expression vector pTrc99A.
NcoI-BamHI site. First, a restriction enzyme site for introducing a vector was introduced by performing PCR using a mutant oligo primer. ATG of Met as a translation starting point
For the codon, NcoI site (CCATGG) and stop codon TGA
A BamHI site (GGATCC) was introduced 84 bp downstream of the site. At this time, the design was made so that the amino acid next to the start codon was not changed. The sequences of the PCR primers for introducing restriction enzyme sites are as shown below. Sense primer DP03: 5'-ATCGC CCATGG GCATGAACGAAAACGTCTC-3 '(SEQ ID NO: 29) NcoI antisense primer DP13: 5'-GAG GGATCC TATAACAACTGAGGCAGCG-3' (SEQ ID NO: 30) BamHI

By performing PCR using these primers, a gene fragment having a restriction enzyme site at the end was amplified. The polymerase used for PCR is said to be superior to Taq DNA polymerase, Pfu DNA polymerase, etc. in the accuracy of DNA synthesis and amplification efficiency.TOYOBO KOD DN
A polymerase (Barnes, WM (1994) Proc. Natl. Aca
d. Sci. USA 91, 2216-2220) was employed. The composition and cycle during the PCR reaction are as shown below.

[0069]

The PCR was performed at 98 ° C. for 30 seconds, 67 ° C. for 30 seconds and 74 ° C.
This was performed for 25 cycles with the reaction of 30 seconds as one cycle. After PCR, cut with restriction enzymes NcoI and BamHI,
Collected after 0.8% agarose electrophoresis. NcoI-BamHI gene fragment was converted to pTrc99A (Amann, E. et al. (1988) Gene 69,
301-305) into the NcoI-BamHI vector. The expression plasmid thus obtained was named pDPm3. These were also sequenced to confirm the sequence of the site linked to the vector.

As a result, it was confirmed that the ORF of the prenyltransferase was definitely inserted and ligated to the NcoI site without frame shift. Thereafter, Escherichia coli DH5α was expressed with this expression plasmid pDPm3.
Was transformed. Escherichia coli DH5α (pDPm3 / DH5α), which carries the expression plasmid pDPm3, was obtained from the Institute of Biotechnology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture).
Has been deposited as FERM BP-6259.

(2) Mass expression of prenyltransferase in E. coli E. coli transformed with the large expression plasmid pDPm3 was
LB medium containing μg / ml ampicillin (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose)
And cultured at 37 ° C. overnight. Next, this cell culture solution 1m
l of M9 nutrient medium containing 50 μg / ml ampicillin (0.2% M9
(Salt, 0.2% glycerol, 0.2% yeast extract) was inoculated into 100 ml and cultured at 30 ° C. When the turbidity reaches A600 = 0.6 to 0.8, isopropyl β-D-thiogalactopyranoside (I
PTG) to a final concentration of 1 mM, and
Overnight.

The culture is centrifuged (4 ° C., 1,000 × g, 10 minutes)
After washing, the cells were washed with 50 mM potassium phosphate buffer (pH 7.2), and the obtained cells were dissolved in lysis buffer (50 mM potassium phosphate buffer (pH 7.2), 5 mM EDTA, 1 mM β-mercaptoethanol, 1 mM PMSF ) And sonicated ((ultrasound 10 seconds-ice-cooled 2 minutes) x 10 cycles) to disrupt the post-bacterial cells. Ultrasonic treatment is SONIFIE manufactured by BRANSON
R250 was used. Centrifuge the solution after crushing (4 ° C, 15,000
× g, 30 minutes), and recovered as a crude enzyme extract.

Next, the prenyltransferase activity was measured as follows. Using an appropriate amount of crude enzyme extract,
A 200 μl reaction solution containing various allylic primers and [ ^1-14C ] IPP (54 or 57 Ci / mol; Amersham) was prepared from the reaction solution having the composition shown below, and incubated at 37 ° C for 1 hour. The enzyme reaction was performed. Thereafter, 200 μl of a saturated saline solution and 1 ml of a saturated saline solution of n-BuOH were added, and the mixture was stirred well and centrifuged to extract a reaction product. 200 μl of the BuOH layer was taken, 3 ml of Clearsol was added, and the amount of radioactivity in the BuOH extract was measured with a liquid scintillation counter to determine the enzyme activity. Enzyme activity was determined by the amount of IPP incorporated into the reaction product per minute (nmo
l) was defined as one unit. Composition of reaction solution As a result, prenyltransferase activity considered to be derived from a foreign gene was confirmed in Escherichia coli transformed with pDPm3 induced by IPTG (Table 2).

[0075]

[Table 2] Even in E. coli transformed with the same pDPm3, no significant prenyltransferase activity was confirmed in E. coli not induced by IPTG. This indicates that the expression of this prenyltransferase activity is under the strong control of the trc promoter.

(3) Analysis of Reaction Product by Reversed-Phase TLC Next, the hydrolysis product of prenyl diphosphate produced by this prenyltransferase by acid phosphatase was analyzed by reversed-phase thin-layer liquid chromatography (TLC). Note that acid phosphatase is available from Boehringer Mannheim.
Reverse phase thin layer chromatography plate
LKC18 from Whatman Chemical Separation was used. After performing the reaction using the crude enzyme extract, n-butanol (n-BuO
H), the reaction product (prenyl diphosphate) extracted was hydrolyzed to the corresponding prenol by acid phosphatase in a reaction solution having the following composition (Fujii, H. et al.
(1982) Biochim. Biophys. Acta. 712, 716-718).

[0077]

The hydrolysis reaction was performed at 37 ° C. overnight. After completion of the reaction, 1 ml and 3 ml of saturated saline solution were added, and the mixture was stirred to extract prenol with pentane. After recovering the pentane layer and washing with H ₂ O, the pentane extract concentrated with a centrifugal evaporator was developed with reversed-phase TLC (LKC-18) to identify the reaction product (developing solvent: acetone:
_{H 2 O = 19: 1)} . The positions of various prelenols used as reference samples were visualized by exposure to iodine vapor. The TLC plate was exposed to the Fuji imaging plate, and this was
The position of radioactive prenol was detected by analyzing with an AS-2000 bioimage analyzer. Figure 14 shows the results.
Shown in

The prenyltransferase activities confirmed in the host Escherichia coli are FPP synthase, OPP synthase and undecaprenyl diphosphate synthase. In E. coli transformed with pDPm3, decaprenyl diphosphate ( DPP) was generated (Fig.
14B). Therefore, it was revealed that the gene of the present invention is a DPP synthase gene. In addition, the substrate specificity of the allylic primer was examined for this DPP synthase. The unit of the enzyme activity is the same as described above. As a result, although GPP has some activity, it still has F
The activity with PP was maximal (Table 3, enzyme activity 54.4). Furthermore, when EEE-geranylgeranyl diphosphate (trans-GGPP) and ZEE-geranylgeranyl diphosphate (cis-GGPP) were used as substrates, cis-GGPP had almost no activity, whereas trans-GGPP had strong activity. The activity confirmed that the enzyme of the present invention was a type E chain extender.

[0080]

[Table 3]

(4) Confirmation of large-scale expression of prenyltransferase Confirmation of large-scale expression of the enzyme using an expression plasmid was performed by using a crude enzyme extract and SDS polyacrylamide gel electrophoresis (12.5% using RESEP GEL Co., Ltd.). Analyzed by SDS-PAGE), the position of the band stained with Coomassie Brilliant Blue R-250 was used as the molecular weight marker (SDS-PAGE Molecular Weight).
Standards, Broad Range BIORAD). The method is basically Laemmli's method (Laemmli,
UK, (1970) Nature 227,680-685). As a result, a band presumed to be expressed in large amounts at around 36 kDa was confirmed in the 15,000 × g precipitated fraction (FIG. 15, lane 6).

(5) Analysis of Ubiquinone Side Chain Length Ubiquinone was extracted from Escherichia coli transformed with pDPm3, and the chain length of the isoprene side chain was analyzed. The method of ubiquinone extraction is as follows. First, 0.3 g of wet cells were suspended in 2 ml of a methanol-0.3% NaCl solution (10: 1 V / V) (hereinafter referred to as “extraction solution”) and subjected to ultrasonic treatment (30 minutes × 4 times). . Next, 1 ml of an extraction solution was added, and hexane extraction was performed twice. After washing with an extraction solution to remove hexane, the residue was dissolved in 1 ml of ethanol and subjected to HPLC.
The HPLC was from Hitachi, Ltd., and the column was LiChrosorb®
P-18 (5 μm) (MERCK) was used. The elution solvent was EtOH (99.8
%) At 1 ml / min and detection at 275 nm. As a result,
As shown in FIG. 16 and Table 4, the vector plasmid pTrc99A
Only ubiquinone-8 (UQ-8) was detected in E. coli transformed only with ubiquinone, but about 20% of ubiquinone was replaced by ubiquinone-10 (UQ-10) in E. coli transformed with pDPm3. Furthermore, about 70% of the total E. coli transformed with pDPm3 cultured with induction with IPTG was replaced with UQ-10.

[0083]

[Table 4] This confirmed that the isolated gene encoded decaprenyl diphosphate synthase. Also, Escherichia coli originally did not have the ability to produce ubiquinone-10, but by transforming Escherichia coli with this gene, it became possible for Escherichia coli to produce ubiquinone-10 (FIG. 17).

[0084]

Industrial Applicability According to the present invention, production of prenyl diphosphate synthase, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and prenyl diphosphate synthase Methods and methods for producing ubiquinone-10 by heterologous microorganisms are provided. The enzyme and gene of the present invention are useful in that they can be used for enzyme production, ubiquinone-10 production, and the like.

[0085]

[Sequence list]

SEQUENCE LISTING <110> TOYOTA JIDOSHA KABUSIKI KAISHA <120> DECAPRENYL DIPHOSPHATE SYNTHETASE GENE <130> P98-0161 <140><141><150> JP 97/251675 <151> 1997-09-17 <160> 30 <170> PatentIn Ver. 2.0 <210> 1 <211> 1002 <212> DNA <213> Paracoccus denitrificans <220><221> CDS <222> (1) .. (999) <400> 1 atg ggc atg aac gaa aac gtc tcc aag ccg ctc gac cgg ctc tcc gtg 48 Met Gly Met Asn Glu Asn Val Ser Lys Pro Leu Asp Arg Leu Ser Val 1 5 10 15 gaa ctg gcc ggg gat atg gac cgg gtc aat gcg ctg atc cgc gag cgc A Glu Le Gly Asp Met Asp Arg Val Asn Ala Leu Ile Arg Glu Arg 20 25 30 atg gcc agc cgc cac gcc ccc cgc att ccg gaa gtg acc gcg cat ctg 144 Met Ala Ser Arg His Ala Pro Arg Ile Pro Glu Val Thr Ala His Leu 35 40 45 gtc gag gcc ggc ggc aag cgg ctg cgg ccg atg ctg gtg ctg gcg gcg 192 Val Glu Ala Gly Gly Lys Arg Leu Arg Pro Met Leu Val Leu Ala Ala 50 55 60 gcg cgg ctg tgc ggc tat cag ctg ctg gcc gcg 240 Ala Arg Leu Cys Gly Tyr Gln Gly Asn Ser His Val Leu Leu Ala Ala 65 70 75 80 gcg gtc gag ttc atc cat acc gcg acg ctt ctg cac gac gac gtg gtc 288 Ala Val Glu Phe Ile His Thr Ala Thr Leu Leu His Asp Asp Val Val 85 90 95 gat gaa agc cag cag cgg cgc ggc cgg ccg acg gcc ctt ctg tgg 336 Asp Glu Ser Gln Gln Arg Arg Gly Arg Pro Thr Ala Asn Leu Leu Trp 100 105 110 gac aac aag tcc agc gtg ctg gtc ggc gac tac ctg ttc gcg cgc agc 384 Asp Asn Lys Ser Ser Val Leu Val Tyr Leu Phe Ala Arg Ser 115 120 125 ttc cag ctg atg gcg gat acg gaa agc atg cag gtc atg cgc atc ttg 432 Phe Gln Leu Met Ala Asp Thr Glu Ser Met Gln Val Met Arg Ile Leu 130 135 140 gcc aat gcc agcg acc atc gcc gag ggc gag gtg ctg cag ctg acc 480 Ala Asn Ala Ser Ala Thr Ile Ala Glu Gly Glu Val Leu Gln Leu Thr 145 150 155 160 gcc gcg cag gac gtc tcg acc acc gag gac acc tat atc cag atc gtg 528 Ala Ala Gln Asp Val Ser Thr Thr Glu Asp Thr Tyr Ile Gln Ile Val 165 170 175 cgc ggc aag aca gcg gcg ctg ttt tcc gcc gcg acc gag gcg ggg gcg 576 Arg Gly Lys Thr Ala Ala Leu Phe Ser Ala Ala Thr Glu Aly Ala 180 185 190 gtg gtg gcc ggc gcc gac ccg gcg gtg cag cag gcg ctg ttc gac tat 624 Val Val Ala Gly Ala Asp Pro Ala Val Gln Gln Ala Leu Phe Asp Tyr 195 200 205 ggc gat gcg ctg ggg atg gc tt gac gac ctg ctg gat 672 Gly Asp Ala Leu Gly Ile Ala Phe Gln Ile Val Asp Asp Leu Leu Asp 210 215 220 tac ggc ggc tcg acc acg acc atc ggc aag aac gtc ggc gac gat ttc 720 Tyr Gly Gly Ser Thr Thr Gly Lys Asn Val Gly Asp Asp Phe 225 230 235 240 cgc gag cgc aag ctg acg ctg ccg gtg atc aag gcc atc gcc cgc gcc 768 Arg Glu Arg Lys Leu Thr Leu Pro Val Ile Lys Ala Ile Ala Arg Ala 245 250 255 gac gcc gag cgc gcc ttc tgg gaa cgc acc atc ggc cag ggc cgg 816 Asp Glu Ala Glu Arg Ala Phe Trp Glu Arg Thr Ile Gly Gln Gly Arg 260 265 270 cag gac gag gcc gac ctg gcc acc gcc ctg gg atc gc atg 864 Gln Asp Glu Ala Asp Leu Ala Thr Ala Leu Glu Ile Leu Arg Arg Arg 275 280 285 gag gcg ctg gag gcc gcc cgc gcc gat gcg atc gcc tgg gcc ggc cgt 912 Glu Ala Leu Glu Ala Ala Tla Ala Gly Arg 290 295 300 gcc aag gcc gcg ctg caa gcc gcg ccc gac cag ccc ctg cgc cgc atc 960 Ala Lys Ala Ala Leu Gln Ala Ala Pro Asp Gln Pro Leu Arg Arg Ile 305 310 315 320 ctg gcg gc gg gct g gtg gtc tcg cgc ctg tcc tga 1002 Leu Ala Asp Leu Ala Asp Phe Val Val Ser Arg Leu Ser 325 330 <210> 2 <211> 333 <212> PRT <213> Paracoccus denitrificans <400> 2 Met Gly Met Asn Glu Asn Val Ser Lys Pro Leu Asp Arg Leu Ser Val 1 5 10 15 Glu Leu Ala Gly Asp Met Asp Arg Val Asn Ala Leu Ile Arg Glu Arg 20 25 30 Met Ala Ser Arg His Ala Pro Arg Ile Pro Glu Val Thr Ala His Leu 35 40 45 Val Glu Ala Gly Gly Lys Arg Leu Arg Pro Met Leu Val Leu Ala Ala 50 55 60 Ala Arg Leu Cys Gly Tyr Gln Gly Asn Ser His Val Leu Leu Ala Ala 65 70 75 80 Ala Val Glu Phe Ile His Thr Ala Thr Leu Leu His Asp Asp Val Val 85 90 95 Asp Glu Ser Gln Gln Arg Arg Gly Arg Pro Thr Ala Asn Leu Leu Trp 100 105 110 Asp Asn Lys Ser Ser Val Leu Val Gly Asp Tyr Leu Phe Ala Arg Ser 115 120 125 Phe Gln Leu Met Ala Asp Thr Glu Ser Met Gln Va l Met Arg Ile Leu 130 135 140 Ala Asn Ala Ser Ala Thr Ile Ala Glu Gly Glu Val Leu Gln Leu Thr 145 150 155 160 Ala Ala Gln Asp Val Ser Thr Thr Glu Asp Thr Tyr Ile Gln Ile Val 165 170 175 Arg Gly Lys Thr Ala Ala Leu Phe Ser Ala Ala Thr Glu Ala Gly Ala 180 185 190 Val Val Ala Gly Ala Asp Pro Ala Val Gln Gln Ala Leu Phe Asp Tyr 195 200 205 Gly Asp Ala Leu Gly Ile Ala Phe Gln Ile Val Asp Asp Leu Leu Asp 210 215 220 Tyr Gly Gly Ser Thr Thr Thr Ile Gly Lys Asn Val Gly Asp Asp Phe 225 230 235 240 Arg Glu Arg Lys Leu Thr Leu Pro Val Ile Lys Ala Ile Ala Arg Ala 245 250 255 Asp Glu Ala Glu Arg Ala Phe Trp Glu Arg Thr Ile Gly Gln Gly Arg 260 265 270 270 Gln Asp Glu Ala Asp Leu Ala Thr Ala Leu Glu Ile Leu Arg Arg Arg 275 280 285 Glu Ala Leu Glu Ala Ala Arg Ala Asp Ala Ile Ala Trp Ala Gly Arg 290 295 300 Ala Lys Ala Ala Leu Gln Ala Ala Pro Asp Gln Pro Leu Arg Arg Ile 305 310 315 320 Leu Ala Asp Leu Ala Asp Phe Val Val Ser Arg Leu Ser 325 330 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Saccharomyces cerevisiae, heptaprenyl diphosph
ate synthetase from Bacillus stearothermophilus o
r octaprenyl diphosphate synthetase from Escherich
ia coli. <220><221> PEPTIDE <222> 1 <223> Xaa represents Gly or Glu <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Micrococcus luteus or Saccharomyces cerevisia
e, heptaprenyl diphosphate synthetase from Bacill
us stearothermophilus, or octaprenyl diphosphate s
ynthetase from Escherichia coli. <220><221> PEPTIDE <222> 1 <223> Xaa represents Thr or Met <220><221> PEPTIDE <222> 3 <223> Xaa represents Thr or Ser <220><221> PEPTIDE <222> 5 <223> Xaa represents Val, Ile or Leu <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Micrococcus luteus or Saccharomyces cerevisia
e, heptaprenyl diphosphate synthetase from Bacill
us stearothermophilus, or octaprenyl diphosphate s
ynthetase from Escherichia coli. <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of heptaprenyl diphosphate synthetase
from Bacillus stearothermophilus. <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Saccharomyces cerevisiae. <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Micrococcus luteus or octaprenyl diphosphate s
ynthetase from Escherichia coli. <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of heptaprenyl diphosphate synthetase
from Bacillus stearothermophilus. <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of octaprenyl diphosphate synthetase f
rom Escherichia coli. <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of heptaprenyl diphosphate synthetase
from Bacillus stearothermophilus. <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of octaprenyl diphosphate synthetase f
rom Escherichia coli. <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Designed peptide based on preserved amino ac
id sequence of hexaprenyl diphosphate synthetase f
rom Micrococcus luteus or Saccharomyces cerevisia
e, heptaprenyl diphosphate synthetase from Bacill
us stearothermophilus, or octaprenyl diphosphate s
ynthetase from Escherichia coli. <220><221> PEPTIDE <222> 3 <223> Xaa represents Leu, Ile or Met <220><221> PEPTIDE <222> 6 <223> Xaa represents Tyr or Phe <220><221> PEPTIDE <222> 7 <223> Xaa represents Asn or Thr <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Saccharomyces cerevisiae or octaprenyl
diphosphate synthetase from Escherichia coli. <400> 14 srcggwaarc rbatbcgtcc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Micrococcus luteus or Saccharomyces cer
evisiae, heptaprenyl diphosphate synthetase from B
acillus stearothermophilus, or octaprenyl diphosph
ate synthetase from Escherichia coli. <220><221> modified base <222> 3 <223> n represents a, g, c or t <220><221> modified base <222> 9 <223> n represents a, g, c or t <220><221> modified base <222> 12 <223> n represents a, g, colt <220><221> modified base <222> 15 <223> n represents a, g, c or t <400> 15 ayngckwcnc tnbtncacga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Saccharomyces cerevisiae or octaprenyl
diphosphate synthetase from Escherichia coli. <220><221> modified base <222> 3 <223> n represents a, g, c or t <220><221> modified base <222> 6 <223> n represents a, g, colt <220><221> modified base <222> 12 <223> n represents a, g, colt <220><221> modified base <222> 18 <223> n represents a, g, c or t <400> 16 ggnggnaarc gnathcgncc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Micrococcus luteus or Saccharomyces cer
evisiae, heptaprenyl diphosphate synthetase from B
acillus stearothermophilus, or octaprenyl diphosph
ate synthetase from Escherichia coli. <220><221> modified base <222> 3 <223> n represents a, g, c or t <220><221> modified base <222> 6 <223> n represents a, g, colt <220><221> modified base <222> 9 <223> n represents a, g, c or t <220><221> modified base <222> 12 <223> n represents a, g, c or t <400> 17 gcntcnctnc tncaygacga 20 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <223> Designed oligonucleotide based on preserved
amino acid sequence ofheptaprenyl diphosphate synt
hetase from Bacillus stearothermophilus. <220><221> modified base <222> 3 <223> n represents a, g, c or t <220><221> modified base <222> 12 <223> n represents a, g, colt <220><221> modified base <222> 15 <223> n represents a, g, c or t <400> 18 gcngayttrm gnmgngg 17 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Saccharomyces cerevisiae. <220><221> modified base <222> 3 <223> n represents a, g, c or t <220><221> modified base <222> 6 <223> n represents a, g, colt <220><221> modified base <222> 9 <223> n represents a, g, c or t <400> 19 ytngcnggng ayttyttrtt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Micrococcus luteus or Saccharomyces cer
evisiae, heptaprenyl diphosphate synthetase from B
acillus stearothermophilus, or octaprenyl diphosph
ate synthetase from Escherichia coli. <400> 20 ktrwaatcga gwahrtcrtc 20 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofhexaprenyl diphosphate synth
etase from Micrococcus luteus or octaprenyldiphosp
hate synthetase from Escherichia coli. <220><221> modified base <222> 1 <223> n represents a, g, colt <220><221> modified base <222> 10 <223> n represents a, g, colt <220><221> modified base <222> 16 <223> n represents a, g, colt <400> 21 narytgyaan arytcncc 18 <210> 22 <211> 18 <212> DNA <213><220><223> Designed oligonucleotide based on preserved
amino acid sequence ofheptaprenyl diphosphate synt
hetase from Bacillus stearothermophilus. <220><221> modified base <222> 4 <223> n represents a, g, c or t <220><221> modified base <222> 7 <223> n represents a, g, colt <220><221> modified base <222> 10 <223> n represents a, g, colt <220><221> modified base <222> 13 <223> n represents a, g, c or t <400> 22 datnagnagn gcngtytt 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofoctaprenyl diphosphate synth
etase from Escherichia coli. <400> 23 rtcrtcdaty aaytgraa 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofheptaprenyl diphosphate synt
hetase from Bacillus stearothermophilus. <220><221> modified base <222> 7 <223> n represents a, g, c or t <400> 24 raartcnard atrtcrtc 18 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on preserved
amino acid sequence ofoctaprenyl diphosphate synth
etase from Escherichia coli. <220><221> modified base <222> 7 <223> n represents a, g, colt <220><221> modified base <222> 10 <223> n represents a, g, colt <220><221> modified base <222> 19 <223> n represents a, g, c or t <400> 25 rtcrtcnccn acrttyttnc c 21 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on sequence o
f 312 to 386 of pRC14. <400> 26 ccggccgacg caaacctt 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide based on complement
ary sequence of 592 to 609 of pRC14. <400> 27 ctgctgcacc gccgggtc 18 <210> 28 <211> 1219 <212> DNA <213> Paracoccus denitrificans <220><221> CDS <222> (151) .. (1149) ) <400> 28 10gatcccctcg gccgccagca ggtcggcggc gcgggtgatg agaagcgggt cggtggtgcg 60 cagaagctct ttcatgacat gggaaagtta cgcggctgtt gcgcatgtgt ccatggcgtg 120 gcaatggctg gcggcgaaag gggatcgctg atg ggc atg aac gaa aac gtc tcc 174 Met Gly Met Asn Glu Asn Val Ser 1 5 aag ccg ctc gac cgg ctc tcc gtg gaa ctg gcc ggg gat atg gac cgg 222 Lys Pro Leu Asp Arg Leu Ser Val Glu Leu Ala Gly Asp Met Asp Arg 10 15 20 gtc aat gcg ctg atc cgc gag cgc atg gcc agc cgc cac gcc ccc cgc 270 Val Asle Ala Le Glu Arg Met Ala Ser Arg His Ala Pro Arg 25 30 35 40 att ccg gaa gtg acc gcg cat ctg gtc gag gcc ggc ggc aag cgg ctg 318 Ile Pro Glu Val Thr Ala His Leu Val Glu Ala Gly Gly Lys Arg Leu 45 50 55 cgg ccg atg ctg gtg ctg gcg gcg gcg cgg ctg tgc ggc tat cag ggg 366 Arg Pro Met Leu Val Leu Ala Ala Ala Arg Leu Cys Gly Tyr Gln Gly 60 65 70 aac agc cat gtg ctg ctg gcc gcg gcg gtc gag ttc atc cat acc gcg 414 Asn Ser His Val Leu Leu Ala Ala Ala Val Glu Phe Ile His Thr Ala 75 80 85 acg ctt ctg cac gac gac gtg gtc gat gaa agc cag cag cgg c462 Thr Leu Leu His Asp Asp Val Val Asp Glu Ser Gln Gln Arg Arg Gly 90 95 100 cgg ccg acg gcc aac ctt ctg tgg gac aac aag tcc agc gtg ctg gtc 510 Arg Pro Thr Ala Asn Leu Leu Trp Asp Asn Lys Ser Ser Val Leu Val 105 110 115 120 ggc gac tac ctg ttc gcg cgc agc ttc cag ctg atg gcg gat acg gaa 558 Gly Asp Tyr Leu Phe Ala Arg Ser Phe Gln Leu Met Ala Asp Thr Glu 125 130 135 agc atg cag gtc atg cg gcc aat gcc agc gcc acc atc gcc 606 Ser Met Gln Val Met Arg Ile Leu Ala Asn Ala Ser Ala Thr Ile Ala 140 145 150 gag ggc gag gtg ctg cag ctg acc gcc gcg cag gac gtc tcg acc acc 654 Glu Gly Glu Val Leu Gln Leu Thr Ala Ala Gln Asp Val Ser Thr Thr 155 160 165 gag gac acc tat atc cag atc gtg cgc ggc aag aca gcg gcg ctg ttt 702 Glu Asp Thr Tyr Ile Gln Ile Val Arg Gly Lys Thr Ala Ala Leu Phe 170 175 180 tcc g cc gcg acc gag gcg ggg gcg gtg gtg gcc ggc gcc gac ccg gcg 750 Ser Ala Ala Thr Glu Ala Gly Ala Val Val Ala Gly Ala Asp Pro Ala 185 190 195 200 gtg cag cag gcg ctg ttc gac gg gg gat g gcc ttc 798 Val Gln Gln Ala Leu Phe Asp Tyr Gly Asp Ala Leu Gly Ile Ala Phe 205 210 215 cag atc gtg gac gac ctg ctg gat tac ggc ggc tcg acc acg acc atc 846 Gln Ile Val Asp Asp Leu Leu Asp Tyr Ser Thr Thr Thr Ile 220 225 230 ggc aag aac gtc ggc gac gat ttc cgc gag cgc aag ctg acg ctg ccg 894 Gly Lys Asn Val Gly Asp Asp Phe Arg Glu Arg Lys Leu Thr Leu Pro 235 240 245 gtg atc aag gcc atc cgc gcc gac gag gcc gag cgc gcc ttc tgg 942 Val Ile Lys Ala Ile Ala Arg Ala Asp Glu Ala Glu Arg Ala Phe Trp 250 255 260 gaa cgc acc atc ggc cag ggc cgg cag gac gag gcc gac ctg gcc acc 990 Glu Ile Gly Gln Gly Arg Gln Asp Glu Ala Asp Leu Ala Thr 265 270 275 280 280 gcg ctg gag atc ctg cgc cgc cgc gag gcg ctg gag gcc gcc cgc gcc 1038 Ala Leu Glu Ile Leu Arg Arg Arg Glu Ala Ala Ala Lea 285 290 295 gat gcg atc gcc tgg gcc ggc cgt gcc aag gcc gcg ctg caa gcc gcg 1086 Asp Ala Ile Ala Trp Ala Gly Arg Ala Lys Ala Ala Leu Gln Ala Ala 300 305 310 ccc gac cag ccc ctg cgcg cg ctg gcg gat ttc gtg 1134 Pro Asp Gln Pro Leu Arg Arg Ile Leu Ala Asp Leu Ala Asp Phe Val 315 320 325 10gtc tcg cgc ctg tcc tgaccaaagc ccccgcacaa atgaaaaagc ccggcgcatg 1189 Val Sertgcctgct Sertgcctgcc Sergcc 211> 30 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide having a NcoI site. <400> 29 atcgcccatg ggcatgaacg aaaacgtctc 30 <210> 30 <211> 28 <212> DNA <213> Artificial Sequence <220><223> Designed oligonucleotide having a BamHI sit
e. <400> 30 gagggatcct ataacaactg aggcagcg 28

[Sequence listing free text] SEQ ID NO: 3: hexaprenyl diphosphate synthase from Saccharomyces cerevisiae,
A peptide designed from the conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli. Sequence number 3: Xaa represents Gly or Glu (location: 1). SEQ ID NO: 4: Hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Peptide designed from the conserved amino acid sequence. SEQ ID NO: 4: Xaa is Thr or
Represents Met (location: 1). SEQ ID NO: 4: Xaa is Thr or
Represents Ser (location: 3). Sequence number 4: Xaa is Val, Il
represents e or Leu (location: 5). SEQ ID NO: 5: hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Peptide designed from the conserved amino acid sequence. SEQ ID NO: 6: peptide designed from conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 7: Peptide designed from the conserved amino acid sequence of hexaprenyl diphosphate synthase from Saccharomyces cerevisiae. SEQ ID NO: 8: Peptide designed from the conserved amino acid sequence of hexaprenyl diphosphate synthase from Micrococcus luteus or octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 9: peptide designed from conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 10: Peptide designed from the conserved amino acid sequence of octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 11: Peptide designed from the conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 12: Peptide designed from the conserved amino acid sequence of octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 13: hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Peptide designed from the conserved amino acid sequence. Sequence number 13: Xaa is Leu, Il
Represents e or Met (location: 3). Sequence number 13: Xaa is Ty
represents r or Phe (location: 6). SEQ ID NO: 13: Xaa is As
represents n or Thr (location: 7). SEQ ID NO: 14: oligonucleotide designed from conserved amino acid sequence of hexaprenyl diphosphate synthase from Saccharomyces cerevisiae or octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 15: hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Oligonucleotide designed from conserved amino acid sequence. SEQ ID NO: 15: n represents a, g, c, or t (location: 3)
SEQ ID NO: 15: n represents a, g, c, or t (location: 9).
SEQ ID NO: 15: n represents a, g, c or t (location: 1
2). SEQ ID NO: 15: n represents a, g, c or t (location:
15). SEQ ID NO: 16: oligonucleotide designed from conserved amino acid sequence of hexaprenyl diphosphate synthase from Saccharomyces cerevisiae or octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 16: n is
represents a, g, c or t (location: 3). SEQ ID NO: 16: n is
represents a, g, c or t (location: 6). SEQ ID NO: 16: n is
represents a, g, c or t (location: 12). SEQ ID NO: 16: n
Represents a, g, c or t (location: 18). SEQ ID NO: 17: hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Oligonucleotide designed from conserved amino acid sequence. SEQ ID NO: 17: n
Represents a, g, c or t (location: 3). SEQ ID NO: 17: n
Represents a, g, c or t (location: 6). SEQ ID NO: 17: n
Represents a, g, c or t (location: 9). SEQ ID NO: 17: n
Represents a, g, c or t (location: 12). SEQ ID NO: 18: oligonucleotide designed from conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 18: n is a,
Represents g, c or t (location: 3). SEQ ID NO: 18: n is a,
Represents g, c or t (location: 12). SEQ ID NO: 18: n is
represents a, g, c or t (location: 15). SEQ ID NO: 19: oligonucleotide designed from conserved amino acid sequence of hexaprenyl diphosphate synthase from Saccharomyces cerevisiae. SEQ ID NO: 19: n is a, g, c or
Represents t (location: 3). SEQ ID NO: 19: n is a, g, c or
Represents t (location: 6). SEQ ID NO: 19: n is a, g, c or
Represents t (location: 9). SEQ ID NO: 20: hexaprenyl diphosphate synthase from Micrococcus luteus or Saccharomyces cerevisiae, heptaprenyl diphosphate synthase from Bacillus stearothermophilus or octaprenyl diphosphate synthase from Escherichia coli Oligonucleotide designed from conserved amino acid sequence. SEQ ID NO: 21: oligonucleotide designed from conserved amino acid sequence of hexaprenyl diphosphate synthase from Micrococcus luteus or octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 21: n is a,
Represents g, c or t (location: 1). SEQ ID NO: 21: n is a,
Represents g, c or t (location: 10). SEQ ID NO: 21: n is
represents a, g, c or t (location: 16). SEQ ID NO: 22: oligonucleotide designed from conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 22: n is a,
Represents g, c or t (location: 4). SEQ ID NO: 22: n is a,
represents g, c or t (location: 7). SEQ ID NO: 22: n is a,
Represents g, c or t (location: 10). SEQ ID NO: 22: n is
represents a, g, c or t (location: 13). SEQ ID NO: 23: oligonucleotide designed from conserved amino acid sequence of octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 24: oligonucleotide designed from conserved amino acid sequence of heptaprenyl diphosphate synthase from Bacillus stearothermophilus. SEQ ID NO: 24: n is a,
represents g, c or t (location: 7). SEQ ID NO: 25: oligonucleotide designed from conserved amino acid sequence of octaprenyl diphosphate synthase from Escherichia coli. SEQ ID NO: 25: n represents a, g, c, or t (location: 7). SEQ ID NO: 25: n represents a, g, c, or t (location: 10). SEQ ID NO: 25: n represents a, g, c, or t (location: 19). SEQ ID NO: 26: oligonucleotide designed based on sequence of positions 312 to 386 of pRC14 SEQ ID NO: 27: oligonucleotide designed based on complementary sequence of positions 592 to 609 of pRC14 SEQ ID NO: 29: oligonucleotide designed to have NcoI site SEQ ID NO: 30: oligonucleotide designed to have BamHI site.

[Brief description of the drawings]

FIG. 1 is a diagram showing the biosynthesis of an isoprenoid compound.

FIG. 2 is a diagram showing a prenyl diphosphate biosynthesis pathway in P. denitrificans.

FIG. 3 is a diagram showing the design of PCR primers.

FIG. 4 is a photograph showing the result of acrylamide gel electrophoresis.

FIG. 5 is a diagram comparing the homology of amino acid sequences.

FIG. 6 is a diagram showing the design of PCR primers.

FIG. 7 is an electrophoretic photograph showing the results of Southern hybridization.

FIG. 8 is an electrophoretic photograph showing the results of Southern hybridization.

FIG. 9 is an electrophoretic photograph showing the results of Southern hybridization.

FIG. 10 is a diagram showing the structure of plasmid p11A1.

FIG. 11 is a view showing an open reading frame contained in a plasmid p11A1.

FIG. 12 is a diagram comparing the amino acid sequences of various prenyltransferases.

FIG. 13 is a schematic diagram showing the structure of a gene contained in the upstream and downstream regions of the gene of the present invention.

FIG. 14 is a photograph of a reversed-phase thin-layer liquid chromatograph.

FIG. 15 is a photograph showing the result of SDS-polyacrylamide gel electrophoresis.

FIG. 16 shows the results of analyzing the side chain of quinone.

FIG. 17 is a view showing a ubiquinone production ratio in each cell.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 28, 1998

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

[Correction contents]

FIG. 4

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 7

[Correction method] Change

[Correction contents]

FIG. 7

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Fig. 8

[Correction method] Change

[Correction contents]

FIG. 8

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] Fig. 9

[Correction method] Change

[Correction contents]

FIG. 9

[Procedure amendment 5]

[Document name to be amended] Drawing

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG. 14

[Procedure amendment 6]

[Document name to be amended] Drawing

[Correction target item name] FIG.

[Correction method] Change

[Correction contents]

FIG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9/12 C12R 1:19) (C12P 7/66 C12R 1 : 19)

Claims (7)

[Claims] 1. A recombinant protein of the following (a) or (b): (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2; (b) a protein comprising an amino acid sequence wherein at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Protein having phosphate synthase activity 2. A gene encoding the following recombinant protein (a) or (b). (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2; (b) a protein comprising an amino acid sequence wherein at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Protein having phosphate synthase activity 3. It comprises a base sequence represented by SEQ ID NO: 1.
The gene according to claim 2. 4. A recombinant vector comprising the gene according to claim 2 or 3. A transformant transformed by the recombinant vector according to claim 4. 6. A method for producing decaprenyl diphosphate synthase, comprising culturing the transformant according to claim 5 in a medium, and collecting decaprenyl diphosphate synthase from the resulting culture. 7. A method for producing ubiquinone-10, comprising extracting ubiquinone-10 from the transformant according to claim 5.