WO2002053751A1 - Polypeptides, polynucleotides, recombinant nucleic acids, recombinants, and process for producing prenyl alcohol - Google Patents

Polypeptides, polynucleotides, recombinant nucleic acids, recombinants, and process for producing prenyl alcohol Download PDF

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WO2002053751A1
WO2002053751A1 PCT/JP2001/011223 JP0111223W WO02053751A1 WO 2002053751 A1 WO2002053751 A1 WO 2002053751A1 JP 0111223 W JP0111223 W JP 0111223W WO 02053751 A1 WO02053751 A1 WO 02053751A1
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gene
phosphate
prenyl
phosphatase
alcohol
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PCT/JP2001/011223
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French (fr)
Japanese (ja)
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Kenro Tokuhiro
Nobuhiko Muramoto
Yukio Yamada
Osamu Asami
Masana Hirai
Chikara Ohto
Shusei Obata
Masayoshi Muramatsu
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Kabushiki Kaisha Toyota Chuo Kenkyusho
Toyota Jidosha Kabushiki Kaisha
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Priority to JP2002555256A priority Critical patent/JP4002833B2/en
Publication of WO2002053751A1 publication Critical patent/WO2002053751A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to a method for producing a polypeptide, a polynucleotide, a recombinant nucleic acid, a recombinant, and prenyl alcohol. More specifically, the present invention relates to a phosphatase that is effective when it is desired to selectively mass-produce prenyl alcohols having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, It controls the expression of enzymes involved in the biosynthesis of prenyl alcohol in living cells and recombinants into which such a polynucleotide has been introduced, and prenyl alcohol (particularly having a biologically active trans isomeric structure). In particular, the present invention relates to a method for efficiently mass-producing, for example, F0H, GG0H) having 15 or more carbon atoms.
  • Background art is a method
  • the isoprenoid biosynthetic pathway is thought to be universally present in a variety of species.
  • the biosynthetic pathways for isopentenyl diphosphate ( ⁇ ), the basic skeletal unit of isoprenoid compounds, are mainly the mevalonate pathway that exists in eukaryotes and the non-mevalonate pathway that mainly exists in prokaryotes (DXP pathway). The two routes are known.
  • IPP In the mevalonic acid pathway, starting with acetyl CoA, acetoacetyl CoA, hydroxymethyl dartaryl CoA (HMG-CoA), mevalonic acid, menolonic acid 5-phosphoric acid (MVP), mevalonic acid 5-diphosphate After (MVPP), IPP with 5 carbon atoms, which is an active isoprene unit, is biosynthesized.
  • IPP is biosynthesized via 1-deoxy-D-xylulose 5-phosphate, which is formed by the condensation of pyruvate and glyceraldehyde 3-phosphate.
  • IPP is converted to dimethylaryl diphosphate (DMAPP) by an isomerization reaction.
  • D. MAPP is successively polycondensed to IPP and trans-form (E-form), resulting in C10 gera ernilic acid (GPP), C15 funaresyl diphosphate (FPP), C20 geranylgeranyl diphosphate (GGPP) ⁇ ⁇ ⁇ Combined in the order of '
  • IPP is sequentially polycondensed to cis-form (Z-form) with all-trans-form FPP and GGPP, whereby ndecabrenyl diphosphoric acid, dehydrorisyl silicic acid, etc. are biosynthesized.
  • prenyl diphosphoric acid The above IPP, DMAPP, GPP, FPP, GGPP, pendecaprenyl diphosphoric acid, dehydrorisyl diphosphoric acid, etc. are collectively referred to as prenyl diphosphoric acid.
  • prenyl diphosphates highly useful (biologically active) all-trans pure products are suitable for production by biosynthesis and are difficult to control by chemical synthesis.
  • prenyl diphosphates are separated by diphosphoric acid by the action of phosphatase (or by relaying prenyle phosphate in which one phosphate is separated from prenyl diphosphate, one more phosphate is separated). Being) produces the corresponding prenyl alcohol.
  • prenyl diphosphoric acid and prenyl-phosphoric acid are collectively referred to as “prenyl phosphoric acid”.
  • prenyl phosphinates / prenyl alcohols those having 15 or more carbon atoms are particularly useful.
  • FPP, GGPP, etc. which are prenyl diphosphoric acids having 15 or more carbon atoms
  • phthalnesol (F0H) having 15 carbon atoms which is a dephosphorized oxide thereof
  • geranylgeraniol (GG0H) having 20 carbon atoms are phytol, vitamin, etc.
  • E 'It is a synthetic substrate for vitamin K and ubiquinone (CoQ).
  • Heptabrenyl alcohol having 35 carbon atoms, decaprenyl alcohol having 50 carbon atoms, and the like are also useful as pharmaceutical intermediates, and the above-mentioned FPP, GGPP, F0H, GG0H, etc. can be used as a synthesis starting material.
  • prenyl phosphoric acid when prenyl phosphoric acid is compared with prenyl alcohol, prenyl phosphoric acid generally has relatively high water solubility, but has a disadvantage that extraction and purification are not easy because of partial non-polar groups. It is more advantageous to produce a prenyl alcohol that can be easily extracted and purified with a non-polar organic solvent such as pentane. Furthermore, prenyl alcohol is required for chemical synthesis of compounds having a farnesyl group and a geranylgerael group. Are used.
  • prenyl alcohols having a biologically active trans-isomeric structure especially those having 15 or more carbon atoms (for example, all trans- Efficient large-scale production of sole, hexaprenyl alcohol, heptabrenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, pendecaprenyl alcohol, F0H, GG0H) It is desired to provide a method for doing this.
  • JP-B-63-17437 discloses a method for producing polyprenyl alcohol by culturing a Pseudomonas strain.
  • Japanese Patent Application Laid-Open No. Hei 10-248575 discloses that a gene for clothing G-CoA (hydroxymethyldaltaryl-CoA) reductase involved in the process of converting acetyl CoA to MVP or MVPP is yeast (Can dida util is).
  • a method for producing carotenoids by introducing the method into carotenoids is disclosed.
  • JP-A-5-115298 discloses a method for increasing the accumulation of sterols in plants by increasing the copy number of the HMG-CoA reductase gene.
  • 5-192184 discloses that in a mutant yeast having a deficiency in sterol biosynthetic enzyme expression, accumulation of squalene sterol is increased by increasing the expression level of a HMG-CoA reductase gene.
  • a method is disclosed.
  • JP-A-10-155497 discloses a carotenoid production method using a fermentation method with a microorganism into which a GGPP synthase gene (crtE) has been introduced.
  • crtE GGPP synthase gene
  • JP-A-53-127889 discloses a method for producing prenyl alcohol in vitro using phosphatase isolated from potato roots. But, This method is not a method utilizing biosynthesis, and there is no disclosure of the use of a phosphatase gene.
  • phosphatases with prenyl phosphate dephosphorylation activity have been reported on prenyl virophosphatase (EC 3.1.7.1) present in rat (Rattus norvegicus) liver microsomes (Bansal, VS et. Al.). (1994) Arch. Biochera. Biophys. 315 (2), 393-399), but not isolated. Report that diasylglycerol diphosphate phosphatase encoded by the DPP1 gene of yeast cerevisia e) acts on IPP, GPP, FPP and GGPP (Faulkner, A. et. Al (1999) J, Biol. Chem.
  • An object of the present invention is to provide a phosphatase which is effective when it is desired to selectively mass-produce prenyl alcohol having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, By controlling the expression of enzymes involved in prenyl alcohol biosynthesis in living cells, recombinants into which such a polynucleotide has been introduced, prenyl alcohol (particularly having a biologically active trans isomeric structure, An object of the present invention is to provide a method for efficiently mass-producing, for example, FOH, GG0H) having 15 or more carbon atoms. Disclosure of the invention
  • the first invention of the present application is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOS: 1 to 6, and having a phosphatase activity having substrate specificity to prenyl linic acid having 15 or more carbon atoms.
  • the polypeptide of the first invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Therefore, the polypeptide of the first invention enables efficient selective production of prenyl alcohols having 15 or more carbon atoms, such as FOH, GG0H, heptaprenyl alcohol, decaprenyl alcohol and the like.
  • the second invention of the present application relates to a prenyl phosphate having an amino acid sequence in which 5 or less amino acids are substituted, deleted or added in the amino acid sequence shown in any one of SEQ ID NOS: 1 to 6, and having 15 or more carbon atoms. It is a polypeptide that exhibits phosphatase activity with substrate specificity.
  • At least one of the following formulas (1) to (3) is used. It is a polypeptide that contains a phosphatase motif and has phosphatase activity with substrate specificity to prenyl phosphate having 15 or more carbon atoms.
  • the inventors of the present invention have found that (3) to (4) in the third invention A conserved sequence common to these polypeptides, defined as 3), was found. The details will be described later with reference to FIG. 1, but this conserved sequence is considered to be a sequence (phosphatase motif) necessary for expressing a phosphatase activity with substrate specificity to prenyl phosphate having 15 or more carbon atoms. . Therefore, it is considered that the polypeptide according to the third invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
  • any one or more amino acid residues of a specified type are subjected to conservative substitution.
  • Polypeptide here, the “amino acid residue whose type is specified” means an amino acid residue represented by a symbol other than “X” in the above formulas (1) to (3).
  • a “conservative substitution” also refers to the replacement of a given amino acid residue with another chemically or functionally similar amino acid such that the function of the polypeptide remains substantially substantially unchanged. Substitution with a residue.
  • Examples of chemically or functionally similar amino acids include hydrophobic amino acids (Ala, lie, Leu, Phe, Pro, Trp, Val, Met), polar but uncharged amino acids (Asn, Cys, Gin, Gly, Ser, Thr, Tyr), basic amino acids (Arg, His, Lys), acid amino acids (Asp, Glu), etc. (by Corn Corn et al., Translated by Nobuo Tazaki et al., “Corn Stamp” Biochemistry 5th Edition, Tokyo Chemical Dojin, p56-58, 1988).
  • the polypeptide according to the fourth invention has the same number of carbon atoms as the polypeptide according to the third invention, since any one or more amino acid residues specified in the phosphatase motif have undergone conservative substitution. It is considered to be a phosphatase with substrate specificity for 15 or more prenyl phosphates.
  • the fifth invention of the present application is a polynucleotide encoding a phosphatase having the base sequence shown in any one of SEQ ID NOs: 7 to 12 and having substrate specificity to prenyl phosphate having 15 or more carbon atoms. is there.
  • the polynucleotide encoded by the polynucleotide according to the fifth invention is a phosphatase having substrate specificity for prenyl phosphoric acid having 15 or more carbon atoms. Therefore, by introducing or enhancing the expression of the polynucleotide of the fifth invention into a living cell or organism, a phosphatase having a substrate specificity for prenyl phosphate having 15 or more carbon atoms is produced in the living cell or organism, Prenyl alcohols with 15 or more carbon atoms can be produced effectively.
  • the sixth invention of the present application has a nucleotide sequence that hybridizes to a nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12 or a nucleotide sequence complementary thereto under predetermined stringent conditions. It is a polynucleotide encoding a phosphatase having substrate specificity for prenyl phosphoric acid having 15 or more carbon atoms.
  • a seventh invention of the present application is a polynucleotide encoding any of the polypeptides according to the first to fourth inventions.
  • An eighth invention of the present application is a recombinant nucleic acid containing the polynucleotide according to any one of the fifth to seventh inventions.
  • the eighth invention provides a powerful means for introducing the polynucleotide according to the fifth invention to the seventh invention into a living cell or an organism.
  • any one of the recombinant nucleic acids according to the eighth invention is introduced. Is a recombinant.
  • prenyl alcohol having 15 or more carbon atoms can be more selectively produced.
  • a tenth invention of the present application is the recombinant, wherein the host cell of the recombinant according to the ninth invention is any of a fungus ycetes), an ascomycetes (Ascomycetes), and a unicellular eukaryote.
  • any of fungi Esumycetes
  • ascomycetes Ascomycetes
  • unicellular eukaryotes can be preferably used.
  • the eleventh invention of the present application is a recombinant wherein the host cell of the recombinant according to the ninth invention is a yeast.
  • yeast can be preferably used as the host of the recombinant according to the ninth invention.
  • the 12th invention of the present application is a recombinant wherein the yeast according to the 11th invention is a yeast of the genus Saccharomyces.
  • yeast according to the eleventh invention for example, yeast of the genus Saccharomyces can be preferably used.
  • a thirteenth invention of the present application is the yeast according to the eleventh invention, wherein the yeast is Saccharomyces cerevisiae YPH499, YPH500, A451, W303-1A, W303-1B, or a strain derived therefrom. A recombinant.
  • yeast according to the eleventh invention for example, preferably used are Saccharomyces' cerevisiae) YPH499 strain, YPH500 strain, A451 strain, W303-1A strain, W303-1B strain and strains derived therefrom. Can be.
  • the fourteenth invention of the present application is a method for producing pryl alcohol by culturing a host cell into which a phosphatase gene has been introduced or having enhanced expression, and collecting prenyl alcohol from the culture.
  • the present inventor has proposed various prenyl diphosphates from the mevalonate pathway starting from acetyl CoA or the non-mevalonate pathway starting from pyruvate.
  • the biophosphorylation of phosphatase gene into living cells Isomeric structures for example, all trans forms of geranyl pharynesol, hexapreninole alcohol, heptaprenyl alcohol, octapreninoreal alcohol, nonapreninoleanolecole, decaprenyl alcohol, pendecaprenyl
  • the method of the fourth invention is also useful when it is desired to increase the overall production of prenyl alcohol having 5 or more carbon atoms.
  • the inventors of the present invention have found that, in an example using yeast as a host, the productivity of prenyl alcohols having 15 or more carbon atoms (eg, all-transformation F0H, GG0H) can be improved by introducing or enhancing the expression of a phosphatase gene. It has been successfully increased about 40 times. That is, the fourteenth invention provides a method for efficiently mass-producing prenyl alcohol, particularly trans-type prenyl alcohol, especially trans-type prenyl alcohol having 15 or more carbon atoms.
  • the fifteenth invention of the present application relates to a method for producing prenyl alcohol, comprising culturing host cells into which a phosphatase gene and a gene of an enzyme involved in the prenyl diphosphate biosynthesis pathway have been introduced or having enhanced expression, and collecting prenyl alcohol from the culture. is there.
  • the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway are introduced or enhanced in host cells, that is, the phosphatase gene is introduced or enhanced in expression and the prenyl diphosphate biosynthesis pathway is enhanced
  • the productivity of prenyl alcohol may be further increased. Even if only the gene encoding the enzyme involved in the prenyl diphosphate biosynthesis pathway is introduced or enhanced, its contribution to the efficient mass production of prenyl alcohols (particularly prenyl alcohols having 15 or more carbon atoms, such as F0H and GG0H) is not Limited.
  • a sixteenth invention of the present application is a method for producing prenyl alcohol, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway according to the fifteenth invention is the following gene of (4) and / or (5): is there.
  • acetyl-CoA synthase gene acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, Mevalonate diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP (2-C-methyl-D-erythritol 4 -phosphate) Citidylinoletransferase gene, CDP-ME (4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol) kinase gene and MECDP (2-C-methyl-D -erythritol 2, 4-cyclodiphosphate) At
  • genes listed in (4) of the 16th invention are particularly preferable as the genes to be introduced or enhanced in expression together with the phosphatase gene in the 15th invention.
  • the genes listed in (16) of the 16th invention are also preferable.
  • a seventeenth invention of the present application is the phosphatase gene according to the fourteenth invention to the sixteenth invention, wherein the phosphatase gene has a substrate specificity to a prenyl phosphate having 15 or more carbon atoms in a hydrolysis activity for a phosphate compound.
  • This is a method for producing prenyl alcohol, which is a gene that encodes lysine.
  • the phosphatase gene to be introduced or enhanced in expression is a gene encoding a phosphatase having substrate specificity to prenyl phosphate having 15 or more carbon atoms
  • prenyl having 15 or more carbon atoms The effect is particularly significant for the purpose of efficient mass production of alcohol.
  • prenyl alcohols prenyl alcohols having various biologically active isomeric structures exemplified in the section of “Function and Effect of the Fourteenth Invention” can be particularly preferably exemplified.
  • the eighteenth invention of the present application is directed to a method of the present invention, wherein the phosphatase gene according to the fourteenth invention to the sixteenth invention is any of the above-described fifth to eighth inventions. It is a manufacturing method.
  • the phosphatase gene according to the fifth to eighth inventions can be preferably used as the phosphatase gene introduced or enhanced in the fourteenth to sixteenth inventions.
  • a nineteenth invention of the present application is the phosphatase gene according to the fourteenth invention to the sixteenth invention, wherein the phosphatase gene has a substrate specificity to preninolenic acid having 15 or more carbon atoms in a hydrolysis activity on a phosphate compound.
  • This is a method for producing prenyl alcohol which is a gene encoding a phosphatase other than phosphatase.
  • the phosphatase gene to be introduced or enhanced in expression is a gene encoding a phosphatase other than a phosphatase having substrate specificity to pre-phosphate having 15 or more carbon atoms, Various prenyl alcohols having a carbon number ranging from 5 to 15 can be effectively obtained.
  • the 20th invention of the present application is the method for producing prenyl alcohol, wherein the host cell according to the 14th invention to the 19th invention is any one of the host cells according to the 10th invention to the 13th invention. is there.
  • any of the host cells according to the tenth to thirteenth inventions can be preferably used as the host cells in the fourteenth to nineteenth inventions.
  • the twenty-first invention of the present application is a method for producing prenyl alcohol, wherein the prenyl alcohol according to the fourteenth to twenty-fifth inventions has 15 or more carbon atoms.
  • prenyl alcohol having 5 or 10 carbon atoms can be collected, but prenyl alcohol having 15 or more carbon atoms is relatively produced. It is large in amount, easy to extract and purify with a non-polar organic solvent, and has high utility as described above. Therefore, it is particularly effective to collect prenyl alcohol having 15 or more carbon atoms. All prenyl alcohols having 15 or more carbon atoms can be collected, or one or more of them can be selectively collected.
  • the twenty-second invention of the present application is the prenyl alcohol according to the twenty-first invention, wherein the prenyl alcohol is fuarnesol, geranylgeranol, geranylfurnesol, hexaprenyl alcohol, heptaprenyl alcohol, octaprenilanoleanol,
  • This is a method for producing prenyl alcohol, which is one or more prenyl alcohols selected from nonaprenylanolecol, decaprenyl alcohol, pendecapreninoleanololecol and dodecaprenyl alcohol.
  • prenyl alcohol having 15 or more carbon atoms collected in the twenty-first invention one or more prenyl alcohols selected from the above prenyl alcohols are particularly preferably exemplified.
  • the twenty-third invention of the present application is the method for producing prenyl alcohol wherein the prenyl alcohol according to the twenty-second invention to the twenty-second invention is an all-trans type.
  • FIG. 1 is a view showing a conserved sequence of each polypeptide according to the present invention.
  • FIG. 2 shows the results of quantification of prenyl alcohol production.
  • the polypeptide according to the present invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
  • An example of a preferable criterion for such a phosphatase can be defined as follows. That is, when the specific activity for (A) shown in the enzyme activity measurement system using only prenyl phosphate (A) having 15 or more carbon atoms as a substrate is 100,
  • the specific activity to (A) when (B) coexists is 80 or more, more preferable. Or more preferably 85 or more, more preferably 90 or more.
  • a typical example of the polypeptide having substrate specificity to prenyl phosphate having 15 or more carbon atoms according to the present invention is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOS: 1 to 6.
  • the present inventors have found for the first time that these polypeptides are phosphatases having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is a phosphatidic acid phosphatase homolog gene present in the genomic DNA of Arabidopsis thaliana (Rabidopsis thaliana) [Gen Bank accession No .: AC006200 (complement (1677 8.. 17686)), This gene was named AtPAPl].
  • SEQ ID NO: 7 shows the nucleotide sequence of AtPAPl.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is a phosphatidic acid phosphatase homolog gene present in genomic DNA of L thaliana [Gen Bank accession No .: AC00 7591 (3610... 5006); Phosphatase encoded by “AtPAP2”.
  • SEQ ID NO: 8 shows the nucleotide sequence of AtPAP2.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 3 is a phosphatase encoded by the gene DPP1 of Saccharomyces cerevisiae (Toke DA et al (1998) J. Biol. Chem. 273 (6 ), 3278-3284).
  • Gen Bank accession No. of DPP1 is NC 001136, and its nucleotide sequence is shown in SEQ ID NO: 9.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is a phosphatase encoded by the yeast gene LPP1 (Toke DA et al (1998) J. Biol. Chem. 273 (23), 14331-14338).
  • the LPP1 Gen Bank accession No. is NC001136, and its nucleotide sequence is shown in SEQ ID NO: 10.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 5 is a phosphatase encoded by the gene PAP2 of rat norve gicus (Brindley DN et al (1998) J. Biol. Chem. 273 (38), 24281-24284) .
  • the PAP2 Gen Bank accession number is U90556, and its nucleotide sequence is shown in SEQ ID NO: 11.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 6 is found in norvegicus jfr gene Dri42. (Bari la D et al (1996) J. Biol.
  • the polypeptides of SEQ ID NO: 1 to SEQ ID NO: 6 are distinguished by the notation of each of the genes encoding them (that is, for example, “the notation of DPPl j represents the polypeptide of SEQ ID NO: 3).
  • the conserved sequences found in each of these polypeptides are shown, and the amino acid sequence common to these conserved sequences is shown in the bottom line of FIG.
  • 1 is selected from amino acid residues S or T
  • 2 is selected from amino acid residues V or T
  • 3 is selected from amino acid residues G or A
  • X is an arbitrary one.
  • Nl represents an arbitrary number (for example, 34 to 39) of arbitrary amino acid residues
  • n2 represents an arbitrary number (for example, 38 to 46) of arbitrary amino acid residues. Representing the Roh acid residues.
  • At least one or more of the sequence portions (1) to (3) defined in the third invention are: Alternatively, it is considered that all of them are sequences (phosphatase motifs) necessary for expressing a phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
  • one or more of the specified amino acid residues is a mutant that has undergone the “conservative substitution” according to the above definition.
  • Polypeptides are also considered to exhibit substrate-specific phosphatase activity for prenyl phosphates having 15 or more carbon atoms.
  • polypeptide according to the present invention is an amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6, which has an amino acid sequence in which 5 or less amino acids have been substituted, deleted or added, and Phospho with substrate specificity for more than 15 prenyl phosphates It is a polypeptide that exhibits an activityse activity.
  • 5 or less amino acids to be substituted, deleted or added may be all amino acids at positions adjacent to each other in the amino acid sequence.
  • the above-mentioned five or less amino acids may be partially adjacent amino acids in the amino acid sequence.
  • any of the above five or less amino acids may be amino acids that are not adjacent to each other in the amino acid sequence.
  • the above-listed polypeptides can be obtained directly from the cells of each of the above-mentioned source organisms by conventional enzyme isolation means, or the DNA encoding each polypeptide can be introduced into a host. Thus, a large amount can be obtained in the same manner.
  • the phosphatase encoded by the phosphatase gene to be introduced or enhanced in expression is not limited as long as it acts on at least prenyl phosphate. That is, from acid phosphatase with low substrate specificity to alkaline phosphatase, etc., to phosphatase having substrate specificity for prenyl phosphinate, and further to phosphatase having substrate specificity for prenyl phosphine having 15 or more carbon atoms. Phosphatases of varying levels of substrate specificity are included without limitation.
  • a phosphatase having a higher substrate specificity particularly a phosphatase having a substrate specificity to prenyl phosphate having 15 or more carbon atoms.
  • the phosphatase having high substrate specificity for prenyl phosphate include the polypeptides according to the first to fourth inventions.
  • Preferable examples include yeast, animal or plant-derived polypeptides having equivalent functions.
  • the polynucleotide refers to single-stranded, double-stranded or triple-stranded DNA and Z or RNA.
  • the term “recombinant nucleic acid” refers to a polynucleotide prepared for recombination.
  • the polynucleotide according to the present invention has a substrate of prenyl phosphate having 15 or more carbon atoms.
  • a typical example of the polynucleotide according to the present invention is a polynucleotide having a base sequence shown in any one of SEQ ID NOS: 7 to 12. It has been found for the first time by the present inventors that these encode polypeptides having phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 7 is AtPAPl.
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 8 is AtPAP2.
  • AtPAPl is known to be located in the second section of chromosome II published in the Arabidopsis thaliana genome project (Lin et al (1997) Nature 402, p. 767-768), and AtPAP2 is located on chromosome I. It is known that it is located in the BAC library F9L1 (F9L1.2 (Accession No., AC007591)).
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 9 is DPP1.
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 10 is LPP1 described above.
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 11 is PAP2.
  • the polynucleotide having the nucleotide sequence of SEQ ID NO: 12 is Dri42 described above.
  • polynucleotide according to the present invention is a part of the conserved sequence represented by the sequence notation of the multiple alignment shown in FIG. 1 (part of the sequence portion (1) to (3) defined in the third invention). Or at least one of the above) or a polypeptide having an amino acid sequence that includes the entire amino acid sequence, or a polypeptide in which at least one of the amino acid residues specified therein has been conservatively substituted.
  • the coding polynucleotide is a part of the conserved sequence represented by the sequence notation of the multiple alignment shown in FIG. 1 (part of the sequence portion (1) to (3) defined in the third invention). Or at least one of the above) or a polypeptide having an amino acid sequence that includes the entire amino acid sequence, or a polypeptide in which at least one of the amino acid residues specified therein has been conservatively substituted.
  • Still another representative example of the polynucleotide according to the present invention has a nucleotide sequence that hybridizes under a predetermined stringent condition with the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12, and has a carbon number of It is a polynucleotide encoding a phosphatase having substrate specificity for 15 or more prenyl phosphates.
  • hybridize under stringent conditions refers to an appropriate hybridization method such as colony hybridization, plaque hybridization, or Southern blot hybridization.
  • the other polynucleotide (thigh) can hybridize to one polynucleotide (DNA) or a fragment of the polynucleotide under the following conditions. That is, one polynucleotide or a fragment of the polynucleotide immobilized on the filter is subjected to hybridization of the other polynucleotide at a predetermined temperature (X ° C) in the presence of 0.7 to 1 M NaCl.
  • ° C.” is at least 50 ° C. or more, more preferably 60 ° C. or more, and further preferably 65 ° C. or more.
  • the above-listed polynucleotides can be obtained from the above-mentioned source organisms by conventional isolation means, or may be obtained from various unicellular organisms such as prokaryotes and animal and plant cells. Available methods well known to those skilled in the art include hybridization technology (Southern 1975 J. Mol. Biol. 98: 503, Maniatis et al. Molecular Cloning Cold Spring Harbor Laboratory Pres) and PCR technology (Saiki et al. Science 239: 487 (1988)).
  • a polynucleotide having a high homology to a known gene or a part thereof as a probe or an oligonucleotide hybridizing to a part of the nucleotide sequence as a primer It is easy to isolate and clone the phosphatase gene from the polynucleotide. It is also easy to isolate the phosphatase using the characteristic phosphatase activity as an index and clone the phosphatase gene from its amino acid sequence.
  • the phosphatase gene introduced or enhanced in the method for producing prenyl alcohol according to the fourteenth invention is not limited as long as it is a gene encoding a phosphatase that acts on at least prenyl phosphate. That is, a variety of substances, from acid phosphatase with low substrate specificity to alkaline phosphatase, to phosphatase having substrate specificity for prenyl phosphate, and phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Phos with a high level of substrate specificity A gene encoding a datase is included without limitation.
  • a gene encoding a phosphatase having a higher substrate specificity particularly a phosphatase having a substrate specificity to prenyl phosphate having 15 or more carbon atoms.
  • the polynucleotides according to the fifth to seventh inventions can be preferably exemplified.
  • Yeast, plant or animal-derived polynucleotides having equivalent functions can also be preferably exemplified.
  • a gene encoding the above-mentioned non-specific phosphatase more preferably a gene encoding a phosphatase having substrate specificity for prenyrphosphate, more preferably a prenylphosphate having 15 or more carbon atoms
  • a gene encoding a phosphatase having specific substrate specificity is introduced into a host cell or its expression is enhanced.
  • introducing a phosphatase gene or enhancing expression has the following meaning. That is, “introducing a phosphatase gene” includes all cases where the phosphatase gene is introduced into a host cell as a recombinant nucleic acid. At least the case where the phosphatase gene is introduced by a vector and the case where the phosphatase gene is introduced by homologous recombination of a polynucleotide using a PCR fragment or the like are included. More preferably, the phosphatase gene is introduced by an expression vector that enhances expression.
  • the expression “enhancement of the expression of the phosphatase gene J” includes all cases in which the expression of the phosphatase gene is consequently enhanced.
  • the expression of the phosphatase gene is combined with a recombinant nucleic acid together with a means for enhancing the expression.
  • the expression of the phosphatase gene is enhanced by adjusting the culture conditions of the host cell (adding an inducer of gene expression, etc.). In this case, etc. are included.
  • At the same time as introducing or enhancing the expression of the various phosphatase-encoding genes at least one gene among the enzymes involved in the prenyl diphosphate biosynthesis pathway is introduced or enhanced in the host cell. can do.
  • a gene encoding a phosphatase and a gene encoding an enzyme involved in the prenyl diphosphate biosynthesis pathway The mode of simultaneous introduction or expression enhancement is arbitrary.For example, these genes may be simultaneously or separately introduced or enhanced by using the same or different means as described above, or as described later.
  • Genes of enzymes involved in the prenyl diphosphate biosynthetic pathway that may be simultaneously introduced or enhanced in expression as a linked gene include, for example, the gene for the oxamethylxartalaryl CoA reductase, Preferred examples include at least one gene selected from germline and geranirgerinirnic acid synthase genes.
  • acetyl CoA synthase gene acetyl CoA-acetyl transferase gene, hydroxymethyldanoletaryl CoA synthase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate decarboxylase gene, isopentene Selected from luniphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene and MECDP synthase gene Preferred examples include at least one gene. .
  • genes encoding the above various phosphatases can be linked to genes of enzymes involved in the prenyl diphosphate biosynthesis pathway, and introduced or enhanced in host cells in the same manner as described above.
  • Preferred examples of the gene of an enzyme involved in the prenyl diphosphate biosynthetic pathway include at least one gene selected from, for example, a huanesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene.
  • acetyl-CoA synthase gene acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethyldaltharyl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalon Acid diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP cityryltransferase gene, CDP-ME kinase gene Alternatively, at least one gene selected from MECDP synthase genes can also be preferably exemplified.
  • the gene linked in this way is also called a “phosphatase linked gene”.
  • the linked gene is a gene in which two or more genes are linked so that they can be expressed as one open reading frame (0RF), and the expressed polypeptide becomes a fusion protein.
  • an artificial nucleotide sequence is used so that the three-dimensional structure in which each gene translation product functions properly can be obtained in the connecting region between the linked genes.
  • Linker sequence can be freely introduced. Examples include Gly Gly Gly Ser and a linker sequence encoding a peptide sequence of Gly Gly Gly Gly Ser. These sequences were used as a linker sequence linking the H and L chains when expressing antibodies in E. coli (Huston J. S. et al (1988) Proc. Natl. Acad.
  • the expression vector or the polynucleotide fragment having the expression function of the gene to be introduced is linked to various polynucleotide fragments that enhance the expression of the phosphatase gene or phosphatase-ligating gene to be introduced.
  • the expression vector may include a transcription promoter, a transcription terminator, a marker gene for selecting a recombinant, and an enhancer.
  • Plant expression vectors may more preferably contain a T-DNA region.
  • a general method for constructing an expression vector for example, a gene fragment prepared by a PCR method or the like can be incorporated into an expression vector by a known method using an appropriate restriction enzyme and ligase.
  • the cerevi s iae is often used in the case of the host "YEpl3", “YEp24”, “YCp50”, r p YES2j, r p RS414j , "P RS415J, r P RS416J,” pRS413 , PRS404, pRS405, pRS406, pRS403j,
  • the expression vector may contain a transcription promoter for constitutively or inducibly expressing phosphatase.
  • a transcription promoter for constitutively or inducibly expressing phosphatase.
  • Used alcohol dehydrogenase genes ADH1 and ADH2
  • TDH3 triosephosphate dehydrogenase gene
  • GAL1, GAL7, GAL10 genes associated with galactose catabolism
  • PH05 acid phosphatase gene
  • CUP1 meta-mouth thionein gene
  • transcription promoters such as trp, lac, trc, and tac can be used.
  • transcription promoters for the constant expression include, for example, the cauliflower mosaic virus 35S promoter (Odel et al. 1985 Nature 313: 810) and the rice actin promoter (Zhang et al. 1991). Plant Cell 3: 3 155), corn ubiquitin promoter (Cornejo et al 1993 Plant Mol. Biol. 23: 567), and various other types can be used without limitation.
  • Promoters for inducible expression include transcription known to be expressed by exogenous factors such as infection or invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, irradiation with ultraviolet light, and spraying of specific compounds. Promoters and the like.
  • a recombinant nucleic acid for introducing or enhancing the expression of a phosphatase gene or a phosphatase-linked gene does not necessarily have to have a vector function, but may be any one that can perform genomic integration, for example.
  • a phosphatase gene or a phosphatase-linked gene linked to an expression vector or other recombinant nucleic acid can contain a base sequence region that adds a localization signal to the vicinity of a lipid membrane to phosphatase.
  • Prenyl phosphate (especially prenyl phosphate having 15 or more carbon atoms) is hydrophobic and is located near the cell membrane of host cells and in the cell membrane of various organs such as the Golgi body and endoplasmic reticulum when the host cell is a eukaryotic cell. Tends to be localized at higher concentrations. Therefore, by adding a localization signal such as a hydrophobic peptide to phosphatase, the probability of contact between phosphatase and prenyl phosphinate as a substrate can be improved, and the productivity of prenyl alcohol can be increased. In particular, when the phosphatase gene originally encodes a substrate non-specific phosphatase, the addition of the above signal to the phosphatase is effective.
  • Representative localization signals include various known hydrophobic signal peptides. You. As is well known, it is also possible to select a phosphatase localization site in a host cell by selecting a localization signal.
  • As a general method for constructing an expression vector or other recombinant nucleic acid with the addition of a nucleotide sequence region for adding a localization signal to a phosphatase gene or a phosphatase-linked gene for example, various known signals may be used.
  • a gene domain encoding a peptide and a phosphatase gene or a phosphatase-linked gene are prepared by PCR or the like, and these genes are ligated by a known method using an appropriate restriction enzyme and ligase. The gene can be incorporated into a suitable expression vector.
  • the type of host cell is not limited.
  • prokaryotes, fungi (Eumvcetes), ascomycetes (Ascomvcetes), unicellular eukaryotes, or living cells of animals or plants can be arbitrarily selected.
  • Live tissue culture cells of multicellular organisms including plant tissue culture cells and animal tissue culture cells can also be arbitrarily selected.
  • the “plant” includes algae, bryophytes, ferns, gymnosperms and angiosperms.
  • animal cells or animal tissue culture cells the term “animal” includes sponges, coelenterates, nematodes, mollusks, arthropods, arthropods, and various vertebrates.
  • living cells or living tissue culture cells of animals or plants include suspension culture cells of animals and plants, tissue culture cells of animal and plant organs such as viscera, leaves, and roots, and callus in plants.
  • Hosts include, but are not limited to, yeast belonging to the genus Saccharomyces ⁇ ⁇ , such as ⁇ cerevi siae, Pichia pastoris (Pichia pastris), ⁇ colli, ⁇ col i, Bacillus subtilis, Bacillus Bacteria belonging to the genus Escherichia or Bacillus, such as previs ac il lus brevis; Aspergillus oryzae; filamentous fungi belonging to the genus Aspergillus, such as Aspergillus nigar; mori), animal cells such as COS cells or CH0 cells, and plant cells.
  • yeast of the genus Saccharomyces such as ⁇ cerevi siae, Pichia pastoris (Pichia pastris), ⁇ colli, ⁇ col i, Bacillus subtilis, Bacillus Bacteria belonging to the genus Escherichia or Bacillus, such as previs ac il lus brevis
  • ⁇ cerevis iae YPH499 (ATCC76625, MATa, ura3-52, lys2-801, ade2-101, trpl-delta63, his3-delta200, leu2-deltal), YPH500 (ATCC76626, MATalpha, ura3 -52, lys2-801, ade2-101, trpl-delta63, his3-delta200, leu2-deltal), A451 (ATCC200589, MATalpha, canl, leu2, trpl, ura3, aro7), W303-1A (ATCC208353, MATalpha , leu2-3, leu2-112, Ms3-11, ade2-1, ura3-1, trpl-1, canl-100) or W303-IB MATa, leu2-3, leu2-112, his3-11, ade2-1 haploid of W303 (ATCC201239)) can be particularly preferably exemp
  • a recombinant nucleic acid suitable for the host can be used in various modes as described above. More specifically, as a method for introducing a foreign gene into Escherichia coli, several established methods such as the Hanahan method can be used. Several well-established methods, such as the lithium method, can be used for introducing a foreign gene into yeast. In introducing the phosphatase gene, it can be integrated into the chromosome of the host cell, or can be retained in the cell as a plasmid or the like.
  • the method for producing prenyl alcohol according to the present invention is advantageous for improving the productivity of prenyl alcohol in general, the productivity of prenyl alcohol having 10 or more carbon atoms, particularly, prenyl alcohol having 15 or more carbon atoms is remarkably improved.
  • the method of the fifteenth invention is particularly effective when the improvement in the productivity of prenyl alcohols having 15 or more carbon atoms, particularly F0H and GG0H in the method for producing prenyl alcohol is generally compared. Further, the method of the thirteenth invention and the method of the nineteenth invention also improve the productivity of prenyl alcohol.
  • the method for producing prenyl alcohol according to the present invention has an advantage that an industrially useful trans prenyl alcohol can be produced.
  • the “trans form” means a trans form in prenyl alcohol having 5 carbon atoms and an all trans form in prenyl alcohol having 10 or more carbon atoms.
  • Factors such as selection of the method of introducing the plasmid and the method of constructing a polynucleotide suitable for the method, selection of the type and concentration of the medium or the additive thereto, and selection of the culture or growth conditions for the recombinant, may affect the production of prenyl alcohol. May affect quantity.
  • prenyl alcohol When collecting prenyl alcohol from a culture, collect prenyl alcohol in general, or a specific prenyl alcohol such as prenyl alcohol having 15 or more carbon atoms or only one or more of them. Whether to selectively collect more than one kind of prenyl alcohol can be arbitrarily determined depending on the purpose of the method for producing a prenyl alcohol.
  • the produced prenyl alcohol can be directly extracted from the culture using an appropriate organic solvent, in addition to the usual extraction from cell debris.
  • an appropriate organic solvent such as an yeast is used as the host
  • at least a portion of the prenyl alcohol may remain in the host cell or on the cell surface, but the cell membrane or cell wall is destroyed, and Extraction can be easily performed through various known operations such as extraction with a non-polar organic solvent such as pentane and pentane. Examples Hosts, vectors, etc.
  • JM109 competent cells (Takara) were used as a host.
  • cloning a DNA fragment by introducing it into a pRS vector or pALTER vector,! : _ Col i SURE2 supercompetent cells (Stratagene) or ⁇ col i HB101 competent cells (Takara) were used as hosts.
  • Y. cerevisiae strains YPH499, A451, and W303-1B were used as the host for gene transfer.
  • the CYClt fragment one of the CYC1 transcription terminators, was prepared by PCR.
  • a PCR primer having the nucleotide sequence shown in SEQ ID NO: 13 (Xhol-TcyclFW), a PCR primer having the nucleotide sequence shown in SEQ ID NO: 14 (Kpnl-TcyclRV), and a PCR primer having the nucleotide sequence shown in SEQ ID NO: 15 ( Apal-TcyclRV) was used according to the combination of the Xhol-TcyclFW and Kpnl-TcyclRV and the combination of the Xhol-TcyclFW and Apal-TcyclRV, and PYES2 was used as a ⁇ type.
  • the reaction solution 0.
  • the two amplified DNAs were cut with restriction enzymes Xhol and Kpnl or Xhol and Apal, respectively, and a 260 bp DNA fragment was purified by agarose gel electrophoresis. These DNA fragments are called CYClt-XK and CYCltXA.
  • CYClt- ⁇ was inserted into the Xhol-Kpnl cleavage site of PRS405, and CYCltXA was inserted into the Xhol-ApaI cleavage site of pRS404 and pRS406. These are called pRS405Tcyc, pRS404Tcyc and pRS406Tcyc, respectively.
  • a primer having the nucleotide sequence shown in SEQ ID NO: 16 (Sacl-Ptdh3FW), a primer having the nucleotide sequence shown in SEQ ID NO: 17 (SacII-Ptdh3RV), a primer having the nucleotide sequence shown in SEQ ID NO: 18 (Sacl-Ptef2FW) and a primer (SacII-Ptef2RV) having the nucleotide sequence shown in SEQ ID NO: 19.
  • Sacl-Ptdh3FW and SacII-Ptdh3RV were used as primers, and for amplification of TEF2p, Sacl-Ptef2FW and SacII-Ptef 2RV were used as primers.
  • type I cerevisiae genomic DNA was used.
  • the reaction solution contains 0.46 ii g of ⁇ cerevisiae genomic DNA, lOOpmol DNA, IX ExTaq buffer (Takara), 20 strokes 1 dNTP, 0.5 U ExTaq DNA polymerase (Takara), 1 ⁇ L perfect match polymerase enhancer 100 A ju L solution was prepared, and PCR was performed as follows.
  • the amplified DNA was cut with restriction enzymes Sacl and SacII, and a 680 bp, 400 b DNA fragment was purified by agarose gel electrophoresis. These are called TDH3p and TEF2p.
  • the 1.5 kbp fragment containing the 2 ⁇ replication origin (2 ⁇ ori) was purified by agarose gel electrophoresis. And blunt-ended with Klenow enzyme. This DNA fragment is called S ⁇ OriSN.
  • pRS404Tcyc, pRS405Tcyc ⁇ Insert 2 ⁇ u0riSN into the Nael cleavage site obtained by treating pRS406Tcyc with BAP (bacterial alkaline phosphatase, Takara), introduce it into coliSURE2, and transform it into plasmid DNA. .
  • BAP bacterial alkaline phosphatase, Takara
  • Drall and EcoRI, Hpal, or with Pstl and PvuII were cut with the restriction enzymes Drall and EcoRI, Hpal, or with Pstl and PvuII, and then subjected to agarose gel electrophoresis to check for the presence of 2 ⁇ and its orientation.
  • PRS404Tcyc, pRS405Tcyc, pRS406Tcyc i, and plasmids with the same orientation as pYES2, p ⁇ 2 ⁇ ori inserted, are called pRS434Tcyc 2 ⁇ Ori, pRS435Tcyc 2 ⁇ Ori, RS436Tcyc 2 ⁇ Ori, and pRS405Tcyc
  • the plasmid into which 2 ⁇ ori was inserted in the opposite direction is called pRS445Tcyc 2 ⁇ 0.
  • pRS434GAP and PRS434TEF were obtained from pRS434Tcyc 2 ⁇ ori, pRS435GAP from RS435Tcyc 2 ⁇ Ori, pRS436GAP power from pRS436Tcyc 2 Ori power, and RS445Tcyc 2 ⁇ Ori power from pRS445GAP power, respectively.
  • the YEp-type expression vectors derived from each pRS vector prepared here are collectively referred to as pRS expression vectors.
  • the ⁇ _cerevisiae cDNA was transformed into type III by PCR, and an approximately 3.2 kbp fragment of ⁇ cerevisiae HMG1 gene was amplified.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 20 and a primer having the nucleotide sequence shown in SEQ ID NO: 21. After the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pT7Blue-T by T / A ligation. The prepared plasmid is called PT7HMG1.
  • the nucleotide sequence of PT7HMG1 was determined using the 373A DNA sequencer (Perkin Elmer). Of 12 PCR errors Was issued. The notation of these PCR errors is, for example, when the notation “c203t” is used, the start of the base sequence registered in SGD (Saccharomyces Genome Database, htt: // genome-www. Stanford. Edu / Sac charomyces) This indicates that the 203rd base “c” when the a of the codon atg is the first base is changed to the base “t” due to an error. In other cases, the same notation is used.
  • substitution mutations of S68F, L607S, and H909R were introduced into the encoded amino acid residues of c203t, t820c, and a2726g, respectively.
  • the notation of these substitution mutations is, for example, when “S 68F” is written, in the amino acid sequence based on the nucleotide sequence registered in the above SGD, the 68th amino acid residue “S” is replaced by “F” due to an error. ”. In other cases, the same notation is used.
  • Other PCR errors were silent mutations in which the encoded amino acid residues were unchanged.
  • the amino acid substitution mutation caused by the PCR error was corrected by site-directed mutagenesis as follows.
  • the site-specific mutation was carried out by the method described in the Protocols and applications guide, third edition, 1996 Promega, ISB 1-882274-57-1, published by Promega.
  • pT7HMGl was digested with restriction enzymes Smal, ApaLl, and Sail, and a 3.2 kbp HMG1 fragment was prepared by agarose gel electrophoresis. These fragments were inserted into the Smal-Sail cleavage site of PALTER-1 to generate pALHMGl.
  • nucleotide sequence of the above mutagenicoligo HM Gl (190-216) is shown in SEQ ID NO: 22
  • nucleotide sequence of HMG1 (1807-1833) is shown in SEQ ID NO: 23
  • nucleotide sequence of HMG1 (2713-2739) is shown in SEQ ID NO: 23. This is shown in SEQ ID NO: 24, respectively.
  • pAL Called HMG106 The plasmid whose sequence in HMG1 has been modified is called pAL Called HMG106.
  • pALHMG106 was digested with restriction enzymes Smal and Sail, a 3.2kbp HMGl gene fragment was purified by agarose gel electrophoresis. This was inserted into the Smal-Sall cleavage site of pRS434GAP and pRS434TE.
  • the physical map of the subcloned plasmid was checked by mapping the restriction enzymes XhoI, Spel, Nael, and Sphl, and confirming the base sequence of the border region of the inserted 3.2 kbp HMG1 gene fragment. Then, we selected the plasmids that could be produced as planned. The selected plasmids are called pRS434GAP-HMGl and pRS434TEF-HMGl, respectively.
  • An expression vector for a gene in which a part of the HMG1 coding region was deleted was prepared by the following method.
  • a BamHI-Sall fragment containing HMG1 was prepared from pT7HMGl and inserted into the BamHI-XhoI cleavage site of pYE S2 to prepare an expression vector pYES-HMGl.
  • a PCR primer primer HMG1 (1191-1165)
  • a PCR primer (primer HMG1 (1267-1293)) having the nucleotide sequence shown in SEQ ID NO: 26
  • pYES- A PCR method was performed using HMGl as type III.
  • the PCR amplification product was blunt-ended with Klenow enzyme, cyclized again by self-ligation, introduced into E. coli JM109, transformed, and plasmid DNA was prepared.
  • the 373A DNA sequencer confirmed that the reading frame of the amino acid upstream and downstream of HMG1 in the obtained plasmid DNA was not displaced, and that no amino acid substitution due to a PCR error occurred near the binding site. .
  • the prepared plasmid is called pYHMG044.
  • the S. cerevisiae cDNA library was converted into type III, and an approximately 0.9 kbp fragment of the IDI1 gene of S. cerevisiae was amplified.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 27 and a primer having the nucleotide sequence shown in SEQ ID NO: 28.
  • IDI1 is in pT7Blue-T LacZ was introduced in the opposite direction.
  • the prepared plasmid DNA is called pT7IDIl.
  • a 0.9 kbp fragment cut out from PT7IDI1 by BamHI-Sall was prepared and inserted into pRS435GAP at the BamHI-Sall cleavage site.
  • the ⁇ _cerevisiae cDNA library was converted into a type II, and an approximately l. Okbp fragment of the BTS1 gene of S. cerevisi was amplified.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 29 and a primer having the nucleotide sequence shown in SEQ ID NO: 30.
  • the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pT7Blue-T by T / A ligation. When the base sequence of the cleaved fragment was determined and compared with the SGD sequence, no PCR errors were found.
  • the prepared plasmid DNA is called PT7BTS1.
  • pYESGGPS was digested with restriction enzymes BamHI and Mlul, and the Okbp fragment was purified by agarose gel electrophoresis. This was inserted into the BamHI-Mlul cleavage site of pRS435GAP and pRS445GAP. These plasmids referred to as pRS43 5 GAP- BTS1, P RS445GAP- BTS1.
  • the ⁇ cerevisiae genomic DNA was converted into type III by PCR, and an approximately 0.9 kbp fragment of the ⁇ cerevisiae gene DPP1 was amplified.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 31 and a primer having the nucleotide sequence shown in SEQ ID NO: 32. After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into pCR2.1-T0P0 by T / A ligation. When the base sequence of the cloned fragment was determined and compared with the SGD sequence, no PCR errors were found. Created The plasmid DNA is called pCR-DPPl.
  • a 0.9 kbp fragment cut out from pCR-DPP1 by SacII-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP and pRS436GAP.
  • the plasmids thus prepared are called pRS434GAP-DPP1 and pRS436GAP-DPP1.
  • ⁇ _Cerevisiae genomic DNA was converted into type III by PCR, and an approximately 0.9 kbp fragment of the S. cerevisiae gene LPP1 was amplified.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 33 and a primer having the nucleotide sequence shown in SEQ ID NO: 34.
  • the PCR fragment was purified by agarose gel electrophoresis and cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cleaved fragment was determined and compared with the SGD sequence, there were no PCR errors.
  • the prepared plasmid DNA is called pCR-LPP1.
  • a 0.9 kbp fragment cut out from pCR-LPP1 by SacI I-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP and pRS436GAP.
  • the plasmids thus prepared are called pRS434GAP-LPP1 and pRS436GAP-LPPl.
  • PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 35 and a primer having the nucleotide sequence shown in SEQ ID NO: 36.
  • a rat liver cDNA library was prepared from rat liver-derived PolyA + RNA (CL0NTECH) using a Marathon cDNA Amplification Kit (CL0NTECH) as type I DNA. After the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of GenBanK, there was no PCR error. The resulting plasmid DNA is called pCR-Dri42.
  • a 0.9 kb P fragment cut out from CR-Dri42 by BamHI-Sail was prepared and inserted into the BaraHI-Sail cleavage site of pR S434GAP and pRS436GAP.
  • the prepared plasmid is called pRS434GAP-Dri42 pRS436GAP-Dri42.
  • PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 37 and a primer having the nucleotide sequence shown in SEQ ID NO: 38.
  • type I DNA rat liver cDNA library was prepared from the above-mentioned PolyA + RNA (CL0NTECH) using Marathon cDNA Amplification Kit (CLONTECH). After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cleaved fragment was determined and compared with the sequence of GenBanK, there was no PCR error. The prepared plasmid DNA is called pCR-PAP2.
  • a 0.9 kbp fragment cut out from pCR-PAP2 by BamHI-EcoRI was prepared and inserted into the BamHI-EcoRI cleavage site of pRS434GAP and pRS436GAP.
  • the prepared plasmids are called PS434GAP-PAP2 and pRS436GAP-PAP2.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 39 and a primer having the nucleotide sequence shown in SEQ ID NO: 40.
  • Type DNA DNA heat-treat 40 ⁇ L of L-thaliana cDNA Uni-ZAP XR Library (STRATAGENE) at 100 ° C for 10 minutes, then extract with phenol, extract with black-mouth form, precipitate with ethanol, and perform ⁇ ⁇ ⁇ TE A solution dissolved in a buffer was used.
  • the obtained PCR fragment was purified by agarose gel electrophoresis, and cloned into pCR2.1-TOP0 by T / A ligation.
  • the generated plasmid DNA is called pCR-AtPAPl.
  • the nucleotide sequence of the cleaved AtPAPl was determined and was as shown in SEQ ID NO: 1.
  • a 0.9 kbp fragment cut out from pCR-AtPAPl by BamHI-Xhol was prepared and inserted into the BamHI-Xhol cleavage site of pRS434GAP and pRS436GAP.
  • the prepared plasmids are called PRS434GAP-AtPAPl and pRS436GAP-AtPAPl.
  • PCR primers include a primer having the nucleotide sequence shown in SEQ ID NO: 41 and a primer having SEQ ID NO: 4 This is a primer having the nucleotide sequence shown in 2.
  • type DNA DNA heat-treat 40 ⁇ L of thaliana cDNA Uni-ZAP XR Library at 100 ° C for 10 minutes, then extract with phenol, extract with clonal form, precipitate with ethanol, and treat with ⁇ TE buffer. The dissolved one was used.
  • pCR-AtPAP2 The generated plasmid DNA is called pCR-AtPAP2.
  • the 0. 9 kbp fragment cut from P CR-AtPAP2 by BamHI- Xhol was prepared and inserted pRS434GAP, the BamHI-XhoI cleavage site of PRS436GAP.
  • the prepared plasmids are called pRS434GAP-AtPAP2 and pRS436GAP-AtPAP2.
  • Frozen EZ yeast transformation kit (Zymo Research) For transformation of S. cereviae.
  • lwg of plasmid DNA was used.
  • the transformed cells were transferred to an SD selective plate medium (1.7 g / l Yeast Nitrogen Base without amino acid (Difco), 20 g / l glucose (Wako), 0.77 g / l Complete Supplement Mixture (Bio 101), 20 g).
  • SD selective plate medium 1.7 g / l Yeast Nitrogen Base without amino acid (Difco), 20 g / l glucose (Wako), 0.77 g / l Complete Supplement Mixture (Bio 101), 20 g).
  • / l Agar (Wako) excluding amino acids and / or nucleotides corresponding to the selectable marker gene), and purify the recombinant by purifying the grown colonies on another SD selective plate medium. And used for subsequent experiments.
  • the transformed ⁇ cereviae was selected from SD selection medium (1.7 g / l Yeast Nitrogen Base without ammo acid (Diico), 20 g / l glucose, 0.77 g / l Complete Supplement Mixture (Bio 101)) according to the marker gene.
  • SD selection medium 1.7 g / l Yeast Nitrogen Base without ammo acid (Diico), 20 g / l glucose, 0.77 g / l Complete Supplement Mixture (Bio 101)
  • 25 L of the preculture was added to 2.5 mL of YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, pH 7) or SG selective medium (Glucose component of SD selective medium (Replaced by toose)) and cultured at 30 ° C.
  • the preculture is SG selective medium
  • the cells were washed with physiological saline so that glucose components were not introduced.
  • adenine hemisulfate was added to the culture medium to a concentration of 40 ⁇ g / mU.
  • the pentane-extracted fraction was separated, identified, and quantified using an HP6890 / 5973 GC / MS system (Hewlett-Packard, Wilmington, DE).
  • the column used was HP-5MS (0.25 mm ⁇ 30 m, film thickness 0.25 ⁇ ), and the analysis conditions were as follows.
  • MS zone temperature MS Quad 150 ° C, MS source 230 ° C, mass scan range 35 -200.
  • Injection conditions automatic injection mode, sample volume 2 ML , split ratio 1/20, carrier gas; helium l.OmL / min, solvent delay 2 min.
  • Heating setting 115 ° C 90 seconds / 250 ° C (70 ° C / min) 1 250 ° C 2min-1 300 ° C (70 ° CZ) 1 300 ° C 7 min. Post time 0.
  • the PRS436GAP-DPP1, RS436GAP-LPPl, pRS436GAP-Dri42, pRS436GAP-PAP2, pRS436GAP-AtPAPl and pRS436GAP-AtPAP2 vectors were each introduced into ⁇ _cerevisiae A451 for transformation.
  • Each of the obtained recombinants was subjected to 5 mL of SD selective culture. 0D e in the ground. . was collected to about 1 and the cells were collected by centrifugation.
  • 300 extraction buffers 50 mM Tris-HC1 (pH 7.5), lOmM ⁇ -mercaptoethanol, 2ra EDTA, Ira PMSF, 10 ⁇ g / L apro
  • GGPPase activity Measurements of GGPP phosphatase activity (GGPPase activity), 5 ⁇ u M of GGPP reaction puffer one containing (2. LKB containing [3 H] GGPP of q) (final concentration 0. 1M citrate (pH6. 0) , 5mM
  • An appropriate amount of the crude enzyme fraction was added to EDTA), and the reaction was carried out at 37 ° C. for 30 minutes (total of 100 ⁇ l). After the reaction, alcohol K0H solution (90% ethanol ': 40% ⁇ 0 ⁇ (1: 1))
  • the reaction was stopped by adding 100 ⁇ 100. After adding 500 ml of n-hexane, the mixture was vigorously stirred with a vortex mixer for 1 minute to extract the generated [ 3 H] GG0H into the hexane layer. 300 / i L of the hexane layer was fractionated, and the radioactivity was measured with a liquid scintillation counter. A calibration curve was prepared using 0 to 500 mU / mL alkaline phosphatase (calf intestine, Boehringer), and the radioactivity determined above was converted to an enzyme activity value (Unit).
  • the protein concentration in the crude enzyme fraction was measured by using BSA as a standard protein by Bio-Rad Protein Assay (Bio-Rad), and the specific activity of GGPPase (U / mg protein) in the crude enzyme solution was determined.
  • Table 1 shows the relative activity when the GGPPase specific activity of the wild strain is set to 1.
  • the phosphatase gene-introduced strain had a 2.4 to 34.8-fold increase in GGPPase specific activity compared to the wild-type strain, confirming that the phosphatase gene was introduced to express GGPPase activity.
  • Reaction buffer containing 5 ⁇ GGPP including 2.lkBq [ 3 H] GGPP
  • partially purified enzyme [final concentration 0.1 M citrate (pH 6.0), 5 mM EDTA], additional substrate other than GGPP
  • ⁇ ATP 100 glucose 6-phosphate (G6P), 100 ⁇ sodium pyrophosphate (NaPP), 100 ⁇ ⁇ -glycerol phosphate ( ⁇ -GP), 100 ⁇ ⁇ -nitrophenyl phosphoric acid ( ⁇ ) , ⁇
  • MVP 100 ⁇ M MVPP, 100 ⁇ M IPP, 100 ⁇ M DMAPP, 100 M GPP, 5 ⁇ M FPP or 5 ⁇ M GGPP were added, and the reaction was carried out at 37 ° C. for 30 minutes. Then, 100 ⁇ alcohol K0H solution was added to stop the reaction, 500 ⁇ uL of hexane was added, and the mixture was vigorously stirred for 1 minute with a vortex mixer to extract the generated [ 3 H] GG0H.
  • the specific activity of GGPPase was determined by collecting 300 / iL of the hexane layer and measuring the radioactivity with a liquid scintillation counter. Table 2 summarizes the relative activities when the specific activity of GGPPase when no additional substrate other than [ 3 H] GGPP was added (+ none) was 100%.
  • TpNPP (100 //) 100 100 100 100, 100 100 100
  • prenyl phosphate precursors MVP, MVPP
  • IPP or DMAPP with 5 carbon atoms or GPP with 10 carbon atoms were added as additional substrates, even when 20 times the amount of GGPP was added but no additional substrate was added.
  • phosphatase activity for GGPP was maintained at 90% or more. That is, it was confirmed that the phosphatase activity of the phosphatase used for the prenyl phosphate precursor and the prenyl phosphate compound having 10 or less carbon atoms was lower than the phosphatase activity for GGPP.
  • the gene may be assembled into an appropriate vector or DNA fragment, introduced into a host, and expressed in vivo.
  • prenyl diphosphate biosynthesis pathway (mevalonate pathway, non-mevalonate pathway).
  • HMG-CoA hydroxymethyldaltarinole CoA
  • reductase is known to be the rate-limiting enzyme in the mevalonate pathway (Dimster — Denk, D. et al (1994) Mol. Biol Cel l. 5, 655-665.).
  • IPP isopenthl diphosphate isomerase
  • the GGPP synthase gene (eg, ⁇ _cereviae BTS1) is an enzyme that synthesizes GGPP, a precursor of GG0H.
  • GGPP synthase gene By enhancing the expression of the GGPP synthase gene, enhancement of GGPP production capacity can be expected. Therefore, by combining the introduction or enhancement of these genes and the introduction or enhancement of the phosphatase gene according to the present invention, higher prenyl alcohol productivity can be expected.
  • PRS435GAP-BTS1 was introduced into ⁇ _cerevisiae W303-1B, and the phosphatase gene according to the present invention was further transformed with respect to the obtained recombinant.
  • the incorporated expression vectors pRS436GAP-DPP1, pRS436GAP-LPP1, pRS436GAP-Dri42, pRS436GAP-PAP2, pRS436GAP-AtPAPl or pRS436GAP-AtPAP2 were introduced.
  • Table 3 shows the results of measuring the GGOH productivity of each recombinant and host.
  • GGOH production was not detected in W303-1B, but GGPP synthase gene was introduced.
  • GGOH power '; 0.2 mg / L was produced.
  • GGPP was produced by increasing the activity of the GGPP synthase and this GGPP was converted to GG0H by the intrinsic dephosphorylation activity of the yeast.
  • the productivity of GG0H was improved 5.7 to 17 times as compared with the case where only the GGPP synthase gene was introduced.
  • PRS434TEF-HMG1, pRS445GAP-BTS1 or RS435GAP-IDIl was introduced into cerevisiae YPH499. Separately, two plasmids, pRS434TEF-HMGl and pRS445GAP-BTS1, were introduced into YPH499.
  • PRS434TEF-H G1 pRS445GAP-BTS1, pRS436GAP-DPP1 1.41
  • PRS434TEF-HMG1, pRS445GAP-BTS1, pRS436GAP-LPP1 2.37
  • PRS434TEF-H G1 pRS445GAP-BTS1 , P RS436GAP-AtPAP1 2.26
  • GGOH was not detected in YPH499. GG0H was not detected when only the IPP- ⁇ isomerase gene was introduced, but GG0H production was increased to some extent (GQ0H 0.05 mg / L) by introducing only the HMG-CoA reductase gene. Gene transfer GGOH production also increased to some extent (GG0H 0, 2 mg / L).
  • AURGG101 AUR1 :: AUR1-C
  • PYHMG044 was introduced into AURGG101 (referred to as 15-2), and pRS435GAP-BTS1 was further introduced into 15-2 to obtain pRS435GAP-BTS 1 / 15-2.
  • pRS435GAP- BTS 1 / 15- 2 to pRS434GAP - DPP l, pRS434GAP-LPPl was introduced pRS434GAP-Dri42, P RS434GAP-PAP2 , PRS436GAP-AtPAP l or pRS434GAP- AtPAP2.
  • Table 5 shows the results of measuring the prenyl alcohol productivity of each recombinant and host.
  • DPP1 and GGPP synthase were determined by PCR.
  • a gene linked to the gene BTS1 (hereinafter referred to as "DPGGJ") was prepared.
  • the PCR product when S-DPP1-1 having the nucleotide sequence shown in SEQ ID NO: 43 as the primer 1 and DPP-2 having the nucleotide sequence shown in SEQ ID NO: 44 as the primer 1 was used as # 1.
  • the PCR product obtained when BTS-3 having the nucleotide sequence shown in SEQ ID NO: 45 as primer 1 and X-BTS-4 having the nucleotide sequence shown in SEQ ID NO: 46 as primer 2 was used was # 2.
  • the obtained PCR product was purified by agarose gel electrophoresis. Next, Annealing and extension reaction was performed under the following conditions.
  • 2nd PCR was performed under the following conditions.
  • 1XK0D buffer T0Y0B0
  • 0.2mM dNT Ps 1 mM MgCl 2
  • 0.5U KOD DNA polymerase T0Y0B0
  • 20pmol S- DPP - 1 20ol X - BTS- 4
  • lO includes a 2 ⁇ ⁇ Annealing and extension reaction L
  • the reaction solution was prepared, and 2nd PCR reaction was performed as follows.
  • the resulting PCR product was purified by agarose gel electrophoresis (this is called SX-DPGG). After digesting SX-DPGG with restriction enzymes SacII and Xhol, pRS434GAP Insertion into the acl-Xhol cleavage site yielded pRS434GAP-DPGG. DNA sequencing between the structural gene portion of pRS434GAP-DPGG and the vicinity of the restriction enzyme site confirmed that the nucleotide sequence was as designed.
  • an expression vector for the phosphatase-linked gene DPGG in which a linker sequence (5 ′ ggtggtggttct 3 ′) was introduced into the binding region between DPP1 and BTS1 was prepared.
  • a gene obtained by ligating the FPP synthase gene ERG20 (Gen Bank access ion No. NC001142, 105006.. Was prepared.
  • 1st PCR was performed using S-DPP13A having the nucleotide sequence of SEQ ID NO: 47 as primer 1 and DPP-2 as primer-2 to obtain PCR product # 3.
  • 1st PCR was performed using DPF3 having the nucleotide sequence shown in SEQ ID NO: 48 as primer 1 and X-FPS4 having the nucleotide sequence shown in SEQ ID NO: 49 as primer 2, to obtain a PCR product # 4.
  • These PCR products were purified by agarose gel electrophoresis.
  • Annealing and extension reactions were performed using PCR products # 3 and # 4, and agarose gel electrophoresis was performed on a portion of the obtained PCR products.
  • the linked genes of PCR products # 3 and # 4 (about 1.9 kb) was confirmed to be synthesized.
  • 2nd PCR was performed using Annealing and extension reaction solution, S-DPP13A and X-FPS4, and the resulting PCR product was purified by agarose gel electrophoresis (this is called SX-DPF).
  • GGPPase activity of YPH499 transfected with phosphatase-linked genes DPF and DPGG When DPF is expressed, a fusion enzyme of the phosphatase encoded by DPP1 and the pharmacophorase synthase encoded by ERG20 is expressed, and when DPGG is expressed, the phosphatase encoded by DPP1 and BTS1 encode It is considered that a fusion enzyme with geranylgera-lunirate synthase is expressed. Therefore, it was examined whether GGPPase activity was expressed even when the linked genes DPF and DPGG were expressed.
  • PRS434GAP-DPP 1, pRS434GAP-DPF and pRS434GAP-DPGG were each introduced into ⁇ cerevisiae YPH499 for transformation, and the GGPPase specific activity of the obtained recombinant was determined in the section of ⁇ Expression of phosphatase activity ''. The measurement was performed in the same manner as described above. Table 6 shows the results.
  • the GGPPase specific activity of the strain expressing DPP1 alone was about 10 times that of the wild-type strain.
  • the GGPPase specific activity of the strain expressing DPF and DPGG was about 4 times that of the wild type. That is, in the strain expressing DPF and DPGG, the GGPPase activity was significantly lower than that in the case where DPP1 was expressed alone, but the GGPPase specific activity was significantly increased as compared with the wild-type strain.
  • HMG- CoA reductase gene P RS434GAP- HMG1 expression is base Kuta one HMG1 were introduced into A451 (referred to as AH1), was introduced more PRS435GAP- DPF to AH1. The recombinant thus obtained, and A451 and AH1 were cultured in a YM medium, and the F0H productivity was measured. Table 7 shows the results.
  • PRS434GAP-DPF and PRS434GAP-DPGG were respectively introduced into 152 described above.
  • two types of plasmids PRS435GAP-BTS1 and PRS434GAP-DPP1, were separately introduced into 15-2.
  • Table 8 shows the results of measuring the prenyl alcohol productivity by culturing each recombinant and host in an SG selective medium.
  • AURGG101 No production of prenyl alcohol was detected with A451, and the amount of prenyl alcohol produced by AURGG101 was very small.
  • AURGG101 the HMG044 gene was introduced 15-2, F0H, GGOH has been produced to some extent (F0H 8.5 mg / L, GG0H 2.2 mg / L).
  • the productivity was improved 7.8-fold in F0H and 2.6-fold in 660 ⁇ 1 compared to 15-2 (F0H 66.7 mg / L, GGOH 5 7mg / L).
  • the strain (PRS434GAP-DPGG / 15-2) in which the connecting gene DPGG was introduced into 15-2 was cultured in a 5 L capacity fermenter (MSJ-U2W; Marubishi Biohenge), and its productivity was measured. It was decided to further confirm the effect of introducing DPGG on productivity improvement.
  • the medium was prepared by mixing 50 g of galactose (Nacalai), 100 g of paptopeptone (DIFC0), 50 g of yeast extract (DIFC0), 50 ml of soybean oil (Nacalai), 5 ml of Adecanol LG109, and 51 tap water.
  • the cells were sterilized with a jar armament (121 ° C, 20 minutes) and used for culture.
  • Figure 2 shows the results of quantifying the amount of prenyl alcohol produced by collecting the culture solution over time.
  • Cloning of a gene encoding a phosphatase encoding (non-specific) phosphatase having no substrate specificity in the activity of hydrolyzing a phosphate ester compound and production of an expression vector were performed by the following methods. ⁇ Cerevisiae acid phosphatase gene PH03 (GenBank accession No. NC001134, complement (427657.. 429060)) and alkaline phosphatase gene PH08 (GenBank access ion No. NC001136, complement (1418537.. 1420237)) as non-specific phosphatases was used.
  • PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 50 and a primer having the nucleotide sequence shown in SEQ ID NO: 51.
  • the resulting plasmid DNA is called pCR-PH03.
  • a 1.4 kbp fragment cut out from pCR-PH03 by SacII-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP.
  • the prepared plasmid is called pRS434GAP-13 ⁇ 403.
  • ⁇ cerevisiae YPH499 genomic DNA as a ⁇ type, an approximately 1.7 kb P fragment of the ⁇ _ cerevisiae gene PH08 was amplified by PCR.
  • the PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 52 and a primer having the nucleotide sequence shown in SEQ ID NO: 53.
  • the PCR fragment was purified by agarose gel electrophoresis and cloned into pCR2.1-TOP0 by T / A ligation. When the base sequence of the cleaved fragment was determined and compared with the SGD sequence, no PCR errors were found.
  • the prepared plasmid DNA is called pCR-PH08.
  • pRS434GAP-PH08 A 1.7 kbp fragment cut out from pCR-PH08 by SacII-Smal was prepared and inserted into the SacII-Smal cleavage site of pRS434GAP. The resulting plasmid is called pRS434GAP-PH08.
  • Acid phosphatase activity was measured as follows with reference to the method of Ferminan et al. (E. Ferminan, et al. (1997) Microbiol. 143, 2615-2625).
  • pRS434GA P-PH03 was introduced into the above 15-2, and the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. Wash the cells twice with saline, and add 300 ⁇ l of extraction buffer (10 mM citrate (pH 4.3), 10 mM ⁇ -mercaptoethanol, 1 mM PMSF, 10 ⁇ g / L aprotinin, 10 ⁇ g / L leupeptin.
  • the reaction was stopped by adding 50 ⁇ L of a 1 M aqueous NaOH solution, and the absorbance at 405 nm was measured.
  • a calibration curve was prepared using 0 to 0.03 U / mL acid phosphatase (Roche Diagnostics), and the absorbance at 405 nm determined above was converted to an enzyme activity value (Unit).
  • the protein concentration in the crude enzyme fraction was determined by Bio-Rad Protein Assay using BSA as a standard protein, and the specific activity of the acid phosphatase (U / mg protein) in the crude enzyme fraction was determined.
  • Alkaline phosphatase activity was determined by the method of Dhamija et al. (S. S. Dhamija, et al.
  • PRS434GAP-PH08 was introduced into the above 15-2, and the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. Wash the cells twice with physiological saline, and add 300 ⁇ L of extraction buffer (100 mM TrisHC1 (pH 8.5), ImM MgCl 2 , 10 mM ⁇ -mercaptoethanol N ImM PMSF, 10 ⁇ g / L aprotinin, 10 ⁇ g / L leupeptin, 1 ⁇ g / L pepstatin).
  • extraction buffer 100 mM TrisHC1 (pH 8.5), ImM MgCl 2 , 10 mM ⁇ -mercaptoethanol N ImM PMSF, 10 ⁇ g / L aprotinin, 10 ⁇ g / L leupeptin, 1 ⁇ g / L pepstatin).
  • a calibration curve was prepared using alkaline phosphatase (calf intestine, Roche Diagnostics) at 0 to 0.3 U / mL, and the absorbance at 405 nm obtained in the above was converted to an enzyme activity value (Unit). Specific phosphatase specific activity (U / mg protein) was determined in the same manner as for acid phosphatase.
  • the GGPPase specific activity of the crude enzyme fraction obtained here was 60 mM as the reaction buffer.
  • the non-specific phosphatase specific activity (acid phosphatase specific activity or alkaline phosphatase specific activity) measured by the above method is hereinafter referred to as pNPPase specific activity.
  • the pNPPase specific activity, GGPPase specific activity, and prenyl alcohol productivity (cultured in SG selective medium) of 15-2 were introduced with expression vectors incorporating the acid phosphatase gene PH03 and the alkaline phosphatase gene PH08, respectively. It was measured. Table 9 shows the results. In Table 9, relative values are shown with the host as 1. In the GGPPase specific activity section of the table, in the + PNPP column, ⁇ of pNPP was added as an additional substrate other than GGPP in the same manner as described in the section ⁇ Substrate specificity of phosphatases ''. The column of -pNPP shows the value of the GGPPase specific activity measured without adding pNPP.
  • the present inventors have found that the amino acid sequence of a phosphatase capable of improving the productivity of F0H and GG0H contains the phosphatase motif shown in FIG. We speculate that this phosphatase motif is related to the substrate specificity for prenyl phosphates having at least 5 carbon atoms.
  • a gene encoding a phosphatase having a substrate specificity of prenyl phosphinate having at least 15 carbon atoms and a gene encoding an enzyme involved in a prenyl diphosphate biosynthetic pathway was linked to F0H and GG0H production.
  • the productivity of F0H and GG0H was further improved.
  • the productivity of F0H and GG0H was improved several times compared to the case where they were incorporated into separate plasmids without introduction.
  • Non-specific phosphatases are considered to react with any phosphate compound in the cells due to lack of substrate specificity. For this reason, it also reacts with phosphate esters other than prenyl phosphate in the cells (for example, ATP which is considered to be present in large amounts in the cells). It is presumed that the productivity of prenyl alcohol did not improve because the number of non-specific phosphatases capable of producing prenyl alcohol by substantially reacting with prenyl phosphinate was very limited.
  • the present invention enables efficient mass production of prenyl alcohols, particularly, prenyl alcohols having a biologically active all-trans form and having 15 or more carbon atoms.

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Abstract

Polypeptides respectively having the amino acid sequences represented by SEQ ID NOS:1 to 6 and showing a phosphatase activity having a substrate-specificity for a prenylphosphoric acid having 15 or more carbon atoms; polypeptides having amino acid sequences functionally equivalent thereto; polynucleotides respectively having the base sequences represented by SEQ ID NOS:7 to 12 and encoding the above polypeptides; polynucleotides encoding a phosphatase having a substrate-specificity for a prenylphosphoric acid having 15 or more carbon atoms; recombinant nucleic acids containing these polynucleotides as described above; recombinants having these recombinant nucleic acids transferred thereinto; and a process for efficiently producing on a mass scale a trans-prenyl alcohol having 15 or more carbon atoms which comprises culturing cells into which a phosphatase gene having a substrate-specificity for a prenylphosphoric acidhaving 15 or more carbon atoms transferred thereinto or in which the expression of this gene has been potentiated, and then obtaining the prenyl alcohol from the culture medium.

Description

明 細 書 ί ポリぺプチド、 ポリヌクレオチド、 組換え核酸、 組換え体、 プレ ニルアルコールの製造方法 技術分野  Description 製造 Method for producing polypeptides, polynucleotides, recombinant nucleic acids, recombinants, prenyl alcohol
本発明は、 ポリペプチド、 ポリヌクレオチド、 組換え核酸、 組換え体及ぴプ レニルアルコールの製造方法に関する。 更に詳しくは、 本発明は、 炭素数 15以上 のプレニルアルコールを選択的に大量生産したい場合に有効であるホスファター ゼ、 かかるホスファターゼをコードするポリヌクレオチド、 このようなポリヌク レオチドを含む組換え核酸、 このようなポリヌクレオチドが導入された組換え体、 及び、 生細胞におけるプレニルアルコールの生合成に関わる酵素の発現をコント ロールしてプレニルアルコール （特に生物学的に活性なトランス型の異性体構造 を持ち、 とりわけ炭素数 15以上の例えば F0H , GG0H) を効率的に大量生産する方 法に関する。 背景技術  The present invention relates to a method for producing a polypeptide, a polynucleotide, a recombinant nucleic acid, a recombinant, and prenyl alcohol. More specifically, the present invention relates to a phosphatase that is effective when it is desired to selectively mass-produce prenyl alcohols having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, It controls the expression of enzymes involved in the biosynthesis of prenyl alcohol in living cells and recombinants into which such a polynucleotide has been introduced, and prenyl alcohol (particularly having a biologically active trans isomeric structure). In particular, the present invention relates to a method for efficiently mass-producing, for example, F0H, GG0H) having 15 or more carbon atoms. Background art
ィソプレノィ ド生合成経路は多様な生物種に普遍的に備わると考えられる。 イソプレノイ ド化合物の基本骨格単位であるイソペンテニルニリン酸 （ΙΡΡ ) の 生合成経路として、 主に真核生物に存在するメバロン酸経路と、 主に原核生物に 存在する非メバロン酸経路 （DXP 経路） の二つの経路が知られている。  The isoprenoid biosynthetic pathway is thought to be universally present in a variety of species. The biosynthetic pathways for isopentenyl diphosphate (ΙΡΡ), the basic skeletal unit of isoprenoid compounds, are mainly the mevalonate pathway that exists in eukaryotes and the non-mevalonate pathway that mainly exists in prokaryotes (DXP pathway). The two routes are known.
メバロン酸経路では、 ァセチル CoA を出発物質として、 ァセトァセチル CoA 、 ヒ ドロキシメチルダルタリル CoA (HMG-CoA ) 、 メバロン酸、 メノ ロン酸 5-リ ン酸 （MVP ) 、 メバロン酸 5 -二リン酸 （MVPP) を経て、 活性イソプレン単位であ る炭素数 5 の IPP が生合成される。 非メバロン酸経路では、 ピルビン酸とグリセ ルアルデヒ ド 3 -リン酸が縮合して生じる 1-デォキシ- D- キシルロース 5-リン酸を 経由して、 IPP が生合成される。 非メバロン酸経路に関する知見は徐々に蓄積さ れつつあるが、 関与する酵素やそれをコードする遺伝子等の幾つかは未だ不明で ある。 In the mevalonic acid pathway, starting with acetyl CoA, acetoacetyl CoA, hydroxymethyl dartaryl CoA (HMG-CoA), mevalonic acid, menolonic acid 5-phosphoric acid (MVP), mevalonic acid 5-diphosphate After (MVPP), IPP with 5 carbon atoms, which is an active isoprene unit, is biosynthesized. In the non-mevalonate pathway, IPP is biosynthesized via 1-deoxy-D-xylulose 5-phosphate, which is formed by the condensation of pyruvate and glyceraldehyde 3-phosphate. Although knowledge on the non-mevalonate pathway is gradually accumulating, some of the enzymes involved and the genes encoding them are still unknown. is there.
IPP は異性化反応によりジメチルァリル二リン酸 （DMAPP ) に変換される。 D . MAPP は IPP と トランス型 （E 型） に順次重縮合することにより、 炭素数 10のゲラ エルニリン酸 （GPP ) 、 炭素数 15のフアルネシル二リン酸 （FPP ) 、 炭素数 20の ゲラニルゲラニルニリン酸 （GGPP) · · '等の順に合成される。 又、 全トランス 型の FPP , GGPPに対して、 シス型 （Z 型） に IPP が順次重縮合することにより、 ゥンデカブレニルニリン酸， デヒ ドロ ドリ シルニリン酸等が生合成される。  IPP is converted to dimethylaryl diphosphate (DMAPP) by an isomerization reaction. D. MAPP is successively polycondensed to IPP and trans-form (E-form), resulting in C10 gera ernilic acid (GPP), C15 funaresyl diphosphate (FPP), C20 geranylgeranyl diphosphate (GGPP) · · · Combined in the order of ' In addition, IPP is sequentially polycondensed to cis-form (Z-form) with all-trans-form FPP and GGPP, whereby ndecabrenyl diphosphoric acid, dehydrorisyl silicic acid, etc. are biosynthesized.
上記 IPP , DMAPP , GPP , FPP , GGPP, ゥンデカプレニルニリン酸， デヒ ド ロ ドリシルニリン酸等が、 プレニルニリン酸と総称される。 これらのプレニルニ リン酸の内、 有用性の高い （生物学的に活性な） 全トランス型の純品は生合成に より製造することに適しており、 化学合成では制御が難しい。  The above IPP, DMAPP, GPP, FPP, GGPP, pendecaprenyl diphosphoric acid, dehydrorisyl diphosphoric acid, etc. are collectively referred to as prenyl diphosphoric acid. Of these prenyl diphosphates, highly useful (biologically active) all-trans pure products are suitable for production by biosynthesis and are difficult to control by chemical synthesis.
又、 これらのプレニルニリン酸は、 ホスファターゼの作用により二リン酸が 分離されて （あるいは、 プレニルニリン酸から 1個のリン酸が分離されたプレニ ルーリン酸を中継して、 更に 1個のリン酸が分離されて） 、 相当するプレニルァ ルコールを生成する。 本明細書において、 以下、 プレニルニリン酸とプレニルー リン酸とを一括して指す時は 「プレニルリン酸」 と言う。  In addition, these prenyl diphosphates are separated by diphosphoric acid by the action of phosphatase (or by relaying prenyle phosphate in which one phosphate is separated from prenyl diphosphate, one more phosphate is separated). Being) produces the corresponding prenyl alcohol. Hereinafter, in this specification, prenyl diphosphoric acid and prenyl-phosphoric acid are collectively referred to as “prenyl phosphoric acid”.
上記のプレニルリ ン酸ゃプレニルアルコールの内、 特に炭素数 15以上のもの は利用価値が高い。 例えば炭素数 15以上のプレニルニリン酸である FPP ， GGPP等 や、 これらの脱リン酸化物である炭素数 15のフアルネソール （F0H ) , 炭素数 20 のゲラニルゲラ二オール (GG0H) 等は、 フィ トール, ビタミン E ' ビタミン K , ュビキノン （CoQ ) 等の合成基質となる。 炭素数 35のヘプタブレニルアルコール、 炭素数 50のデカプレニルアルコール等も医薬品中間体として有用であり、 合成開 始物質と して上記 FPP , GGPP, F0H， GG0H等を利用できる。  Of the above prenyl phosphinates / prenyl alcohols, those having 15 or more carbon atoms are particularly useful. For example, FPP, GGPP, etc., which are prenyl diphosphoric acids having 15 or more carbon atoms, phthalnesol (F0H) having 15 carbon atoms, which is a dephosphorized oxide thereof, and geranylgeraniol (GG0H) having 20 carbon atoms, are phytol, vitamin, etc. E 'It is a synthetic substrate for vitamin K and ubiquinone (CoQ). Heptabrenyl alcohol having 35 carbon atoms, decaprenyl alcohol having 50 carbon atoms, and the like are also useful as pharmaceutical intermediates, and the above-mentioned FPP, GGPP, F0H, GG0H, etc. can be used as a synthesis starting material.
又、 プレニルリン酸とプレニルアルコールとを対比すると、 一般的にプレニ ルリン酸は水溶性が比較的高い一方、 部分的に非極性基を有するので抽出 ·精製 が容易でないと言う難点があり、 へキサンゃぺンタン等の非極性有機溶媒で容易 に抽出 '精製できるプレニルアルコールを生産する方が有利である。 更に、 ファ ルネシル基ゃゲラニルゲラエル基を有する化合物の化学合成にはプレニルアルコ —ルが用いられる。 Also, when prenyl phosphoric acid is compared with prenyl alcohol, prenyl phosphoric acid generally has relatively high water solubility, but has a disadvantage that extraction and purification are not easy because of partial non-polar groups. It is more advantageous to produce a prenyl alcohol that can be easily extracted and purified with a non-polar organic solvent such as pentane. Furthermore, prenyl alcohol is required for chemical synthesis of compounds having a farnesyl group and a geranylgerael group. Are used.
以上の点から、 生合成経路を利用して、 生物学的に活性なトランス型の異性 体構造を持つ、 特に炭素数 15以上のプレニルアルコール （例えば、 いずれも全ト ランス型の、 ゲラニルフアルネソール， へキサプレニルアルコール， ヘプタブレ ニルァノレコーノレ， ォクタプレニルァノレコール， ノナプレニルァノレコール， デカプ レニルアルコール， ゥンデカプレニルアルコール， F0H , GG0H等） を効率的に大 量生産する方法の提供が望まれる。  In view of the above, utilizing the biosynthetic pathway, prenyl alcohols having a biologically active trans-isomeric structure, especially those having 15 or more carbon atoms (for example, all trans- Efficient large-scale production of sole, hexaprenyl alcohol, heptabrenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, pendecaprenyl alcohol, F0H, GG0H) It is desired to provide a method for doing this.
特公昭 63- 17437号公報には、 シユードモナス株の培養によるポリプレニルァ ルコールの生産方法が開示されているが、 全トランス型ゥンデカプレニルアルコ JP-B-63-17437 discloses a method for producing polyprenyl alcohol by culturing a Pseudomonas strain.
—ルが 3mg/L生産されるだけであって、 有用な F0H や GG0Hの生産は開示されてい ない。 特開平 9- 238692号公報には、 トウダイグサ科の植物細胞の培養による GG0H 及び GGPPの生産方法が開示されているが、 最大で 3. 25mg/L の GG0H生産が報告さ れるに止まる。 天然の微生物や動植物細胞をそのまま培養しても、 プレニルアル コールの効率的な大量生産は期待できない。 —Only 3 mg / L is produced and no useful F0H or GG0H production is disclosed. Japanese Patent Application Laid-Open No. Hei 9-238692 discloses a method for producing GG0H and GGPP by culturing plant cells of the Euphorbiaceae family. However, only GG0H production at a maximum of 3.25 mg / L is reported. Efficient mass production of prenyl alcohol cannot be expected even if natural microorganisms, animal and plant cells are cultured as they are.
特開平 10- 248575 号公報には、 ァセチル CoA の MVP や MVPPへの変換過程に関 与する服 G - CoA (ヒ ドロキシメチルダルタリル- CoA) 還元酵素遺伝子を酵母 (Can dida ut i l is) に導入してカロテノイ ドを生産する方法が開示されている。 特開 平 5- 115298号公報には、 HMG - CoA 還元酵素遺伝子のコピー数を増加することによ つて、 植物中のステロールの蓄積を増加する方法が開示されている。 特開平 5-192 184号公報には、 ステロール生合成酵素の発現に欠損を持つ変異体酵母において、 HMG-CoA 還元酵素遺伝子の発現レベルを上昇させることによりスクアレンおょぴ ステロールの蓄積を増大させる方法が開示されている。 特開平 10- 155497 号公報 には、 GGPP合成酵素遺伝子 （crtE) を導入した微生物による発酵法を用いたカロ テノィ ド生産方法が開示されている。 しかし、 上記した特開平 10 - 248575 号公報， 特開平 5- 115298号公報， 特開平 5- 192184号公報ないし特開平 10- 155497 号公報に 開示された方法は、 プレニルアルコールの効率的な大量生産には結び付かない。 特開昭 53- 127889 号公報には、 ジャガイモの根から分離したホスファタ一ゼを用 いた in vitro でのプレニルアルコールの製造方法が開示されている。 しかし、 この方法は生合成を利用する方法ではないし、 ホスファターゼ遺伝子の利用につ いて何ら開示するところがない。 Japanese Patent Application Laid-Open No. Hei 10-248575 discloses that a gene for clothing G-CoA (hydroxymethyldaltaryl-CoA) reductase involved in the process of converting acetyl CoA to MVP or MVPP is yeast (Can dida util is). A method for producing carotenoids by introducing the method into carotenoids is disclosed. JP-A-5-115298 discloses a method for increasing the accumulation of sterols in plants by increasing the copy number of the HMG-CoA reductase gene. Japanese Patent Application Laid-Open No. 5-192184 discloses that in a mutant yeast having a deficiency in sterol biosynthetic enzyme expression, accumulation of squalene sterol is increased by increasing the expression level of a HMG-CoA reductase gene. A method is disclosed. JP-A-10-155497 discloses a carotenoid production method using a fermentation method with a microorganism into which a GGPP synthase gene (crtE) has been introduced. However, the methods disclosed in JP-A-10-248575, JP-A-5-115298, JP-A-5-192184 and JP-A-10-155497 described above are effective in the efficient mass production of prenyl alcohol. Is not tied to JP-A-53-127889 discloses a method for producing prenyl alcohol in vitro using phosphatase isolated from potato roots. But, This method is not a method utilizing biosynthesis, and there is no disclosure of the use of a phosphatase gene.
一方、 プレニルリン酸脱リン酸化活性のあるホスファターゼに関しては、 ラ ッ ト (Rattus norvegicus) 肝臓ミクロソーム中に存在するプレニルビロホスフ ァターゼ （E. C. 3. 1. 7. 1) の報告 （Bansal, V. S. et. al (1994) Arch. Biochera. Biophys. 315 (2) , 393-399 ) があるが、 単離されていない。 酵母 cerevisia e) の DPP1遺伝子がコードするジァシルグリセロールニリン酸ホスファターゼが、 IPP , GPP , FPP , GGPPに作用する旨の報告 （Faulkner, A. et. al (1999) J, Biol . Chem. 274 (21) , 14831-14837) もあるが、 このホスファタ一ゼがプレニノレ リン酸とその前駆体のリン酸エステル化合物 （MVP や MVPP) との間でどのような 選択的な基質特異性を示すかについて解明していない。 更に、 上記 Faulkner ら の報告の Fi g. 5 によれば、 上記 IPP , GPP , FPP , GGPPの個々に対する相対的な 基質特異性においては、相互に余り選択的とは言えないデータが提示されている。 そして、 上記の Bansalら、 Faulknerらによる 2報は、 専ら酵素学的研究等を目的 とし、 ホスファターゼやその遺伝子の産業的利用を全く示唆しない。  On the other hand, phosphatases with prenyl phosphate dephosphorylation activity have been reported on prenyl virophosphatase (EC 3.1.7.1) present in rat (Rattus norvegicus) liver microsomes (Bansal, VS et. Al.). (1994) Arch. Biochera. Biophys. 315 (2), 393-399), but not isolated. Report that diasylglycerol diphosphate phosphatase encoded by the DPP1 gene of yeast cerevisia e) acts on IPP, GPP, FPP and GGPP (Faulkner, A. et. Al (1999) J, Biol. Chem. 274 (21), 14831-14837), but how does this phosphatase show selective substrate specificity between preninoleric acid and its precursor phosphate compounds (MVP and MVPP)? Has not been clarified. Furthermore, according to Fig. 5 of the above-mentioned report by Faulkner et al., Data showing that the relative substrate specificities of the IPP, GPP, FPP, and GGPP relative to each individual are not very selective. I have. The above two reports by Bansal et al. And Faulkner et al. Are intended solely for enzymatic studies and do not suggest any industrial use of phosphatases or their genes.
なお、 USP 6, 242， 227 号公報では、 FPP， GGPP を脱リン酸する効果の高いホ スファターゼをコードする遺伝子を過剰発現させれば FOH, GG0H の生産性を向上 させることが出来るであろう、 との記載がある。 しかし、 ホスファターゼ遺伝子 の利用に関する実施例は開示されていないし、 炭素数 15以上のプレニルリン酸に 基質特異性のあるホスファタ一ゼに関する記述はない。 更に、 実施例に示したよ うに、 上記のような基質特異性のない非特異的ホスファタ一ゼは FPP， GGPP を脱 リン酸する効果はあるが、 非特異的ホスファタ一ゼをコ一ドする遺伝子を過剰発 現させても FOH, GG0H の生産性を向上させることは出来なかった。  In US Pat. No. 6,242,227, production of FOH and GG0H could be improved by overexpressing a gene encoding a phosphatase having a high dephosphorylation effect on FPP and GGPP. , And are described. However, no examples relating to the use of the phosphatase gene are disclosed, and no description is given of phosphatases having substrate specificity for prenyl phosphates having 15 or more carbon atoms. Further, as shown in the Examples, the non-specific phosphatase having no substrate specificity as described above has an effect of dephosphorylating FPP and GGPP, but a gene encoding the non-specific phosphatase. However, overexpression of FOH and GG0H could not improve the productivity.
微生物等の生細胞を利用した目的物質の生産において、 代謝経路の特定ステ ップを酵素作用の強化等により増強して目的物質の生産性を向上させる方法は、 一般論としては知られている。  In the production of target substances using living cells such as microorganisms, it is generally known that a specific step in the metabolic pathway is enhanced by enhancing the enzymatic action and the like to improve the productivity of the target substance. .
しかし、 生細胞におけるプレニルアルコールの生合成プロセスを人為的にコ ントロールしたプレニルアルコールの効率的な大量生産系の構築は、 未だ報告さ れていない。 又、 プレニルアルコールを生細胞で特異的に合成するには上記生合 成プロセスのどのステップをどのようにコントロールすれば良いか、 未だ明らか となっていない。 更に、 炭素数 15以上のプレニルアルコールを選択的に大量生産 したい場合に有効であるホスファターゼも、 明らかになっていない。 However, construction of an efficient mass production system for prenyl alcohol that artificially controls the biosynthesis process of prenyl alcohol in living cells has not yet been reported. Not. Further, it is not yet clear which step in the biosynthesis process should be controlled and how to specifically synthesize prenyl alcohol in living cells. Furthermore, a phosphatase that is effective for selectively mass-producing prenyl alcohols having 15 or more carbon atoms has not been clarified.
本発明の目的は、 炭素数 15以上のプレニルアルコールを選択的に大量生産し たい場合に有効であるホスファターゼ、 かかるホスファターゼをコ一ドするポリ ヌクレオチド、 このようなポリヌクレオチドを含む組換え核酸、 このようなポリ ヌクレオチドが導入された組換え体、 生細胞におけるプレニルアルコールの生合 成に関わる酵素の発現をコントロールしてプレニルアルコール （特に生物学的に 活性なトランス型の異性体構造を持ち、 とりわけ炭素数 15以上の例えば FOH , GG0 H) を効率的に大量生産する方法を提供することである。 発明の開示  An object of the present invention is to provide a phosphatase which is effective when it is desired to selectively mass-produce prenyl alcohol having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, By controlling the expression of enzymes involved in prenyl alcohol biosynthesis in living cells, recombinants into which such a polynucleotide has been introduced, prenyl alcohol (particularly having a biologically active trans isomeric structure, An object of the present invention is to provide a method for efficiently mass-producing, for example, FOH, GG0H) having 15 or more carbon atoms. Disclosure of the invention
本願の第 1発明は、 配列番号 1〜配列番号 6のいずれかに示すァミノ酸配列 を有し、 炭素数 15以上のプレニルリ ン酸に基質特異性のあるホスファターゼ活性 を示すポリペプチドである。  The first invention of the present application is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOS: 1 to 6, and having a phosphatase activity having substrate specificity to prenyl linic acid having 15 or more carbon atoms.
本願発明者は、 第 1発明のポリぺプチドが炭素数 15以上のプレニルリン酸に 基質特異性のあるホスファターゼであることを発見した。 従って、 第 1発明のポ リペプチドにより、 炭素数 15以上のプレニルアルコール、 例えば FOH , GG0H, へ プタプレニルアルコール， デカプレニルアルコール等を効率良く選択生産するこ とができる。  The present inventor has discovered that the polypeptide of the first invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Therefore, the polypeptide of the first invention enables efficient selective production of prenyl alcohols having 15 or more carbon atoms, such as FOH, GG0H, heptaprenyl alcohol, decaprenyl alcohol and the like.
本願の第 2発明は、 配列番号 1〜配列番号 6のいずれかに示すァミノ酸配列 における 5 個以下のアミノ酸が置換， 欠失又は付加されたアミノ酸配列を有し、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファターゼ活性を示すボ リぺプチドである。  The second invention of the present application relates to a prenyl phosphate having an amino acid sequence in which 5 or less amino acids are substituted, deleted or added in the amino acid sequence shown in any one of SEQ ID NOS: 1 to 6, and having 15 or more carbon atoms. It is a polypeptide that exhibits phosphatase activity with substrate specificity.
第 2発明に係るポリべプチドにおいても、 前記第 1発明と同等の作用 ·効果 を期待することができる。  With the polypeptide according to the second invention, the same operation and effect as those of the first invention can be expected.
本願の第 3発明は、 以下の （1 ) 〜 （ 3 ) 式の内の少なくとも一つのホスフ 1223 ァターゼモチーフを含み、 炭素数 15以上のプレニルリン酸に基質特異性のあるホ スファターゼ活性を示すポリぺプチドである。 According to a third aspect of the present invention, at least one of the following formulas (1) to (3) is used. It is a polypeptide that contains a phosphatase motif and has phosphatase activity with substrate specificity to prenyl phosphate having 15 or more carbon atoms.
( 1 ) KXXXGXXRP (式中、 X は任意のアミノ酸残基から選ばれることを表す）  (1) KXXXGXXRP (where X represents an amino acid residue)
( 2 ) 1XXSGH (式中、 1 はアミノ酸残基 S 又は T から選ばれ、 X は任意のァミノ 酸残基から選ばれることを表す）  (2) 1XXSGH (where 1 is selected from amino acid residues S or T, and X is selected from any amino acid residues)
( 3 ) SR2XDXXHXXXDVXXGXXX3 (式中、 2 はアミノ酸残基 V 又は T から選ばれ、 3 はアミノ酸残基 G 又は A から選ばれ、 X は任意のアミノ酸残基から選ばれること を表す） 。  (3) SR2XDXXHXXXDVXXGXXX3 (wherein, 2 is selected from amino acid residues V or T, 3 is selected from amino acid residues G or A, and X is selected from any amino acid residues).
本願発明者は、 配列番号 1〜配列番号 6に示す炭素数 15以上のプレニルリ ン 酸に基質特異性のあるポリぺプチドのアミノ酸配列を詳細に検討した結果、 第 3 発明において （1 ) 〜 （3 ) として規定した、 これらのポリペプチドに共通した 保存配列を見出した。 その詳細は第 1図に基づいて後述するが、 この保存配列は 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファターゼ活性を発現す るために必要な配列 （ホスファターゼモチーフ） であると考えられる。 従って、 第 3発明に係るポリべプチドは、 炭素数 15以上のプレニルリン酸に基質特異性の あるホスファターゼであると考えられる。  As a result of detailed examination of the amino acid sequence of a polypeptide having substrate specificity to prenylphosphonic acid having 15 or more carbon atoms shown in SEQ ID NOs: 1 to 6, the inventors of the present invention have found that (3) to (4) in the third invention A conserved sequence common to these polypeptides, defined as 3), was found. The details will be described later with reference to FIG. 1, but this conserved sequence is considered to be a sequence (phosphatase motif) necessary for expressing a phosphatase activity with substrate specificity to prenyl phosphate having 15 or more carbon atoms. . Therefore, it is considered that the polypeptide according to the third invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
本願の第 4発明は、 前記第 3発明に係る （1 ) 〜 （3 ) 式で示されるホスフ ァターゼモチーフにおいて、 種類が特定されている任意の 1以上のァミノ酸残基 が同類置換を受けているポリペプチドである。 ここに、 「種類が特定されている アミノ酸残基」 とは、 上記 （1 ) 〜 （3 ) 式において 「X 」 以外の記号で表記さ れているアミノ酸残基を言う。 又、 「同類置換」 とは、 ポリペプチドの機能が全 体的に実質的に不変のまま維持されるように、 所定のァミノ酸残基を化学的また は機能的に類似した別のァミノ酸残基で置換することを言う。 化学的または機能 的に類似したアミノ酸の例示として、 疎水性アミノ酸 （Ala 、 lie 、 Leu 、 Phe 、 Pro 、 Trp、 Val 、 Met ) 同士、 極性だが電荷のないアミノ酸 （Asn 、 Cys 、 Gin 、 Gly 、 Ser 、 Thr 、 Tyr ) 同士、 塩基性アミノ酸 （Arg 、 His 、 Lys ) 同士、 酸 性アミノ酸 （Asp 、 Glu ) 同士、 等が挙げられる （E. E. Cornn ら著、 田宫信雄ら 訳、 「コーン · スタンプ生化学 第 5 版」 東京化学同人刊、 p56- 58、 1988) 。 第 4発明に係るポリぺプチドは、 そのホスファターゼモチーフにおいて特定 されている任意の 1以上のァミノ酸残基が同類置換を受けているため、 第 3発明 に係るポリぺプチドと同様に、 炭素数 15以上のプレニルリン酸に基質特異性のあ るホスファターゼであると考えられる。 In the fourth invention of the present application, in the phosphatase motif represented by the formulas (1) to (3) according to the third invention, any one or more amino acid residues of a specified type are subjected to conservative substitution. Polypeptide. Here, the “amino acid residue whose type is specified” means an amino acid residue represented by a symbol other than “X” in the above formulas (1) to (3). A "conservative substitution" also refers to the replacement of a given amino acid residue with another chemically or functionally similar amino acid such that the function of the polypeptide remains substantially substantially unchanged. Substitution with a residue. Examples of chemically or functionally similar amino acids include hydrophobic amino acids (Ala, lie, Leu, Phe, Pro, Trp, Val, Met), polar but uncharged amino acids (Asn, Cys, Gin, Gly, Ser, Thr, Tyr), basic amino acids (Arg, His, Lys), acid amino acids (Asp, Glu), etc. (by Corn Corn et al., Translated by Nobuo Tazaki et al., “Corn Stamp” Biochemistry 5th Edition, Tokyo Chemical Dojin, p56-58, 1988). The polypeptide according to the fourth invention has the same number of carbon atoms as the polypeptide according to the third invention, since any one or more amino acid residues specified in the phosphatase motif have undergone conservative substitution. It is considered to be a phosphatase with substrate specificity for 15 or more prenyl phosphates.
本願の第 5発明は、 配列番号 7〜配列番号 1 2のいずれかに示す塩基配列を 有し、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼをコ一 ドするポリヌクレオチドである。  The fifth invention of the present application is a polynucleotide encoding a phosphatase having the base sequence shown in any one of SEQ ID NOs: 7 to 12 and having substrate specificity to prenyl phosphate having 15 or more carbon atoms. is there.
本願発明者は、 第 5発明に係るポリヌクレオチドがコ一ドするポリぺプチド 、 炭素数 15以上のプレニルリ ン酸に基質特異性のあるホスファターゼであるこ とを発見した。 従って第 5発明のポリヌクレオチドを生細胞又は生物体に導入又 は発現強化することにより、 該生細胞内又は生物体内で炭素数 15以上のプレニル リン酸に基質特異性のあるホスファターゼを生産させ、 炭素数 15以上のプレニル アルコールを有効に生産することができる。  The present inventor has discovered that the polynucleotide encoded by the polynucleotide according to the fifth invention is a phosphatase having substrate specificity for prenyl phosphoric acid having 15 or more carbon atoms. Therefore, by introducing or enhancing the expression of the polynucleotide of the fifth invention into a living cell or organism, a phosphatase having a substrate specificity for prenyl phosphate having 15 or more carbon atoms is produced in the living cell or organism, Prenyl alcohols with 15 or more carbon atoms can be produced effectively.
本願の第 6発明は、 配列番号 7〜配列番号 1 2のいずれかに示す塩基配列又 はこれと相補的な塩基配列に対して所定のストリンジェントな条件下でハイプリ ダイズする塩基配列を有し、 炭素数 15以上のプレニルリ ン酸に基質特異性のある ホスファタ一ゼをコ一ドするポリヌクレオチドである。  The sixth invention of the present application has a nucleotide sequence that hybridizes to a nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12 or a nucleotide sequence complementary thereto under predetermined stringent conditions. It is a polynucleotide encoding a phosphatase having substrate specificity for prenyl phosphoric acid having 15 or more carbon atoms.
第 6発明に係るポリヌクレオチドにおいても、 前記第 5発明と同等の作用 - 効果を期待することができる。  In the polynucleotide according to the sixth invention, the same action and effect as those of the fifth invention can be expected.
本願の第 7発明は、 前記第 1発明〜第 4発明に係るいずれかのポリべプチド をコードするポリヌクレオチドである。  A seventh invention of the present application is a polynucleotide encoding any of the polypeptides according to the first to fourth inventions.
第 7発明に係るポリヌクレオチドにおいても、 前記第 5発明と同等の作用 · 効果を期待することができる。  In the polynucleotide according to the seventh invention, the same action and effect as those of the fifth invention can be expected.
本願の第 8発明は、 前記第 5発明〜第 7発明に係るいずれかのポリヌクレオ チドを含む組換え核酸である。  An eighth invention of the present application is a recombinant nucleic acid containing the polynucleotide according to any one of the fifth to seventh inventions.
第 8発明によって、 前記第 5発明〜第 7発明に係るポリヌクレオチドを生細 胞又は生物体に導入する有力な手段が提供される。  The eighth invention provides a powerful means for introducing the polynucleotide according to the fifth invention to the seventh invention into a living cell or an organism.
本願の第 9発明は、 前記第 8発明に係るいずれかの組換え核酸が導入されて いる組換え体である。 According to a ninth invention of the present application, any one of the recombinant nucleic acids according to the eighth invention is introduced. Is a recombinant.
第 9発明に係る組換え体によって、 炭素数 15以上のプレニルアルコールを一 層有利に選択生産することができる。  By the recombinant according to the ninth invention, prenyl alcohol having 15 or more carbon atoms can be more selectively produced.
本願の第 1 0発明は、 前記第 9発明に係る組換え体の宿主細胞が、 真菌 ycetes ) , 子嚢菌類 (Ascomycetes ) ， 単細胞真核生物のいずれかである組換え 体である。  A tenth invention of the present application is the recombinant, wherein the host cell of the recombinant according to the ninth invention is any of a fungus ycetes), an ascomycetes (Ascomycetes), and a unicellular eukaryote.
即ち、 第 9発明に係る組換え体の宿主としては、 例えば真菌 (Eumycetes ) , 子嚢菌類 (Ascomycetes ) , 単細胞真核生物のいずれかを好ましく用いることが できる。  That is, as the host of the recombinant according to the ninth invention, for example, any of fungi (Eumycetes), ascomycetes (Ascomycetes), and unicellular eukaryotes can be preferably used.
本願の第 1 1発明は、 前記第 9発明に係る組換え体の宿主細胞が酵母である 耝換え体である。  The eleventh invention of the present application is a recombinant wherein the host cell of the recombinant according to the ninth invention is a yeast.
即ち、 第 9発明に係る組換え体の宿主としては、 例えば酵母を好ましく用い ることができる。  That is, as the host of the recombinant according to the ninth invention, for example, yeast can be preferably used.
本願の第 1 2発明は、 前記第 1 1発明に係る酵母がサッカロミセス属 (Sacch aromycetes ) の酵母である組換え体である。  The 12th invention of the present application is a recombinant wherein the yeast according to the 11th invention is a yeast of the genus Saccharomyces.
即ち、 第 1 1発明に係る酵母としては、 例えばサッカロミセス属 (Saccharom ycetes ) の酵母を好ましく用いることができる。  That is, as the yeast according to the eleventh invention, for example, yeast of the genus Saccharomyces can be preferably used.
本願の第 1 3発明は、 前記第 1 1発明に係る酵母がサッカロミセス .セレビ シェ (Saccharomyces cerevisiae) YPH499株， YPH500株， A451株， W303- 1A株， W303-1B株又はこれらに由来する株である組換え体である。  A thirteenth invention of the present application is the yeast according to the eleventh invention, wherein the yeast is Saccharomyces cerevisiae YPH499, YPH500, A451, W303-1A, W303-1B, or a strain derived therefrom. A recombinant.
即ち、 第 1 1発明に係る酵母としては、 例えば、 サッカロミセス 'セレビシ ェ cerevis iae) YPH499株， YPH500株， A451株， W303- 1A株， W303- 1B株又 はこれらに由来する株を好ましく用いることができる。  That is, as the yeast according to the eleventh invention, for example, preferably used are Saccharomyces' cerevisiae) YPH499 strain, YPH500 strain, A451 strain, W303-1A strain, W303-1B strain and strains derived therefrom. Can be.
本願の第 1 4発明は、 ホスファターゼ遺伝子を導入又は発現強化した宿主細 胞を培養し、 その培養物からプレニルアルコールを採取するプレ-ルアルコール の製造方法である。  The fourteenth invention of the present application is a method for producing pryl alcohol by culturing a host cell into which a phosphatase gene has been introduced or having enhanced expression, and collecting prenyl alcohol from the culture.
本願発明者は、 ァセチル CoA を出発物質とする前記メバロン酸経路、 又はピ ルビン酸を出発物質とする前記非メバロン酸経路より、 各種のプレニルニリン酸 を経由してプレニルアルコールに到るプレニルアルコールの生合成反応に対して 詳細な実験的検討を加えた結果、 生細胞におけるホスファターゼ遺伝子の導入又 は発現強化と言う方法により、 生物学的に活性な異性体構造 （例えば、 いずれも 全トランス型の、 ゲラニルフアルネソール, へキサプレニノレアルコール， ヘプタ プレニルアルコール, ォクタプレニノレアルコーノレ， ノナプレニノレアノレコール， デ カプレニルアルコール， ゥンデカプレニルアルコール， F0H , GG0H等) を持つプ レニルアルコールの効率的な大量生産が可能となることを見出した。 一方、 第ェ 4発明の方法は、 炭素数 5 以上のプレニルアルコール全般の生産量を増量させた い場合にも有用である。 The present inventor has proposed various prenyl diphosphates from the mevalonate pathway starting from acetyl CoA or the non-mevalonate pathway starting from pyruvate. As a result of detailed experimental studies on the biosynthetic reaction of prenyl alcohol via urea to prenyl alcohol, the biophosphorylation of phosphatase gene into living cells Isomeric structures (for example, all trans forms of geranyl pharynesol, hexapreninole alcohol, heptaprenyl alcohol, octapreninoreal alcohol, nonapreninoleanolecole, decaprenyl alcohol, pendecaprenyl It has been found that efficient mass production of prenyl alcohol having alcohol, F0H, GG0H, etc. is possible. On the other hand, the method of the fourth invention is also useful when it is desired to increase the overall production of prenyl alcohol having 5 or more carbon atoms.
本願発明者は、 酵母を宿主とする実施例において、 ホスファターゼ遺伝子の 導入又は発現強化により、現在のところ、炭素数 15以上のプレニルアルコール（例 えば、 全トランス型の F0H ， GG0H) の生産性を約 40倍に高めることに成功してい る。 即ち、 第 1 4発明により、 プレニルアルコール、 特にトランス型のプレニル アルコール、 とりわけトランス型で炭素数 15以上のプレニルアルコールを効率的 に大量生産する方法が提供される。  The inventors of the present invention have found that, in an example using yeast as a host, the productivity of prenyl alcohols having 15 or more carbon atoms (eg, all-transformation F0H, GG0H) can be improved by introducing or enhancing the expression of a phosphatase gene. It has been successfully increased about 40 times. That is, the fourteenth invention provides a method for efficiently mass-producing prenyl alcohol, particularly trans-type prenyl alcohol, especially trans-type prenyl alcohol having 15 or more carbon atoms.
本願の第 1 5発明は、 ホスファターゼ遺伝子とプレニルニリン酸生合成経路 に関与する酵素の遺伝子とを導入又は発現強化した宿主細胞を培養し、 その培養 物からプレニルアルコールを採取するプレニルアルコールの製造方法である。  The fifteenth invention of the present application relates to a method for producing prenyl alcohol, comprising culturing host cells into which a phosphatase gene and a gene of an enzyme involved in the prenyl diphosphate biosynthesis pathway have been introduced or having enhanced expression, and collecting prenyl alcohol from the culture. is there.
第 1 5発明のように、 ホスファターゼ遺伝子とプレニルニリン酸生合成経路 に関与する酵素の遺伝子とを宿主細胞に導入又は発現強化した場合、 即ちホスフ ァターゼ遺伝子を導入又は発現強化すると共にプレニルニリン酸生合成経路にお ける他の任意のステツプに関わる酵素の遺伝子を導入又は発現強化した場合、 プ レニルアルコールの生産性が更に高くなることがある。 プレニルニリン酸生合成 経路に関与する酵素をコードする遺伝子のみを導入又は発現強化しても、 プレニ ルアルコール （特に炭素数 15以上のプレニルアルコール、 例えば F0H , GG0H) の 効率的な大量生産に対する寄与は限定的である。 更にこの場合において、 ホスフ ァターゼ遺伝子とプレニルニリン酸生合成経路に関与する酵素の遺伝子とを連結 して導入又は発現強化することが、 とりわけ好ましい。 本願の第 1 6発明は、 前記第 1 5発明に係るプレニルニリン酸生合成経路に 関与する酵素の遺伝子が、 以下の （4 ) 及び/又は （5 ) の遺伝子であるプレニ ルアルコールの製造方法である。 As in the fifteenth invention, when the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway are introduced or enhanced in host cells, that is, the phosphatase gene is introduced or enhanced in expression and the prenyl diphosphate biosynthesis pathway is enhanced When the gene of any other step-related enzyme is introduced or its expression is enhanced, the productivity of prenyl alcohol may be further increased. Even if only the gene encoding the enzyme involved in the prenyl diphosphate biosynthesis pathway is introduced or enhanced, its contribution to the efficient mass production of prenyl alcohols (particularly prenyl alcohols having 15 or more carbon atoms, such as F0H and GG0H) is not Limited. Further, in this case, it is particularly preferable that the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthetic pathway are linked to introduce or enhance expression. A sixteenth invention of the present application is a method for producing prenyl alcohol, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway according to the fifteenth invention is the following gene of (4) and / or (5): is there.
( 4 ) フアルネシルニリン酸合成酵素遺伝子及ぴゲラ二ルゲラ二ルニリン酸合成 酵素遺伝子から選ばれる少なくとも 1の遺伝子。  (4) At least one gene selected from the pharmacogene synthase gene and the geraniruberinirulinate synthase gene.
( 5 ) ァセチル CoA 合成酵素遺伝子、 ァセチル CoA —ァセチルトランスフユラー ゼ遺伝子、 ヒ ドロキシメチルダルタリル CoA 合成酵素遺伝子、 ヒ ドロキシメチル グルタリル CoA還元酵素遺伝子、 メバロン酸キナーゼ遺伝子、 メバロン酸リン酸 キナーゼ遺伝子、 メバロン酸二リン酸デカルボキシラーゼ遺伝子、 イソペンテ二 ルニリン酸ィソメラーゼ遺伝子、 デォキシキシルロースリン酸リダク トイソメラ ーゼ遺伝子、 デォキシキシルロースリン酸合成酵素遺伝子、 MEP ( 2- C- methyl - D -erythritol 4- phosphate) シチジリノレトランスフェラ一ゼ遺伝子、 CDP- ME ( 4—(c yt idine 5' -diphospho) -2- C-methyl-D- erythritol)キナーゼ遺伝子及び MECDP (2 -C-methyl-D-erythritol 2, 4-cyclodiphosphate) 合成酵素遺伝子から選ばれる少 なくとも 1の遺伝子。  (5) acetyl-CoA synthase gene, acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, Mevalonate diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP (2-C-methyl-D-erythritol 4 -phosphate) Citidylinoletransferase gene, CDP-ME (4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol) kinase gene and MECDP (2-C-methyl-D -erythritol 2, 4-cyclodiphosphate) At least one gene selected from synthase genes.
前記第 1 5発明においてホスファターゼ遺伝子と共に導入又は発現強化する 遺伝子として、 第 1 6発明の （4 ) に列挙する遺伝子が特に好ましい。 第 1 6発 明の （5 ) に列挙する遺伝子も好ましい。  The genes listed in (4) of the 16th invention are particularly preferable as the genes to be introduced or enhanced in expression together with the phosphatase gene in the 15th invention. The genes listed in (16) of the 16th invention are also preferable.
本願の第 1 7発明は、 前記第 1 4発明〜第 1 6発明に係るホスファターゼ遺 伝子が、 リン酸エステル化合物に対する加水分解活性において炭素数 15以上のプ レニルリン酸に基質特異性のあるホスファタ一ゼをコ一ドする遺伝子であるプレ ニルアルコールの製造方法である。  A seventeenth invention of the present application is the phosphatase gene according to the fourteenth invention to the sixteenth invention, wherein the phosphatase gene has a substrate specificity to a prenyl phosphate having 15 or more carbon atoms in a hydrolysis activity for a phosphate compound. This is a method for producing prenyl alcohol, which is a gene that encodes lysine.
第 1 7発明のように、 導入又は発現強化されるホスファターゼ遺伝子が炭素 数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼをコ一ドする遺伝 子である場合、 炭素数 15以上のプレニルアルコールの効率的な大量生産と言う目 的からは、 特に効果が著しい。 これらのプレニルアルコールとして、 前記 「第 1 4発明の作用 .効果」 の欄で例示した各種の生物学的に活性な異性体構造を持つ プレニルアルコールを特に好ましく例示することができる。 本願の第 1 8発明は、 前記第 1 4発明〜第 1 6発明に係るホスファターゼ遺 伝子が、 前記第 5発明〜第 8発明に記載のいずれかのポリぺプチドであるプレ- ルアルコールの製造方法である。 As in the seventeenth invention, when the phosphatase gene to be introduced or enhanced in expression is a gene encoding a phosphatase having substrate specificity to prenyl phosphate having 15 or more carbon atoms, prenyl having 15 or more carbon atoms The effect is particularly significant for the purpose of efficient mass production of alcohol. As these prenyl alcohols, prenyl alcohols having various biologically active isomeric structures exemplified in the section of “Function and Effect of the Fourteenth Invention” can be particularly preferably exemplified. The eighteenth invention of the present application is directed to a method of the present invention, wherein the phosphatase gene according to the fourteenth invention to the sixteenth invention is any of the above-described fifth to eighth inventions. It is a manufacturing method.
即ち、 第 1 4発明〜第 1 6発明において導入又は発現強化されるホスファタ ーゼ遺伝子として、 第 5発明〜第 8発明に係るホスファターゼ遺伝子を好ましく 利用できる。  That is, the phosphatase gene according to the fifth to eighth inventions can be preferably used as the phosphatase gene introduced or enhanced in the fourteenth to sixteenth inventions.
本願の第 1 9発明は、 前記第 1 4発明〜第 1 6発明に係るホスファターゼ遺 伝子が、 リン酸エステル化合物に対する加水分解活性において炭素数 15以上のプ レニノレリン酸に基質特異性のあるホスファターゼ以外のホスファタ一ゼをコ一ド する遺伝子であるプレニルアルコールの製造方法である。  A nineteenth invention of the present application is the phosphatase gene according to the fourteenth invention to the sixteenth invention, wherein the phosphatase gene has a substrate specificity to preninolenic acid having 15 or more carbon atoms in a hydrolysis activity on a phosphate compound. This is a method for producing prenyl alcohol which is a gene encoding a phosphatase other than phosphatase.
第 1 9発明のように、 導入又は発現強化されるホスファターゼ遺伝子が炭素 数 15以上のプレ-ルリン酸に基質特異性のあるホスファタ一ゼ以外のホスファタ ーゼをコードする遺伝子である場合にも、 炭素数が 5〜15の範囲にわたる各種の プレニルアルコールを有効に得ることができる。  As in the nineteenth invention, even when the phosphatase gene to be introduced or enhanced in expression is a gene encoding a phosphatase other than a phosphatase having substrate specificity to pre-phosphate having 15 or more carbon atoms, Various prenyl alcohols having a carbon number ranging from 5 to 15 can be effectively obtained.
本願の第 2 0発明は、 前記第 1 4発明〜第 1 9発明に係る宿主細胞が、 第 1 0発明〜第 1 3発明に記載のいずれかの宿主細胞であるプレニルアルコールの製 造方法である。  The 20th invention of the present application is the method for producing prenyl alcohol, wherein the host cell according to the 14th invention to the 19th invention is any one of the host cells according to the 10th invention to the 13th invention. is there.
即ち、 第 1 4発明〜第 1 9発明における宿主細胞として、 前記第 1 0発明〜 第 1 3発明に係るいずれかの宿主細胞を好ましく用いることができる。  That is, any of the host cells according to the tenth to thirteenth inventions can be preferably used as the host cells in the fourteenth to nineteenth inventions.
本願の第 2 1発明は、 前記第 1 4発明〜第 2 0発明に係るプレニルアルコ一 ルが炭素数 15以上のものであるプレニルアルコールの製造方法である。  The twenty-first invention of the present application is a method for producing prenyl alcohol, wherein the prenyl alcohol according to the fourteenth to twenty-fifth inventions has 15 or more carbon atoms.
上記第 1 4発明〜第 2 0発明に係るプレニルアルコールの製造方法において、 炭素数 5 又は炭素数 10のプレニルアルコールも採取することができるが、 炭素数 1 5以上のプレニルアルコールが相対的に生産量が多く、 非極性有機溶媒による抽 出 '精製が容易であり、 かつ前記のように有用性も高い。 従って炭素数 15以上の プレニルアルコールを採取することが特に有効である。 炭素数 15以上のプレニル アルコールの全てを採取することもできるし、 その内の 1種又は 2種以上を選択 的に採取することもできる。 本願の第 2 2発明は、 前記第 2 1発明に係るプレニルアルコールが、 フアル ネソ—ル, ゲラニルゲラ二オール, ゲラニルフアルネソ一ル, へキサプレニルァ ルコール， ヘプタプレニルアルコール， ォクタプレニノレアノレコール， ノナプレニ ルァノレコール， デカプレニルアルコール， ゥンデカプレニノレアノレコール又はドデ カプレニルアルコールから選ばれる 1種又は 2種以上のプレニルアルコールであ るプレニルアルコールの製造方法である。 In the method for producing a prenyl alcohol according to the fourteenth invention to the twenty-fifth invention, prenyl alcohol having 5 or 10 carbon atoms can be collected, but prenyl alcohol having 15 or more carbon atoms is relatively produced. It is large in amount, easy to extract and purify with a non-polar organic solvent, and has high utility as described above. Therefore, it is particularly effective to collect prenyl alcohol having 15 or more carbon atoms. All prenyl alcohols having 15 or more carbon atoms can be collected, or one or more of them can be selectively collected. The twenty-second invention of the present application is the prenyl alcohol according to the twenty-first invention, wherein the prenyl alcohol is fuarnesol, geranylgeranol, geranylfurnesol, hexaprenyl alcohol, heptaprenyl alcohol, octaprenilanoleanol, This is a method for producing prenyl alcohol, which is one or more prenyl alcohols selected from nonaprenylanolecol, decaprenyl alcohol, pendecapreninoleanololecol and dodecaprenyl alcohol.
即ち、 前記第 2 1発明において採取する炭素数 15以上のプレニルアルコール として、 上記の各プレニルアルコールから選ばれる 1種又は 2種以上のプレニル アルコールが、 特に好ましく例示される。  That is, as the prenyl alcohol having 15 or more carbon atoms collected in the twenty-first invention, one or more prenyl alcohols selected from the above prenyl alcohols are particularly preferably exemplified.
本願の第 2 3発明は、 前記第 1 2発明〜第 2 2発明に係るプレニルアルコー ルが全トランス型であるプレニルアルコールの製造方法である。  The twenty-third invention of the present application is the method for producing prenyl alcohol wherein the prenyl alcohol according to the twenty-second invention to the twenty-second invention is an all-trans type.
即ち、第 1 2〜第 2 2発明に係るプレニルアルコールの製造方法においては、 宿主細胞を用いた生合成であるため、 有用性の高い全トランス型のプレニルアル コールを製造することができる。 このような全トランス型のプレニルアルコール を採取することが、 より好ましい。 図面の簡単な説明  That is, in the method for producing prenyl alcohol according to the twelfth to twenty-second inventions, since biosynthesis is performed using a host cell, highly useful all-trans prenyl alcohol can be produced. It is more preferable to collect such all-trans prenyl alcohol. BRIEF DESCRIPTION OF THE FIGURES
第 1図は本発明に係る各ポリべプチドの保存配列を示す図である。 第 2図は プレニルアルコール生産量の定量結果を示す図である。  FIG. 1 is a view showing a conserved sequence of each polypeptide according to the present invention. FIG. 2 shows the results of quantification of prenyl alcohol production.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
ポリぺプチド  Polypeptide
本願発明に係るポリべプチドは、 炭素数 15以上のプレニルリン酸に基質特異 性のあるホスファターゼである。 かかるホスファターゼの好ましい基準の一例を、 次のように規定できる。 即ち、 炭素数 15以上のプレニルリン酸 （A) のみを基質 とした酵素活性測定系において示された （A) に対する比活性を 100とした場合、 The polypeptide according to the present invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. An example of a preferable criterion for such a phosphatase can be defined as follows. That is, when the specific activity for (A) shown in the enzyme activity measurement system using only prenyl phosphate (A) having 15 or more carbon atoms as a substrate is 100,
( A ) 及びモル比でその 20倍量の炭素数 10以下のプレニルリン酸又はその前駆体(A) and a prenyl phosphoric acid having 10 or less carbon atoms in a molar ratio of 20 times the amount thereof or a precursor thereof
( B ) を共存させた場合における （A ) に対する比活性が、 80以上、 より好まし くは 85以上、 更に好ましくは 90以上を示すことである。 The specific activity to (A) when (B) coexists is 80 or more, more preferable. Or more preferably 85 or more, more preferably 90 or more.
本願発明に係る、 炭素数 15以上のプレニルリン酸に基質特異性のあるポリぺ プチドの代表例が、 配列番号 1〜配列番号 6のいずれかに示すァミノ酸配列を有 するポリペプチドである。 これらのポリペプチドはいずれも、 炭素数 15以上のプ レニルリン酸に基質特異性のあるホスファターゼであることが、 本願発明者によ つて初めて見出された。  A typical example of the polypeptide having substrate specificity to prenyl phosphate having 15 or more carbon atoms according to the present invention is a polypeptide having an amino acid sequence shown in any one of SEQ ID NOS: 1 to 6. The present inventors have found for the first time that these polypeptides are phosphatases having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
配列番号 1に示すアミノ酸配列を有するポリペプチドは、 シロイヌナズナ rabidopsis thal iana) のゲノム D N A中に存在するホスファチジン酸ホスファ ターゼホモログ遺伝子 〔 Gen Bank accession No. ： AC006200 (complement (1677 8. . 17686) ) 、 この遺伝子を AtPAPl と命名した〕 によりコードされるホスファタ —ゼである。 AtPAPl の塩基配列を配列番号 7に示す。 配列番号 2に示すアミノ 酸配列を有するポリペプチドは、 L thal ianaのゲノム D N A中に存在するホス ファチジン酸ホスファターゼホモログ遺伝子 〔 Gen Bank access ion No. ： AC00 7591 (3610. . 5006) 、 この遺伝子を 「 AtPAP2 」 と命名した） によりコードされ るホスファターゼである。 AtPAP2 の塩基配列を配列番号 8に示す。  The polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is a phosphatidic acid phosphatase homolog gene present in the genomic DNA of Arabidopsis thaliana (Rabidopsis thaliana) [Gen Bank accession No .: AC006200 (complement (1677 8.. 17686)), This gene was named AtPAPl]. SEQ ID NO: 7 shows the nucleotide sequence of AtPAPl. The polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is a phosphatidic acid phosphatase homolog gene present in genomic DNA of L thaliana [Gen Bank accession No .: AC00 7591 (3610... 5006); Phosphatase encoded by “AtPAP2”. SEQ ID NO: 8 shows the nucleotide sequence of AtPAP2.
配列番号 3に示すアミノ酸配列を有するポリべプチドはサッカロミセス ·セ レビシェ d cerevi siae) の遺伝子 DPP1 によりコードされるホスファタ一ゼ である ( Toke DA et al (1998) J. Biol. Chem. 273 (6) , 3278-3284) 。 DPP1の Ge n Bank accession No. は NC 001136であり、 その塩基配列を配列番号 9に示す。 配列番号 4に示すアミノ酸配列を有するポリべプチドは酵母の遺伝子 LPP1により コードされるホスファターゼである （Toke DA et al (1998) J. Biol. Chem. 273 (2 3) , 14331-14338) 。 LPP1 の Gen Bank accession No. は、 NC001136であり、 そ の塩基配列を配列番号 1 0に示す。  The polypeptide having the amino acid sequence shown in SEQ ID NO: 3 is a phosphatase encoded by the gene DPP1 of Saccharomyces cerevisiae (Toke DA et al (1998) J. Biol. Chem. 273 (6 ), 3278-3284). Gen Bank accession No. of DPP1 is NC 001136, and its nucleotide sequence is shown in SEQ ID NO: 9. The polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is a phosphatase encoded by the yeast gene LPP1 (Toke DA et al (1998) J. Biol. Chem. 273 (23), 14331-14338). The LPP1 Gen Bank accession No. is NC001136, and its nucleotide sequence is shown in SEQ ID NO: 10.
配列番号 5に示すアミノ酸配列を有するポリペプチドは、 ラット norve gicus) の遺伝子 PAP2 によりコードされるホスファターゼである （Brindley DN et al (1998) J. Biol. Chem. 273 (38) , 24281-24284) 。 PAP2 の Gen Bank acces sion No. は、 U90556であり、 その塩基配列を配列番号 1 1に示す。 配列番号 6に 示すアミノ酸配列を有するポリペプチドは、 norvegicusの jfr伝子 Dri42に よりコードされるホスファターゼである （ Bari la D et al (1996) J. Biol. The polypeptide having the amino acid sequence shown in SEQ ID NO: 5 is a phosphatase encoded by the gene PAP2 of rat norve gicus (Brindley DN et al (1998) J. Biol. Chem. 273 (38), 24281-24284) . The PAP2 Gen Bank accession number is U90556, and its nucleotide sequence is shown in SEQ ID NO: 11. The polypeptide having the amino acid sequence of SEQ ID NO: 6 is found in norvegicus jfr gene Dri42. (Bari la D et al (1996) J. Biol.
Chem. 271 (47) , 29928-29936) 。 Dri42の Gen Bank access ion No. は、 Y07783 であり、 その塩基配列を配列番号 1 2に示す。 Chem. 271 (47), 29928-29936). The Gen Bank access ion No. of Dri42 is Y07783, and its nucleotide sequence is shown in SEQ ID NO: 12.
更に、 配列番号 1〜配列番号 6に示す、 炭素数 15以上のプレニルリン酸に基 質特異性のあるポリべプチドのアミノ酸配列を詳細に検討した結果、 第 1図に示 すように共通して保存された配列を見出した。  Furthermore, as a result of a detailed study of the amino acid sequences of the polypeptides having a substrate specificity of prenyl phosphate having 15 or more carbon atoms shown in SEQ ID NOs: 1 to 6, the common amino acid sequences were as shown in FIG. A conserved sequence was found.
第 1図において、 配列番号 1〜配列番号 6に係る各ポリペプチドは、 それら をコードする前記各遺伝子の表記により区別 （即ち、 例えば 「DPPl j の表記は、 配列番号 3に係るポリペプチドを表す） し、 これらの各ポリペプチドに見出した 保存配列を示した。 又、 第 1図の最下行には、 これらの保存配列に共通するアミ ノ酸配列をマルチプルァライメント方式の配列表記により示した。 このマルチプ ルァライメントの配列表記式において、 1 はアミノ酸残基 S または T から選ばれ、 2 はアミノ酸残基 V または T から選ばれ、 3 はアミノ酸残基 G または A から選ば れ、 X は任意のアミノ酸残基から選ばれることを表す。 nlは任意の個数 （例えば、 34〜39個） の任意のアミノ酸残基を表し、 n2も任意の個数 （例えば、 38〜46個） の任意のァミノ酸残基を表す。  In FIG. 1, the polypeptides of SEQ ID NO: 1 to SEQ ID NO: 6 are distinguished by the notation of each of the genes encoding them (that is, for example, “the notation of DPPl j represents the polypeptide of SEQ ID NO: 3). The conserved sequences found in each of these polypeptides are shown, and the amino acid sequence common to these conserved sequences is shown in the bottom line of FIG. In the sequence notation of this multiple alignment, 1 is selected from amino acid residues S or T, 2 is selected from amino acid residues V or T, 3 is selected from amino acid residues G or A, and X is an arbitrary one. Nl represents an arbitrary number (for example, 34 to 39) of arbitrary amino acid residues, and n2 represents an arbitrary number (for example, 38 to 46) of arbitrary amino acid residues. Representing the Roh acid residues.
従って、 第 1図のマルチプルァライメン卜の配列表記式で示される保存配列 においては、 第 3発明において規定する （1 ) 〜 （3 ) の配列部分の内の少なく とも 1以上の部分が、 又はその全部が、 炭素数 15以上のプレニルリン酸に基質特 異性のあるホスファタ一ゼ活性を発現するために必要な配列 （ホスファターゼモ チーフ） であると考えられる。  Therefore, in the conserved sequence represented by the multiple-partition sequence notation formula in FIG. 1, at least one or more of the sequence portions (1) to (3) defined in the third invention are: Alternatively, it is considered that all of them are sequences (phosphatase motifs) necessary for expressing a phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
又、 第 1図のマルチプルァライメントの配列表記式で示される保存配列にお いて、 特定されたアミノ酸残基の 1以上が前記した定義に係る 「同類置換」 を受 けている変異体であるポリべプチドについても、 炭素数 15以上のプレニルリン酸 に基質特異性のあるホスファターゼ活性を示すと考えられる。  In addition, in the conserved sequence represented by the sequence notation of the multiple alignment in FIG. 1, one or more of the specified amino acid residues is a mutant that has undergone the “conservative substitution” according to the above definition. Polypeptides are also considered to exhibit substrate-specific phosphatase activity for prenyl phosphates having 15 or more carbon atoms.
本願発明に係るポリぺプチドの他の代表例が、 配列番号 1〜配列番号 6に示す アミノ酸配列において、 5 個以下のアミノ酸が置換， 欠失又は付加されたァミノ 酸配列を有すると共に、 炭素数 15以上のプレニルリン酸に基質特異性のあるホス ファタ一ゼ活性を示すポリぺプチドである。 Another typical example of the polypeptide according to the present invention is an amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6, which has an amino acid sequence in which 5 or less amino acids have been substituted, deleted or added, and Phospho with substrate specificity for more than 15 prenyl phosphates It is a polypeptide that exhibits fatase activity.
上記において、 置換， 欠失又は付加される 5 個以下のアミノ酸は、 その全部 がアミノ酸配列において互いに隣接する位置にあるアミノ酸であり得る。 又、 上 記の 5 個以下のアミノ酸は、 その一部がアミノ酸配列において互いに隣接する位 置にあるアミノ酸であり得る。 更に、 上記の 5 個以下のアミノ酸は、 いずれもが アミノ酸配列において互いに隣接しない位置にあるアミノ酸であり得る。  In the above, 5 or less amino acids to be substituted, deleted or added may be all amino acids at positions adjacent to each other in the amino acid sequence. In addition, the above-mentioned five or less amino acids may be partially adjacent amino acids in the amino acid sequence. Furthermore, any of the above five or less amino acids may be amino acids that are not adjacent to each other in the amino acid sequence.
以上に列挙したポリべプチドは、 上記それぞれの起源生物の細胞から常套的 な酵素単離手段によって直接に取得することができ、 又はそれぞれのポリぺプチ ドをコードする DNA を宿主に導入することにより、 同様にして多量に取得するこ とができる。  The above-listed polypeptides can be obtained directly from the cells of each of the above-mentioned source organisms by conventional enzyme isolation means, or the DNA encoding each polypeptide can be introduced into a host. Thus, a large amount can be obtained in the same manner.
第 1 4発明に係るプレニルアルコールの製造方法において、 導入又は発現強 化されるホスファターゼ遺伝子によってコ一ドされるホスファターゼは、 少なく ともプレニルリン酸に対して作用するものである限りにおいて限定されない。 即 ち、 基質特異性の低い酸性ホスファターゼゃアルカリホスファターゼ等から、 プ レニルリ ン酸に基質特異性のあるホスファターゼ、 更には炭素数 15以上のプレニ ルリ ン酸に基質特異性のあるホスファターゼに到る、 多様なレベルの基質特異性 のホスファターゼが限定なく含まれる。 但し、 より高い基質特異性のあるホスフ ァターゼ、 特に炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一 ゼを用いること力 更に好ましい。 プレニルリン酸に対して高い基質特異性のあ るホスファターゼとして、 第 1発明〜第 4発明に係るポリペプチドを好ましく例 示することができる。 同等の機能を有する酵母， 動物又は植物由来のポリぺプチ ドも好ましく例示することができる。 ポリヌクレオチド  In the method for producing prenyl alcohol according to the fourteenth aspect, the phosphatase encoded by the phosphatase gene to be introduced or enhanced in expression is not limited as long as it acts on at least prenyl phosphate. That is, from acid phosphatase with low substrate specificity to alkaline phosphatase, etc., to phosphatase having substrate specificity for prenyl phosphinate, and further to phosphatase having substrate specificity for prenyl phosphine having 15 or more carbon atoms. Phosphatases of varying levels of substrate specificity are included without limitation. However, it is more preferable to use a phosphatase having a higher substrate specificity, particularly a phosphatase having a substrate specificity to prenyl phosphate having 15 or more carbon atoms. Preferable examples of the phosphatase having high substrate specificity for prenyl phosphate include the polypeptides according to the first to fourth inventions. Preferable examples include yeast, animal or plant-derived polypeptides having equivalent functions. Polynucleotide
本願発明において、 ポリヌクレオチドとは、 1本鎖、 2本鎖又は 3本鎖の DNA 及び Z又は RNAを言う。 又、 本発明において、 組換え核酸とは、 組換えのために調製 されたポリヌクレオチドを言う。  In the present invention, the polynucleotide refers to single-stranded, double-stranded or triple-stranded DNA and Z or RNA. In the present invention, the term “recombinant nucleic acid” refers to a polynucleotide prepared for recombination.
本願発明に係るポリヌクレオチドは、 炭素数 15以上のプレニルリン酸に基質 特異性のあるホスファターゼをコ一ドするポリヌクレオチドである。 The polynucleotide according to the present invention has a substrate of prenyl phosphate having 15 or more carbon atoms. A polynucleotide encoding a specific phosphatase.
本願発明に係るポリヌクレオチドの代表例が、 配列番号 7〜配列番号 1 2の いずれかに示す塩基配列を有するポリヌクレオチドである。 これらは、 炭素数 15 以上のプレニルリン酸に基質特異性のあるホスファターゼ活性を示すポリぺプチ ドをコードすることが、 本願発明者によって初めて見出された。  A typical example of the polynucleotide according to the present invention is a polynucleotide having a base sequence shown in any one of SEQ ID NOS: 7 to 12. It has been found for the first time by the present inventors that these encode polypeptides having phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
配列番号 7に示す塩基配列を有するポリヌクレオチドは、 前記 AtPAPl であ る。 配列番号 8に示す塩基配列を有するポリヌクレオチドは、 前記 AtPAP2 であ る。 なお AtPAPl はシロイヌナズナ 'ゲノムプロジェク トで発表された第 II染色 体第 2セクションに位置することが分っており（Lin et al (1997) Nature 402, p. 767-768 ) 、 AtPAP2 は第 I染色体 BAC ライブラリ F9L1 に位置することが分つ ている （ F9L1. 2 (Accession No, AC007591 ) )。  The polynucleotide having the nucleotide sequence of SEQ ID NO: 7 is AtPAPl. The polynucleotide having the nucleotide sequence of SEQ ID NO: 8 is AtPAP2. AtPAPl is known to be located in the second section of chromosome II published in the Arabidopsis thaliana genome project (Lin et al (1997) Nature 402, p. 767-768), and AtPAP2 is located on chromosome I. It is known that it is located in the BAC library F9L1 (F9L1.2 (Accession No., AC007591)).
配列番号 9に示す塩基配列を有するポリヌクレオチドは前記 DPP1 である。 配列番号 1 0に示す塩基配列を有するポリヌクレオチドは前記 LPP1 である。 配 列番号 1 1に示す塩基配列を有するポリヌクレオチドは前記 PAP2 である。 配列 番号 1 2に示す塩基配列を有するポリヌクレオチドは前記 Dri42である。  The polynucleotide having the nucleotide sequence of SEQ ID NO: 9 is DPP1. The polynucleotide having the nucleotide sequence of SEQ ID NO: 10 is LPP1 described above. The polynucleotide having the nucleotide sequence of SEQ ID NO: 11 is PAP2. The polynucleotide having the nucleotide sequence of SEQ ID NO: 12 is Dri42 described above.
本願発明に係るポリヌクレオチドの他の代表例が、 図 1のマルチプルァライ メントの配列表記式で示される保存配列の一部 （第 3発明において規定する （1 ) 〜（3 ) の配列部分の内の少なくとも 1以上の部分） 又は全部を含むアミノ酸配 列を有するポリべプチドをコ一ドし、 又はそれらにおいて特定されたァミノ酸残 基の 1以上が同類置換を受けているポリぺプチドをコ一ドするポリヌクレオチド である。  Another representative example of the polynucleotide according to the present invention is a part of the conserved sequence represented by the sequence notation of the multiple alignment shown in FIG. 1 (part of the sequence portion (1) to (3) defined in the third invention). Or at least one of the above) or a polypeptide having an amino acid sequence that includes the entire amino acid sequence, or a polypeptide in which at least one of the amino acid residues specified therein has been conservatively substituted. The coding polynucleotide.
本願発明に係るポリヌクレオチドの更に他の代表例が、 配列番号 7〜配列番 号 1 2のいずれかに示す塩基配列と所定のストリンジェントな条件下でハイプリ ダイズする塩基配列を有し、 炭素数 15以上のプレニルリン酸に基質特異性のある ホスファターゼをコードするポリヌクレオチドである。  Still another representative example of the polynucleotide according to the present invention has a nucleotide sequence that hybridizes under a predetermined stringent condition with the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12, and has a carbon number of It is a polynucleotide encoding a phosphatase having substrate specificity for 15 or more prenyl phosphates.
上記において 「ストリンジェントな条件下でハイブリダィズする」 とは、 コ ロニーハイプリダイゼーション法， プラークハイプリダイゼーション法又はサザ ンブロットハイプリダイゼーション法等の適宜なハイプリダイゼーション法にお いて、 以下の条件下で一方のポリヌクレオチド （DNA ) 又は該ポリヌクレオチド の断片に対し他方のポリヌクレオチド (腿 ) がハイプリダイズできることを言 う。 即ち、 フィルターに固定化された一方のポリヌクレオチド又は該ポリヌクレ ォチドの断片に対し、 0. 7~ 1Mの NaCl の存在下、 所定温度 （X ° C ) 下で他方 のポリヌクレオチドのハイプリダイゼーションを行った後、 0. 1〜2 倍程度の SSC 溶液 （1倍濃度の SSC溶液の組成は、 150mM塩化ナトリウム， 15mMクェン酸ナト リウムよりなる） を用いて X ° Cの条件下でフィルターを洗浄した場合に、 他方 のポリヌクレオチドを同定できることを言う。 そして Γ χ° C」 とは、 少なく と も 50° C以上であり、 より好ましくは 60° C以上であり、 更に好ましくは 65° C 以上である。 In the above description, “hybridize under stringent conditions” refers to an appropriate hybridization method such as colony hybridization, plaque hybridization, or Southern blot hybridization. And that the other polynucleotide (thigh) can hybridize to one polynucleotide (DNA) or a fragment of the polynucleotide under the following conditions. That is, one polynucleotide or a fragment of the polynucleotide immobilized on the filter is subjected to hybridization of the other polynucleotide at a predetermined temperature (X ° C) in the presence of 0.7 to 1 M NaCl. After that, wash the filter under X ° C using 0.1 to 2 times SSC solution (1X concentration of SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate). Means that the other polynucleotide can be identified. The term “° C.” is at least 50 ° C. or more, more preferably 60 ° C. or more, and further preferably 65 ° C. or more.
以上に列挙したポリヌクレオチドは、 上記した各起源生物から常套的な単離 手段によって取得することができるし、 その他の原核生物等の各種単細胞生物や 動植物細胞から取得できる可能性がある。 当業者に良く知られた利用可能な方法 としてハイプリダイゼーシヨン技術 （ Southern 1975 J. Mol. Biol. 98 : 503、 Maniatis et al. Molecular CloningCold Spring Harbor Laboratory Pres) や PC R 技術 (Saiki et al. Science 239： 487 (1988) ) が挙げられる。 即ち、 当業者に とって、 既知の遺伝子の塩基配列又はその一部をプローブとして、 あるいはかか る塩基配列の一部にハイプリダイズするオリゴヌクレオチドをプライマーとして、 これと高い相同性を持つポリヌクレオチドを単離し、 該ポリヌクレオチドからホ スファターゼ遺伝子をクロ一ユングすることは容易である。 又、 上記した特徴的 なホスファターゼ活性を指標にして該ホスファタ一ゼを単離し、 そのアミノ酸配 列から該ホスファターゼ遺伝子をクローニングすることも容易である。  The above-listed polynucleotides can be obtained from the above-mentioned source organisms by conventional isolation means, or may be obtained from various unicellular organisms such as prokaryotes and animal and plant cells. Available methods well known to those skilled in the art include hybridization technology (Southern 1975 J. Mol. Biol. 98: 503, Maniatis et al. Molecular Cloning Cold Spring Harbor Laboratory Pres) and PCR technology (Saiki et al. Science 239: 487 (1988)). That is, for those skilled in the art, a polynucleotide having a high homology to a known gene or a part thereof as a probe or an oligonucleotide hybridizing to a part of the nucleotide sequence as a primer It is easy to isolate and clone the phosphatase gene from the polynucleotide. It is also easy to isolate the phosphatase using the characteristic phosphatase activity as an index and clone the phosphatase gene from its amino acid sequence.
第 1 4発明に係るプレニルアルコールの製造方法において導入又は発現強化 されるホスファターゼ遺伝子は、 少なくともプレニルリン酸に対して作用するホ スファターゼをコードする遺伝子である限りにおいて、 限定されない。 即ち、 基 質特異性の低い酸性ホスファターゼゃアル力リホスファターゼ等から、 プレニル リン酸に基質特異性のあるホスファターゼ、 更には炭素数 15以上のプレニルリン 酸に基質特異性のあるホスファターゼに到る、 多様なレベルの基質特異性のホス ファターゼをコードする遺伝子が限定なく含まれる。 但し、 より高い基質特異性 のあるホスファターゼ、 特に炭素数 15以上のプレニルリン酸に基質特異性のある ホスファタ一ゼをコ一ドする遺伝子を導入又は発現強化することが、 更に好まし レ、。 プレニルリン酸に対して髙ぃ基質特異性のあるホスファタ一ゼをコ一ドする 遺伝子として、 第 5発明〜第 7発明に係るポリヌクレオチドを好ましく例示でき る。 同等の機能を有する酵母， 植物又は動物由来のポリヌクレオチドも好ましく 例示できる。 The phosphatase gene introduced or enhanced in the method for producing prenyl alcohol according to the fourteenth invention is not limited as long as it is a gene encoding a phosphatase that acts on at least prenyl phosphate. That is, a variety of substances, from acid phosphatase with low substrate specificity to alkaline phosphatase, to phosphatase having substrate specificity for prenyl phosphate, and phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Phos with a high level of substrate specificity A gene encoding a fatase is included without limitation. However, it is more preferable to introduce or enhance the expression of a gene encoding a phosphatase having a higher substrate specificity, particularly a phosphatase having a substrate specificity to prenyl phosphate having 15 or more carbon atoms. As a gene encoding a phosphatase having substrate specificity to prenyl phosphate, the polynucleotides according to the fifth to seventh inventions can be preferably exemplified. Yeast, plant or animal-derived polynucleotides having equivalent functions can also be preferably exemplified.
ホスファターゼ遺伝子の導入又は発現強化  Introduction of phosphatase gene or enhanced expression
上記した非特異的ホスファタ一ゼをコ一ドする遺伝子、 より好ましくはプレ 二ルリン酸に基質特異性のあるホスファタ一ゼをコ一ドする遺伝子、 更に好まし くは炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼをコ一ド する遺伝子が、 宿主細胞に導入又は発現強化される。  A gene encoding the above-mentioned non-specific phosphatase, more preferably a gene encoding a phosphatase having substrate specificity for prenyrphosphate, more preferably a prenylphosphate having 15 or more carbon atoms A gene encoding a phosphatase having specific substrate specificity is introduced into a host cell or its expression is enhanced.
本願発明において 「ホスファターゼ遺伝子を導入又は発現強化」 とは、 以下 の意味である。 即ち、 「ホスファターゼ遺伝子を導入」 とは、 該ホスファターゼ 遺伝子を組換え核酸として宿主細胞に導入する全ての場合を含む。 少なくとも、 ベクターによってホスファターゼ遺伝子を導入する場合や、 PCR フラグメント等 を用いたポリヌクレオチドの相同組換えによってホスファターゼ遺伝子を導入す る場合が含まれる。 より好ましくは、 該ホスファターゼ遺伝子が発現を強化する 発現ベクターによって導入される。 又、 「ホスファターゼ遺伝子を発現強化 J と は、 結果的に該ホスファターゼ遺伝子の発現が強化される全ての場合を含む。 例 えば、 該ホスファターゼ遺伝子をその発現を強化する手段を伴って組換え核酸と して導入する場合、 宿主細胞における該ホスファターゼ遺伝子のコピー数を増強 させる場合、 宿主細胞の培養条件を調整 （遺伝子発現の誘導物質の添加等） する ことにより該ホスファタ一ゼ遺伝子の発現を強化する場合、 等が含まれる。  In the present invention, “introducing a phosphatase gene or enhancing expression” has the following meaning. That is, "introducing a phosphatase gene" includes all cases where the phosphatase gene is introduced into a host cell as a recombinant nucleic acid. At least the case where the phosphatase gene is introduced by a vector and the case where the phosphatase gene is introduced by homologous recombination of a polynucleotide using a PCR fragment or the like are included. More preferably, the phosphatase gene is introduced by an expression vector that enhances expression. The expression “enhancement of the expression of the phosphatase gene J” includes all cases in which the expression of the phosphatase gene is consequently enhanced. For example, the expression of the phosphatase gene is combined with a recombinant nucleic acid together with a means for enhancing the expression. When the copy number of the phosphatase gene in the host cell is increased, the expression of the phosphatase gene is enhanced by adjusting the culture conditions of the host cell (adding an inducer of gene expression, etc.). In this case, etc. are included.
又、 上記各種のホスファタ一ゼをコ一ドする遺伝子を導入又は発現強化する と同時に、 プレニルニリン酸生合成経路に関与する酵素の遺伝子のうち少なく と も 1の遺伝子を宿主細胞に導入又は発現強化することができる。 ホスファターゼ をコードする遺伝子とプレニルニリン酸生合成経路に関与する酵素の遺伝子とを 同時に導入又は発現強化する形態は任意であり、 例えば、 これらの遺伝子を同時 期にかつ別個に上記各種の同一又は別異の手段を利用して導入又は発現強化して も良いし、 後述のように連結遺伝子として同時に導入又は発現強化しても良い プレニルニリン酸生合成経路に関与する酵素の遺伝子としては、 例えばヒ ド 口キシメチルダルタリル Co A還元酵素遺伝子、 フアルネシルニリン酸合成酵素遺 伝子及ぴゲラ二ルゲラ二ルニリン酸合成酵素遺伝子から選ばれる少なく とも 1の 遺伝子を好ましく例示できる。 あるいは、 ァセチル CoA合成酵素遺伝子、 ァセチル CoA —ァセチルトランスフェラーゼ遺伝子、 ヒ ドロキシメチルダノレタリル CoA 合 成酵素遺伝子、 メバロン酸キナーゼ遺伝子、 メバロン酸リン酸キナーゼ遺伝子、 メバロン酸ニリン酸デカルボキシラーゼ遺伝子、 ィソペンテ二ルニリン酸イソメ ラーゼ遺伝子、 デォキシキシルロースリン酸リダク トイソメラーゼ遺伝子、 デォ キシキシルロースリン酸合成酵素遺伝子、 MEPシチジリルトランスフェラーゼ遺 伝子、 CDP- MEキナーゼ遺伝子及ぴ MECDP合成酵素遺伝子から選ばれる少なくとも 1の遺伝子も好ましく例示できる。 . In addition, at the same time as introducing or enhancing the expression of the various phosphatase-encoding genes, at least one gene among the enzymes involved in the prenyl diphosphate biosynthesis pathway is introduced or enhanced in the host cell. can do. A gene encoding a phosphatase and a gene encoding an enzyme involved in the prenyl diphosphate biosynthesis pathway The mode of simultaneous introduction or expression enhancement is arbitrary.For example, these genes may be simultaneously or separately introduced or enhanced by using the same or different means as described above, or as described later. Genes of enzymes involved in the prenyl diphosphate biosynthetic pathway that may be simultaneously introduced or enhanced in expression as a linked gene include, for example, the gene for the oxamethylxartalaryl CoA reductase, Preferred examples include at least one gene selected from germline and geranirgerinirnic acid synthase genes. Alternatively, acetyl CoA synthase gene, acetyl CoA-acetyl transferase gene, hydroxymethyldanoletaryl CoA synthase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate decarboxylase gene, isopentene Selected from luniphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene and MECDP synthase gene Preferred examples include at least one gene. .
又、 上記各種のホスファターゼをコードする遺伝子を、 プレニルニリン酸生 合成経路に関与する酵素の遺伝子と連結して、 上記と同様に宿主細胞に導入又は 発現強化することができる。 プレニルニリン酸生合成経路に関与する酵素の遺伝 子としては、 例えばフアルネシルニリン酸合成酵素遺伝子及びゲラニルゲラニル ニリン酸合成酵素遺伝子から選ばれる少なく とも 1の遺伝子を好ましく例示でき る。 あるいは、 ァセチル CoA合成酵素遺伝子、 ァセチル CoA —ァセチルトランスフ エラーゼ遺伝子、 ヒ ドロキシメチルダルタリル CoA 合成酵素遺伝子、 ヒ ドロキシ メチルダルタリル Co A還元酵素遺伝子、 メバロン酸キナーゼ遺伝子、 メバロン酸 リン酸キナーゼ遺伝子、 メバロン酸二リン酸デカルボキシラーゼ遺伝子、 イソべ ンテニルニリン酸ィソメラーゼ遺伝子、 デォキシキシルロースリン酸リダク トイ ソメラーゼ遺伝子、 デォキシキシルロースリン酸合成酵素遺伝子、 MEPシチジリ ルトランスフェラ一ゼ遺伝子、 CDP- MEキナーゼ遺伝子又は MECDP合成酵素遺伝子 から選ばれる少なく とも 1の遺伝子も好ましく例示できる。 このように連結され た遺伝子を 「ホスファターゼ連結遺伝子」 とも呼ぶ。 なお、 連結された遺伝子とは、 2つ以上の遺伝子を 1つの読み枠（0RF) として発 現できるように連結し、 発現されるポリペプチドが融合タンパク質になるように したものである。 また連結する遺伝子と遺伝子の結合領域に、 それぞれの遺伝子 翻訳産物が適切に機能する立体構造がとれるように人工的なヌクレオチド配列In addition, the genes encoding the above various phosphatases can be linked to genes of enzymes involved in the prenyl diphosphate biosynthesis pathway, and introduced or enhanced in host cells in the same manner as described above. Preferred examples of the gene of an enzyme involved in the prenyl diphosphate biosynthetic pathway include at least one gene selected from, for example, a huanesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene. Alternatively, acetyl-CoA synthase gene, acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethyldaltharyl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalon Acid diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP cityryltransferase gene, CDP-ME kinase gene Alternatively, at least one gene selected from MECDP synthase genes can also be preferably exemplified. The gene linked in this way is also called a “phosphatase linked gene”. The linked gene is a gene in which two or more genes are linked so that they can be expressed as one open reading frame (0RF), and the expressed polypeptide becomes a fusion protein. In addition, an artificial nucleotide sequence is used so that the three-dimensional structure in which each gene translation product functions properly can be obtained in the connecting region between the linked genes.
(リンカ一配列） を自由に揷入することも可能である。 例えば Gly Gly Gly Ser や Gly Gly Gly Gly Ser というペプチド配列をコードするリ ンカ一配列などが挙 げられる。 これらの配列は、 大腸菌で抗体を発現させるときの H 鎖と L 鎖をつな ぐリ ンカ一配列として利用された （Huston J. S. et al (1988) Proc. Natl. Acad.(Linker sequence) can be freely introduced. Examples include Gly Gly Gly Ser and a linker sequence encoding a peptide sequence of Gly Gly Gly Gly Ser. These sequences were used as a linker sequence linking the H and L chains when expressing antibodies in E. coli (Huston J. S. et al (1988) Proc. Natl. Acad.
Sci . USA 85： 5879-5883)。 Sci. USA 85: 5879-5883).
発現ベクター又は導入しようとする遺伝子の発現機能を持つポリヌクレオチ ド断片は、 導入しようとするホスファターゼ遺伝子又はホスファタ一ゼ連結遣伝 子の発現を強化する種々のポリヌクレオチド断片と連結されたものである。 好ま しくは、 発現ベクターは転写プロモーター， 転写ターミネータ一， 組換え体選抜 用マーカー遺伝子， ェンハンサーを含み得る。 植物発現ベクターにおいては、 更 に好ましく T - DNA 領域を含み得る。 一般的な発現ベクターの構築方法として、 例 えば、 PCR 法等で調製した遺伝子断片を、 適当な制限酵素とリガーゼを用いる既 知の方法で発現ベクターに組み込むことができる。  The expression vector or the polynucleotide fragment having the expression function of the gene to be introduced is linked to various polynucleotide fragments that enhance the expression of the phosphatase gene or phosphatase-ligating gene to be introduced. . Preferably, the expression vector may include a transcription promoter, a transcription terminator, a marker gene for selecting a recombinant, and an enhancer. Plant expression vectors may more preferably contain a T-DNA region. As a general method for constructing an expression vector, for example, a gene fragment prepared by a PCR method or the like can be incorporated into an expression vector by a known method using an appropriate restriction enzyme and ligase.
ベクタ一としては、 例えば ^ cerevi s iaeを宿主とする場合に良く利用さ れる 「YEpl3」 ， 「YEp24」 ， 「YCp50」 ， rpYES2j , rpRS414j ， 「PRS415J , rPRS416J , 「pRS413」， 「pRS404」 , 「pRS405」 , 「pRS406」 ， 「pRS403j ,As a vector one, for example ^ the cerevi s iae is often used in the case of the host "YEpl3", "YEp24", "YCp50", r p YES2j, r p RS414j , "P RS415J, r P RS416J," pRS413 , PRS404, pRS405, pRS406, pRS403j,
「 pAUR101」 等、 大腸菌 (Escherichia col i) を宿主とする場合に良く利用され るプラスミ ド 「pSC101」， 「pBR322」， 「pHSG298」， 「pVC18」， 「pVC19」 , 「p Trc99AJ , 「pMal- c2」 , 「pGEX2T」 ， 「pTV118N」， 「pTV119N」 等、 バシルス · ズブチリス (Bac i l lus subt i l i s) を宿主とする場合に良く利用されるプラスミ ド 「pUB110」 ， rpC194j 等を好ましく使用でき、 その他にも 「ρΒΙ 1221」 ， 「pBI 1101」 その他各種のものを限定なく使用できる。 Plasmids “pSC101”, “pBR322”, “pHSG298”, “pVC18”, “pVC19”, “pVC19A”, “pTrc99AJ”, “pMal-”, which are often used when Escherichia coli is used as a host, such as “pAUR101” Plasmids “pUB110”, r p C194j and the like often used when Bacillus subtilis (Bacil lus subtilis) is used as a host, such as “c2”, “pGEX2T”, “pTV118N” and “pTV119N”, can be preferably used. , "ΡΒΙ1221", "pBI 1101" and other various types can be used without limitation.
発現べク'ターは、 ホスファターゼを恒常的又は誘導的に発現させるための転写 プロモーターを含有し得る。 例えば、 ^ cerevisiaeを宿主とする場合に良く禾I 用されるアルコールデヒ ドロゲナ一ゼ遺伝子 （ADH1及ぴ ADH2) , トリオースリン 酸デヒ ドロゲナーゼ遺伝子 （TDH3) , ガラクトース異化に関連する遺伝子 （GAL1, GAL7, GAL10 ) , 酸性ホスファタ一ゼ遺伝子 （PH05) 又はメタ口チォネイン遺伝 子 （CUP1) の転写プロモーターが挙げられる。 大腸菌 （ col i ) での発現用と して trp, lac, trc, tac 等の転写プロモーターを用いることができる。 The expression vector may contain a transcription promoter for constitutively or inducibly expressing phosphatase. For example, if you use ^ cerevisiae as a host, Used alcohol dehydrogenase genes (ADH1 and ADH2), triosephosphate dehydrogenase gene (TDH3), genes associated with galactose catabolism (GAL1, GAL7, GAL10), acid phosphatase gene (PH05) or Examples include the transcription promoter of the meta-mouth thionein gene (CUP1). For expression in E. coli (col), transcription promoters such as trp, lac, trc, and tac can be used.
他にも、 恒常的に発現させるための転写プロモーターとしては、 例えばカリ フラワーモザイクウィルスの 3 5 Sプロモーター （ Odel l et al. 1985 Nature 313 : 810 ) , イネのァクチンプロモーター （ Zhang et al. 1991 Plant Cel l 3 : 1 155) , トウモロコシのュビキチンプロモーター （Cornejo et al 1993 Plant Mol . Biol. 23 : 567) その他各種のものを限定なく使用できる。 誘導的に発現させるた めのプロモーターとしては、 糸状菌， 細菌， ウィルスの感染や侵入、 低温， 高温， 乾燥， 紫外線の照射， 特定化合物の散布等の外因によって発現することが知られ ている転写プロモーター等が挙げられる。  Other transcription promoters for the constant expression include, for example, the cauliflower mosaic virus 35S promoter (Odel et al. 1985 Nature 313: 810) and the rice actin promoter (Zhang et al. 1991). Plant Cell 3: 3 155), corn ubiquitin promoter (Cornejo et al 1993 Plant Mol. Biol. 23: 567), and various other types can be used without limitation. Promoters for inducible expression include transcription known to be expressed by exogenous factors such as infection or invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, irradiation with ultraviolet light, and spraying of specific compounds. Promoters and the like.
ホスファターゼ遺伝子又はホスファターゼ連結遺伝子を導入又は発現強化す るための組換え核酸は、 必ずしもベクター機能を持っていなくても、 例えばゲノ ムインテグレーションすることができれば良い。  A recombinant nucleic acid for introducing or enhancing the expression of a phosphatase gene or a phosphatase-linked gene does not necessarily have to have a vector function, but may be any one that can perform genomic integration, for example.
発現ベクターその他の組換え核酸に連結するホスファターゼ遺伝子又はホス ファターゼ連結遺伝子には、 ホスファターゼに脂質膜近傍への局在化シグナルを 付加する塩基配列領域を含ませることも可能である。  A phosphatase gene or a phosphatase-linked gene linked to an expression vector or other recombinant nucleic acid can contain a base sequence region that adds a localization signal to the vicinity of a lipid membrane to phosphatase.
プレニルリン酸 （特に炭素数 15以上のプレニルリン酸） は疎水性であり、 宿 主細胞の細胞膜近傍や、 宿主細胞が真核細胞である場合のゴルジ体， 小胞体等の 各種細胞器管膜近傍に高濃度に局在する傾向がある。 従ってホスファタ一ゼに疎 水性ぺプチド等の局在化シグナルを付加することにより、 ホスファタ一ゼと基質 たるプレニルリ ン酸との接触確率を向上させ、 プレニルアルコールの生産性を高 め得る。 特にホスファターゼ遺伝子が元々基質非特異的ホスファタ一ゼをコ一ド する遺伝子である場合に、 ホスファターゼに対する上記シグナルの付加が有効で ある。  Prenyl phosphate (especially prenyl phosphate having 15 or more carbon atoms) is hydrophobic and is located near the cell membrane of host cells and in the cell membrane of various organs such as the Golgi body and endoplasmic reticulum when the host cell is a eukaryotic cell. Tends to be localized at higher concentrations. Therefore, by adding a localization signal such as a hydrophobic peptide to phosphatase, the probability of contact between phosphatase and prenyl phosphinate as a substrate can be improved, and the productivity of prenyl alcohol can be increased. In particular, when the phosphatase gene originally encodes a substrate non-specific phosphatase, the addition of the above signal to the phosphatase is effective.
局在化シグナルの代表的なものは、 公知の各種疎水性シグナルぺプチドであ る。 周知のように、 局在化シグナルの選択により宿主細胞内におけるホスファタ 一ゼの局在化部位を選択することも可能である。 ホスファターゼ遺伝子又はホス ファターゼ連結遺伝子に対して局在化シグナルを付加するための塩基配列領域を 付加したもとで発現ベクターその他の組換え核酸を構築する一般的な方法として、 例えば、 公知の各種シグナルぺプチドをコ一ドする遺伝子ドメイン及びホスファ ターゼ遺伝子又はホスファターゼ連結遺伝子を PCR 法等で調製し、 これらの遺伝 子を適当な制限酵素とリガーゼを用いる既知の方法で連結し、 更に連結された遺 伝子を適当な発現ベクターに組込むことができる。 Representative localization signals include various known hydrophobic signal peptides. You. As is well known, it is also possible to select a phosphatase localization site in a host cell by selecting a localization signal. As a general method for constructing an expression vector or other recombinant nucleic acid with the addition of a nucleotide sequence region for adding a localization signal to a phosphatase gene or a phosphatase-linked gene, for example, various known signals may be used. A gene domain encoding a peptide and a phosphatase gene or a phosphatase-linked gene are prepared by PCR or the like, and these genes are ligated by a known method using an appropriate restriction enzyme and ligase. The gene can be incorporated into a suitable expression vector.
組換え核酸の宿主への導入  Introduction of recombinant nucleic acid into host
本願発明に係るホスファターゼ遺伝子を生物体に導入又は発現強化させる場 合、 宿主細胞の種類は限定されない。 例えば、 原核生物， 真菌 (Eumvcetes ) ， 子嚢菌類 (Ascomvcetes ) , 単細胞真核生物， 又は動物あるいは植物の生細胞を 任意に選択できる。 植物組織培養細胞及び動物組織培養細胞を含む多細胞生物の 生組織培養細胞も任意に選択できる。  When introducing or enhancing the expression of the phosphatase gene according to the present invention into an organism, the type of host cell is not limited. For example, prokaryotes, fungi (Eumvcetes), ascomycetes (Ascomvcetes), unicellular eukaryotes, or living cells of animals or plants can be arbitrarily selected. Live tissue culture cells of multicellular organisms including plant tissue culture cells and animal tissue culture cells can also be arbitrarily selected.
上記の植物細胞又は植物組織培養細胞において、 「植物」 とは藻類， 蘚苔類， シダ類， 裸子植物及び被子植物を含む。 上記の動物細胞又は動物組織培養細胞に おいて、 「動物」 とは海綿動物， 腔腸動物， 線形動物， 軟体動物， 節足動物， 刺 皮動物及び各種の脊椎動物を含む。 動物又は植物の生細胞又は生組織培養細胞と しては、 動植物の懸濁培養細胞、 内臓， 葉， 根等の動植物器管の組織培養細胞， 植物におけるカルス等を例示できる。  In the above-mentioned plant cells or plant tissue culture cells, the “plant” includes algae, bryophytes, ferns, gymnosperms and angiosperms. In the above animal cells or animal tissue culture cells, the term "animal" includes sponges, coelenterates, nematodes, mollusks, arthropods, arthropods, and various vertebrates. Examples of living cells or living tissue culture cells of animals or plants include suspension culture cells of animals and plants, tissue culture cells of animal and plant organs such as viscera, leaves, and roots, and callus in plants.
宿主としては、 特に限定されないが、 ^ cerevi siae, ピキア ' パス トリス (Pichia pastris) 等のサッカロミセス属ゃピキア属その他に属する酵母、 ^ col i、 バシルス ·ズブチリス (Baci l lus subtil is) , バシルス ·プレビス ac i l lus brevis) 等のェシエリヒア属又はバシルス属に属する細菌、 ァスペル ギルス · オリゼー (Aspergi l lus oryzae) ， ァスペルギルス ·二ガー (Aspergil lus ni gar) 等のァスペルギルス属に属する糸状菌、 カイコ (Bombyx mori) の 培養細胞， COS細胞又は CH0細胞等の動物細胞、 あるいは植物細胞等が好ましく 例示される。 宿主としては、 特にサッカロミセス属の酵母を好ましく例示できる。 より具 体的には、 ^ cerevis iaeの YPH499 (ATCC76625, MATa, ura3 - 52, lys2- 801, ade2 - 101， trpl-delta63, hi s3-delta200, leu2 - deltal) 、 YPH500 ( ATCC76626, MATalpha, ura3- 52, lys2- 801, ade2- 101, trpl- delta63, his3- delta200， l eu 2 - deltal ) 、 A451 (ATCC200589, MATalpha, canl, leu2， trpl, ura3, aro7) 、 W303-1A (ATCC208353, MATalpha, leu2 - 3, leu2 - 112, Ms3— 11, ade2— 1, ura3 - 1, trpl-1, canl-100) 又は W303 - IB MATa, leu2- 3， leu2- 112， his3 - 11, ade2 - 1 , ura3-l, trpl-1, canl-100 ! haploid of W303 (ATCC201239) ) を、 とりわけ好まし く例示できる。 Hosts include, but are not limited to, yeast belonging to the genus Saccharomyces セ ス, such as ^ cerevi siae, Pichia pastoris (Pichia pastris), ^ colli, ^ col i, Bacillus subtilis, Bacillus Bacteria belonging to the genus Escherichia or Bacillus, such as previs ac il lus brevis; Aspergillus oryzae; filamentous fungi belonging to the genus Aspergillus, such as Aspergillus nigar; mori), animal cells such as COS cells or CH0 cells, and plant cells. Preferred examples of the host include yeast of the genus Saccharomyces. More specifically, ^ cerevis iae YPH499 (ATCC76625, MATa, ura3-52, lys2-801, ade2-101, trpl-delta63, his3-delta200, leu2-deltal), YPH500 (ATCC76626, MATalpha, ura3 -52, lys2-801, ade2-101, trpl-delta63, his3-delta200, leu2-deltal), A451 (ATCC200589, MATalpha, canl, leu2, trpl, ura3, aro7), W303-1A (ATCC208353, MATalpha , leu2-3, leu2-112, Ms3-11, ade2-1, ura3-1, trpl-1, canl-100) or W303-IB MATa, leu2-3, leu2-112, his3-11, ade2-1 haploid of W303 (ATCC201239)) can be particularly preferably exemplified.
本願発明に係るホスファターゼ遺伝子を宿主細胞に導入するに当たり、 前記 のような種々の態様で、 宿主に適合した組換え核酸を利用することができる。 よ り具体的には、 大腸菌への外来遺伝子の導入法は、 ハナハンの方法等の確立され た幾つかの方法を利用できる。 酵母への外来遺伝子の導入法は、 リチウム法等の 確立された幾つかの方法を利用できる。 ホスファターゼ遺伝子の導入においては、 宿主細胞の染色体内に組込むこともできるし、 プラスミ ド等として細胞内に保持 することもできる。  In introducing the phosphatase gene according to the present invention into a host cell, a recombinant nucleic acid suitable for the host can be used in various modes as described above. More specifically, as a method for introducing a foreign gene into Escherichia coli, several established methods such as the Hanahan method can be used. Several well-established methods, such as the lithium method, can be used for introducing a foreign gene into yeast. In introducing the phosphatase gene, it can be integrated into the chromosome of the host cell, or can be retained in the cell as a plasmid or the like.
プレニ アルコールの製造方法  Method for producing pleni alcohol
本願発明に係るプレニルアルコールの製造方法はプレニルアルコール全般の 生産性の向上に有利であるが、 中でも炭素数 10以上のプレニルアルコール、 と り わけ炭素数 15以上のプレニルアルコールの生産性の向上が著しい。 プレニルアル コールの製造方法における炭素数 15以上のプレニルアルコール特に F0H ， GG0Hの 生産性の向上を一般的に対比すると、 第 1 5発明の方法が特に効果が高い。 又、 第 1 3発明の方法や第 1 9発明の方法も、 プレニルアルコールの生産性を向上さ せる。  Although the method for producing prenyl alcohol according to the present invention is advantageous for improving the productivity of prenyl alcohol in general, the productivity of prenyl alcohol having 10 or more carbon atoms, particularly, prenyl alcohol having 15 or more carbon atoms is remarkably improved. . The method of the fifteenth invention is particularly effective when the improvement in the productivity of prenyl alcohols having 15 or more carbon atoms, particularly F0H and GG0H in the method for producing prenyl alcohol is generally compared. Further, the method of the thirteenth invention and the method of the nineteenth invention also improve the productivity of prenyl alcohol.
本願発明に係るプレニルアルコールの製造方法においては、 産業上有用なト ランス型のプレニルアルコールを製造できると言う利点がある。 ここに 「トラン ス型」 とは、 炭素数 5 のプレニルアルコールにおける トランス型と、 炭素数 10以 上のプレニルアルコールにおける全トランス型とを意味する。 一般的に組換え体を利用する目的物の生産法において認められることである 力 S、 本発明のプレニルアルコールの製造方法においても、 導入遺伝子の選択、 導 入すべき宿主の選択、 発現ベクター等の導入手段とそれに適したポリヌクレオチ ドの構築方法の選択、 培地もしくはこれに対する添加物の種類や濃度の選択、 組 換え体の培養条件あるいは生育条件の選択等のファクターが、 プレニルアルコー ルの生産量に影響する場合がある。 The method for producing prenyl alcohol according to the present invention has an advantage that an industrially useful trans prenyl alcohol can be produced. Here, the “trans form” means a trans form in prenyl alcohol having 5 carbon atoms and an all trans form in prenyl alcohol having 10 or more carbon atoms. In the method for producing a prenyl alcohol of the present invention, selection of a transgene, selection of a host to be introduced, expression vector, etc. Factors such as selection of the method of introducing the plasmid and the method of constructing a polynucleotide suitable for the method, selection of the type and concentration of the medium or the additive thereto, and selection of the culture or growth conditions for the recombinant, may affect the production of prenyl alcohol. May affect quantity.
培養物からのプレニルアルコールの採取に当たり、 プレニルアルコール全般 を採取するか、 例えば炭素数 15以上のプレニルアルコール又はその内の 1種又は 2種以上のみを採取する場合のように特定の 1種又は 2種以上のプレニルアルコ ールを選択的に採取するかは、 プレニルアルコール製造方法の実施目的次第で、 任意に決定できる。  When collecting prenyl alcohol from a culture, collect prenyl alcohol in general, or a specific prenyl alcohol such as prenyl alcohol having 15 or more carbon atoms or only one or more of them. Whether to selectively collect more than one kind of prenyl alcohol can be arbitrarily determined depending on the purpose of the method for producing a prenyl alcohol.
生産されたプレニルアルコールは、 通常行われる細胞破砕物からの抽出だけ でなく、 適当な有機溶媒を用いて培養液からも直接に抽出できる。 宿主として酵 母を用いる場合を含め、 利用する宿主の種類によってはプレニルアルコールの少 なく とも一部が宿主細胞内あるいは細胞表面に止まる場合があるが、 細胞膜もし くは細胞壁の破壊や、 へキサンやペンタン等の非極性有機溶媒による抽出等の公 知の各種の操作を経て、 容易に抽出することができる。 実施例 宿主、 ベクター等  The produced prenyl alcohol can be directly extracted from the culture using an appropriate organic solvent, in addition to the usual extraction from cell debris. Depending on the type of host used, including the case where an yeast is used as the host, at least a portion of the prenyl alcohol may remain in the host cell or on the cell surface, but the cell membrane or cell wall is destroyed, and Extraction can be easily performed through various known operations such as extraction with a non-polar organic solvent such as pentane and pentane. Examples Hosts, vectors, etc.
S^. cerevi s iae = E. col i間のシャ トルベクターとして、 pRS404 pRS405 p RS406 (Stratagene) pYES2 ( Invitrogen) および AURlOl ( Takara)を使用 した。 DM 断片を pT7Blue ベクター又は pCR2. 1- T0P0 ベクターへ導入してクロー ユングする際には、 JM109コンビテント細胞 （Takara) を宿主として用 いた。 DNA 断片を pRS ベクター又は pALTERベクタ 導入してクローニングする 際には、 ！：_ col i SURE2スーパーコンビテント細胞 ( Stratagene) 又は ^ col i HB101コンビテント細胞 （Takara) を宿主として用いた。 遺伝子導入用の宿主には、 cerevisiaeの YPH499 、 A451、 W303-1B の各 株を用いた。 PRS404 pRS405 p RS406 (Stratagene) pYES2 (Invitrogen) and AURlOl (Takara) were used as shuttle vectors between S ^. Cerevisiae = E. coli. When the DM fragment was introduced into the pT7Blue vector or pCR2.1-TOP0 vector and cloned, JM109 competent cells (Takara) were used as a host. When cloning a DNA fragment by introducing it into a pRS vector or pALTER vector,! : _ Col i SURE2 supercompetent cells (Stratagene) or ^ col i HB101 competent cells (Takara) were used as hosts. As the host for gene transfer, Y. cerevisiae strains YPH499, A451, and W303-1B were used.
S^. cerevi siaeの cDNA から遺伝子をクローニングする際には、 cerevi s iae DBY746 由来の cDNAライブラリ "Quick-Clone cDNA" ( Clontec) を用いた。 又、 酵母ゲノム DNA 調製用キッ ト "Gen とるくん" （Takara) 添付のプロ トコル に従って、 ^ cerevi s iae YPH499 からゲノム DNA を調製した。  When cloning a gene from cDNA of S ^. Cerevisiae, a cDNA library "Quick-Clone cDNA" (Clontec) derived from cerevisiae DBY746 was used. Genomic DNA was prepared from ^ cerevisiae YPH499 according to the protocol attached to the yeast genomic DNA preparation kit "Gen Toru-kun" (Takara).
col iからのプラスミ ド DNA の調製には、 Wizard PureFect ion Plasmi d DNA Purification System (Promega) 用レヽた。  For preparation of plasmid DNA from coli, we used the Wizard PureFection Plasmid DNA Purification System (Promega).
PC PC
PCR法による遺伝子クローユングは、 特別な記載がない限り、 次の条件で行 つた。 5 μ ί の 10x ExTaq buffer (Takara) 、 4 μ L の 2. 5mM dNTP混合物、 1 μ L の 5U/ L ExTaq (Takara) 、 10 pmol のプライマー 1 (FWプライマー、 以 下同様） 、 10 pmol のプライマー 2 (RVプライマー、 以下同様） 、 及ぴ 0. 5ngの cDNA 又はゲノム DNA を含む 50 L 溶液を調製し、 次のように PCRを行った。 Gene closing by the PCR method was performed under the following conditions unless otherwise specified. 5 μί of 10x ExTaq buffer (Takara), 4 μL of 2.5 mM dNTP mixture, 1 μL of 5 U / L ExTaq (Takara), 10 pmol of primer 1 (FW primer, same as below), 10 pmol of A 50 L solution containing Primer 2 (RV primer, the same applies hereinafter) and 0.5 ng of cDNA or genomic DNA was prepared, and PCR was performed as follows.
94° C一 45秒， 55° C - 1 分， 72° C— 2 分； 30サイクル。 94 ° C-45 seconds, 55 ° C-1 minute, 72 ° C-2 minutes; 30 cycles.
pRS ベクターへの CYC1転写ターミネ一ターの挿入  Insertion of CYC1 transcription terminator into pRS vector
CYC1転写ターミネータ一である CYClt 断片は、 PCR で調製した。 配列番号 1 3に示す塩基配列を持つ PCRプライマー （Xhol- TcyclFW) 、 配列番号 1 4に示す 塩基配列を持つ PCRプライマー （Kpnl-TcyclRV) 、 配列番号 1 5に示す塩基配列 を持つ PCRプライマ一 （Apal- TcyclRV) を、 前記 Xhol - TcyclFW と Kpnl- TcyclR V の組合せと、 前記 Xhol- TcyclFW と Apal- TcyclRV の組合せによって用い、 铸 型として PYES2 を用いた。 反応溶液としては、 0. 1 μ g pYES2、 50pmolプライマ 一 DNA、 IX pfu buffer with MgS04 (Promega) 、 lOnmol dNTP、 1. 5U Pfu DNA p olymerase ( Promega) 、 1 μ L perfect match polymerase enhancer (Stratag ene) を含む 50 μ L 溶液を調製し、 次のように PCRを行った。 The CYClt fragment, one of the CYC1 transcription terminators, was prepared by PCR. A PCR primer having the nucleotide sequence shown in SEQ ID NO: 13 (Xhol-TcyclFW), a PCR primer having the nucleotide sequence shown in SEQ ID NO: 14 (Kpnl-TcyclRV), and a PCR primer having the nucleotide sequence shown in SEQ ID NO: 15 ( Apal-TcyclRV) was used according to the combination of the Xhol-TcyclFW and Kpnl-TcyclRV and the combination of the Xhol-TcyclFW and Apal-TcyclRV, and PYES2 was used as a 铸 type. The reaction solution, 0. 1 μ g pYES2, 50pmol primer one DNA, IX pfu buffer with MgS0 4 (Promega), lOnmol dNTP, 1. 5U Pfu DNA p olymerase (Promega), 1 μ L perfect match polymerase enhancer (Stratag ene) was prepared and a PCR was performed as follows.
95° C— 2 分 95 ° C—2 minutes
95° C— 45秒， 60° C—30秒， 72° C— 1 分； 30サイクル  95 ° C—45 seconds, 60 ° C—30 seconds, 72 ° C—1 minute; 30 cycles
72° C - 5 分, 4° Cス トック 増幅した 2 種の DNA をそれぞれ、 制限酵素 Xholと Kpnl、 又は Xholと Apal を 用いて切断し、 ァガロースゲル電気泳動によって 260bpの DNA 断片を精製した。 これらの DNA 断片を CYClt- XK及び CYCltXAと呼ぶ。 72 ° C-5 minutes, 4 ° C stock The two amplified DNAs were cut with restriction enzymes Xhol and Kpnl or Xhol and Apal, respectively, and a 260 bp DNA fragment was purified by agarose gel electrophoresis. These DNA fragments are called CYClt-XK and CYCltXA.
PRS405の Xhol- Kpnl 切断部位に CYClt- ΧΚ を、 pRS404及び pRS406の Xhol- Apa I切断部位に CYCltXA を揷入した。 これらをそれぞれ、 pRS405Tcyc、 pRS404Tcyc, pRS406Tcycと呼ぶ。  CYClt-ΧΚ was inserted into the Xhol-Kpnl cleavage site of PRS405, and CYCltXA was inserted into the Xhol-ApaI cleavage site of pRS404 and pRS406. These are called pRS405Tcyc, pRS404Tcyc and pRS406Tcyc, respectively.
転写プロモーターの調製  Preparation of transcription promoter
^_ cerevisiaeのゲノム DNA を踌型にして、 PCR により TDH3 転写プロモー タ一 （TDH3P) 及び TEF2 転写プロモーター （TEF2p) を含む DNA 断片を調製した。 ^ _ Cerevisiae genomic DNA as a踌型and the DNA fragment containing the TDH3 transcriptional promoter data one (TDH3 P) and TEF2 transcription promoter (TEF2p) was prepared by PCR.
PCRプライマーとしては、 配列番号 1 6に示す塩基配列を持つプライマー （Sacl- Ptdh3FW) 、 配列番号 1 7に示す塩基配列を持つプライマー （SacII- Ptdh3RV) 、 配列番号 1 8に示す塩基配列を持つ プライマー （Sacl- Ptef2FW) 、 及ぴ配列番号 1 9に示す塩基配列を持つプライマ一 （SacII- Ptef2RV) を用いた。 TDH3pの増幅 用には、 プライマーとして Sacl- Ptdh3FWと SacII - Ptdh3RV を用い、 TEF2pの増幅 用には、 プライマーとして Sacl- Ptef2FW と SacII- Ptef 2RV を用いた。 铸型とし ては cerevisiaeゲノム DNAを用いた。 As a PCR primer, a primer having the nucleotide sequence shown in SEQ ID NO: 16 (Sacl-Ptdh3FW), a primer having the nucleotide sequence shown in SEQ ID NO: 17 (SacII-Ptdh3RV), a primer having the nucleotide sequence shown in SEQ ID NO: 18 (Sacl-Ptef2FW) and a primer (SacII-Ptef2RV) having the nucleotide sequence shown in SEQ ID NO: 19. For amplification of TDH3p, Sacl-Ptdh3FW and SacII-Ptdh3RV were used as primers, and for amplification of TEF2p, Sacl-Ptef2FW and SacII-Ptef 2RV were used as primers. As type I, cerevisiae genomic DNA was used.
反応溶液として、 0. 46 ii g の^ cerevisiaeゲノム DNA、 lOOpmol DNA 、 IX ExTaq buffer (Takara) 、 20画 1 dNTP 、 0. 5U ExTaq DNA polymerase (Takara) 、 1 μ L perfect match polymerase enhancer を含む 100 ju L溶液を調製し、 次の ように PCR を行った。  The reaction solution contains 0.46 ii g of ^ cerevisiae genomic DNA, lOOpmol DNA, IX ExTaq buffer (Takara), 20 strokes 1 dNTP, 0.5 U ExTaq DNA polymerase (Takara), 1 μL perfect match polymerase enhancer 100 A ju L solution was prepared, and PCR was performed as follows.
95° C— 2分 95 ° C—2 minutes
95° C— 45秒， 60° C— 1分， 72° C— 2分； 30サイクル  95 ° C—45 seconds, 60 ° C—1 minute, 72 ° C—2 minutes; 30 cycles
72° C— 4分， 4° 。ス トック 72 ° C—4 minutes, 4 °. Stock
増幅した DNA を制限酵素 Sacl と SacII で切断し、 ァガロースゲル電気泳動 によって 680bp 、 400b の DNA 断片を精製した。 これらを TDH3p、 TEF2p と呼ぶ。  The amplified DNA was cut with restriction enzymes Sacl and SacII, and a 680 bp, 400 b DNA fragment was purified by agarose gel electrophoresis. These are called TDH3p and TEF2p.
2 u DNA複製開始領域の調製  Preparation of 2u DNA replication initiation region
YEp ベクターである pYES2を制限酵素 Sspl と Nhelで切断した後、 2 μ ϋΝΑ 複製開始点 （ 2 μ ori) を含む 1. 5kbpの断片をァガロースゲル電気泳動により精 製し、 Klenow酵素で平滑末端化した。 この DNA 断片を S ^ OriSN と呼ぶ。 After cutting the YEp vector pYES2 with the restriction enzymes Sspl and Nhel, the 1.5 kbp fragment containing the 2 μϋΝΑ replication origin (2 μ ori) was purified by agarose gel electrophoresis. And blunt-ended with Klenow enzyme. This DNA fragment is called S ^ OriSN.
YEp 型発現ベクターの作製  Construction of YEp type expression vector
pRS404Tcyc、 pRS405Tcycヽ pRS406Tcycを BAP (bacterial alkal ine phosphat ase, Takara) によって処理した Nael切断部位に 2 <u 0riSN を挿入し、 col i S URE2に導入して形質転換した後、 プラスミ ド DNA を調製した。 これらを、 制限酵 素 Drall lと EcoRI 、 Hpal、 または、 Pstlと PvuI I により切断した後、 ァガロース ゲル電気泳動し、 2 μ ο の揷入の有無とその向きとをチェックした。 作製した pRS404Tcyc 、 pRS405Tcyc、 pRS406Tcyc iこおレヽて pYES2と同じ向き ίこ 2 μ ori が挿入されたプラスミ ドをそれぞれ、 pRS434Tcyc 2 μ Ori 、 pRS435Tcyc 2 μ Ori 、 RS436Tcyc 2 μ Ori と呼び、 pRS405Tcycにおいて pYES2 と反対向きに 2 μ ori が揷入されたプラスミ ドを pRS445Tcyc 2 μ 0ΐί と呼ぶ。  pRS404Tcyc, pRS405Tcyc ヽ Insert 2 <u0riSN into the Nael cleavage site obtained by treating pRS406Tcyc with BAP (bacterial alkaline phosphatase, Takara), introduce it into coliSURE2, and transform it into plasmid DNA. . These were cut with the restriction enzymes Drall and EcoRI, Hpal, or with Pstl and PvuII, and then subjected to agarose gel electrophoresis to check for the presence of 2 μο and its orientation. PRS404Tcyc, pRS405Tcyc, pRS406Tcyc i, and plasmids with the same orientation as pYES2, pί2μ ori inserted, are called pRS434Tcyc 2μ Ori, pRS435Tcyc 2μ Ori, RS436Tcyc 2μ Ori, and pRS405Tcyc The plasmid into which 2 μ ori was inserted in the opposite direction is called pRS445Tcyc 2 μ 0.
pRS434Tcyc 2 μ Ori 、 pRS435Tcyc 2 μ Ori 、 pRS436Tcyc 2 μ Ori 、 pRS445Tcy c 2 ix Ori 、 の 4種のプラスミ ドの Sacl- SacII切断部位に、 転写プロモーターを含 む断片 TDH3p、 TEF2p を挿入して DNA をクローン化したところ、 pRS434Tcyc 2 μ 0 ri から pRS434GAP , PRS434TEF が、 RS435Tcyc 2 μ Ori から pRS435GAP が、 pRS4 36Tcyc 2 Ori 力 ら pRS436GAP 力 RS445Tcyc 2 μ Ori 力 ら pRS445GAP 力 それ ぞれ得られた。 ここで作製した各 pRSベクター由来の YEp型発現ベクターを総称 して pRS発現ベクターと呼ぶ。  Insert the fragments TDH3p and TEF2p containing the transcription promoter into the Sacl-SacII cleavage site of the four plasmids pRS434Tcyc 2μ Ori, pRS435Tcyc 2μ Ori, pRS436Tcyc 2μ Ori, and pRS445Tcyc2ix Ori. As a result of cloning, pRS434GAP and PRS434TEF were obtained from pRS434Tcyc 2 μori, pRS435GAP from RS435Tcyc 2 μ Ori, pRS436GAP power from pRS436Tcyc 2 Ori power, and RS445Tcyc 2 μ Ori power from pRS445GAP power, respectively. The YEp-type expression vectors derived from each pRS vector prepared here are collectively referred to as pRS expression vectors.
ヒ ドロキシメチルダルタリル CoA 還元酵素遺伝子 HMG1 のサブクローニング および発現ベクターの作製  Subcloning of the hydroxymethyldaltharyl CoA reductase gene HMG1 and construction of an expression vector
PCR 法で、 ^_ cerevis iaeの cDNA を鎵型にして、 ^ cerevi siae HMG1 遺 伝子約 3. 2kbp断片を増幅した。 PCRプライマーは、 配列番号 2 0に示す塩基配列 を持つプライマーと配列番号 2 1に示す塩基配列を持つプライマーである。 PCR 断片をァガロースゲル電気泳動によって精製した後、 pT7Blue- Tへ T/Aライグー シヨンによりクローユングした。 作製したプラスミ ドを PT7HMG1と呼ぶ。  The ^ _cerevisiae cDNA was transformed into type III by PCR, and an approximately 3.2 kbp fragment of ^ cerevisiae HMG1 gene was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 20 and a primer having the nucleotide sequence shown in SEQ ID NO: 21. After the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pT7Blue-T by T / A ligation. The prepared plasmid is called PT7HMG1.
PT7HMG1 を錡型にして 373A DNA sequencer (Perkin Elmer) により塩基配列 を決定したところ、 HMG1内に、 c203t， t426c, tl026c, al l67g, t l248c, gl557a, al605g, tl820c, a2451g, a2726g, t2787c, g2940aの 12ケ所の PCRエラーが検 出された。 これらの PCRエラーの表記法は、 例えば 「 c203t」 と表記した場合、 S GD (Saccharomyces Genome Database , htt ： // genome-www. Stanford. edu/Sac charomyces) に登録されている塩基配列において、 開始コ ドン atgの aを第一塩 基と したときの第 2 0 3番目の塩基 「 c」 がエラーにより塩基 「 t」 となってい ることを示す。 他の場合も同様の表記法である。 これら PCRエラ一のうち、 c203t， t l820c, a2726g については、 コードしているアミノ酸残基にそれぞれ S68F, L60 7S, H909Rの置換変異が導入されていた。 これらの置換変異の表記法は、 例えば「S 68F」 と表記した場合、 上記 SGDに登録されている塩基配列に基づくアミノ酸配列 において、 第 6 8番目のアミノ酸残基 「S」 がエラーにより 「F」 となっている ことを示す。 他の場合も同様の表記法である。 その他の PCRエラーは、 コードし ているアミノ酸残基に変化がないサイレントミユーテーションであった。 The nucleotide sequence of PT7HMG1 was determined using the 373A DNA sequencer (Perkin Elmer). Of 12 PCR errors Was issued. The notation of these PCR errors is, for example, when the notation “c203t” is used, the start of the base sequence registered in SGD (Saccharomyces Genome Database, htt: // genome-www. Stanford. Edu / Sac charomyces) This indicates that the 203rd base “c” when the a of the codon atg is the first base is changed to the base “t” due to an error. In other cases, the same notation is used. Of these PCR errors, substitution mutations of S68F, L607S, and H909R were introduced into the encoded amino acid residues of c203t, t820c, and a2726g, respectively. The notation of these substitution mutations is, for example, when “S 68F” is written, in the amino acid sequence based on the nucleotide sequence registered in the above SGD, the 68th amino acid residue “S” is replaced by “F” due to an error. ”. In other cases, the same notation is used. Other PCR errors were silent mutations in which the encoded amino acid residues were unchanged.
上記 PCRエラーによるアミノ酸置換変異を、 部位特異的変異 （site - directed mutagenesis ) により次のようにして修正した。 部位特異的変異は、 Promega 発丁の Protocol s and appl ications guide, third edit ion, 1996 Promega, IS B 1-882274- 57- 1に記載の方法で行った。 pT7HMGl を制限酵素 Smal , ApaLl , Sail で切断し、 3. 2kbpの HMG1 断片をァガロースゲル電気泳動によって調製した。 これらの断片を PALTER- 1 の Smal - Sai l切断部位に揷入し、 pALHMGl を作製した。 pALHMGl をアルカリ変性させた後、 mutagenic ol igo として HMG1 (190- 216)、 HM Gl (1807 - 1833)、 HMG1 (2713— 2739)の 3種、 Repair oligoとして Amp Repair ol ig o (Promega ) 、 Knock Out ol igo として Tet Knock Out ol igo (Promega ) を アニーリングさせ、 ^ col i ES1301 ( Promega) に導入した。 そして部位特異 的変異が導入されたプラスミ ドを保持する形質転換体を S / g/mLの ampici l in を含む培地で集積培養し、 プラスミ ド DNAを調製した。 上記 mutageni c ol igo HM Gl (190- 216)の塩基配列を配列番号 2 2に、 同 HMG1 (1807- 1833)の塩基配列を配列 番号 2 3に、 同 HMG1 (2713- 2739)の塩基配列を配列番号 2 4に、 それぞれ示す。  The amino acid substitution mutation caused by the PCR error was corrected by site-directed mutagenesis as follows. The site-specific mutation was carried out by the method described in the Protocols and applications guide, third edition, 1996 Promega, ISB 1-882274-57-1, published by Promega. pT7HMGl was digested with restriction enzymes Smal, ApaLl, and Sail, and a 3.2 kbp HMG1 fragment was prepared by agarose gel electrophoresis. These fragments were inserted into the Smal-Sail cleavage site of PALTER-1 to generate pALHMGl. After alkaline denaturation of pALHMGl, HMG1 (190-216), HM Gl (1807-1833), HMG1 (2713-2739) as mutagenic ol igo, Amp Repair ol ig o (Promega), Knock as repair oligo As Out ol igo, Tet Knock Out ol igo (Promega) was annealed and introduced into ^ col i ES1301 (Promega). Then, the transformant having the plasmid into which the site-specific mutation was introduced was integrated and cultured in a medium containing S / g / mL ampicilin to prepare plasmid DNA. The nucleotide sequence of the above mutagenicoligo HM Gl (190-216) is shown in SEQ ID NO: 22, the nucleotide sequence of HMG1 (1807-1833) is shown in SEQ ID NO: 23, and the nucleotide sequence of HMG1 (2713-2739) is shown in SEQ ID NO: 23. This is shown in SEQ ID NO: 24, respectively.
こうして得られたプラスミ ド DNAの塩基配列を 373A DNA sequencer により 決定したところ、 c203t, tl820c, a2726gの 3ケ所のエラーは SGD に登録されて いる配列に修正されていた。 この HMG1 内の配列が修正されたプラスミ ドを、 pAL HMG106 と呼ぶ。 When the nucleotide sequence of the thus obtained plasmid DNA was determined using the 373A DNA sequencer, the three errors c203t, tl820c, and a2726g were corrected to the sequences registered in SGD. The plasmid whose sequence in HMG1 has been modified is called pAL Called HMG106.
pALHMG106 を制限酵素 Smal と Sail で切断した後、 ァガロースゲル電 気泳動によって 3.2kbp の HMGl遺伝子断片を精製した。 これを pRS434GAP、 pRS434TEの Smal-Sall切断部位へ挿入した。サブクローン化したプラスミ ドは、 XhoI、 Spel、 Nael、 Sphlの各制限酵素マッピングと、 揷入された 3.2kbpの HM G1 遺伝子断片のボーダー領域の塩基配列の確認とにより、 物理地図をチェック した。 そして計画通りに作製できたプラスミ ドを選抜した。 選抜したプラスミ ド はそれぞれ、 pRS434GAP-HMGl 、 pRS434TEF-HMGlと呼ぶ。  After pALHMG106 was digested with restriction enzymes Smal and Sail, a 3.2kbp HMGl gene fragment was purified by agarose gel electrophoresis. This was inserted into the Smal-Sall cleavage site of pRS434GAP and pRS434TE. The physical map of the subcloned plasmid was checked by mapping the restriction enzymes XhoI, Spel, Nael, and Sphl, and confirming the base sequence of the border region of the inserted 3.2 kbp HMG1 gene fragment. Then, we selected the plasmids that could be produced as planned. The selected plasmids are called pRS434GAP-HMGl and pRS434TEF-HMGl, respectively.
PYHMG044 発現ベクターの構築  Construction of PYHMG044 expression vector
HMG1コード領域の一部を欠失させた遺伝子の発現ベクターを、 以下の方法 で作製した。 pT7HMGlから HMG1 を含む BamHI-Sall 断片を調製し、 pYE S2 の BamHI-XhoI 切断部位に揷入し、発現ベクター pYES-HMGlを作製した。 配列番号 2 5に示す塩基配列を持つ PCRプライマー （プライマー HMG1 (1191- 1165 ) ) と、 配列番号 2 6に示す塩基配列を持つ PCRプライマー （プライマー HMG1 (1267-1293) ) を用いて、 pYES-HMGl を鍚型にした PCR法を行った。 PCR増幅産物を Klenow酵素で平滑末端にした後、 セルフ一ライゲーションによ り再び環化し、 E. coli JM109 へ導入して形質転換した後、 プラスミ ド DNAを 調製した。 得られたプラスミ ド DNA内における HMG1 の上流、 下流のァミノ 酸の読み枠がずれていないことと、その結合部位近辺に PCRエラーによるアミノ 酸置換が起きていないことを、 373A DNA sequencerで確認した。 作製したプラ スミ ドを pYHMG044 と呼ぶ。  An expression vector for a gene in which a part of the HMG1 coding region was deleted was prepared by the following method. A BamHI-Sall fragment containing HMG1 was prepared from pT7HMGl and inserted into the BamHI-XhoI cleavage site of pYE S2 to prepare an expression vector pYES-HMGl. Using a PCR primer (primer HMG1 (1191-1165)) having the nucleotide sequence shown in SEQ ID NO: 25 and a PCR primer (primer HMG1 (1267-1293)) having the nucleotide sequence shown in SEQ ID NO: 26, pYES- A PCR method was performed using HMGl as type III. The PCR amplification product was blunt-ended with Klenow enzyme, cyclized again by self-ligation, introduced into E. coli JM109, transformed, and plasmid DNA was prepared. The 373A DNA sequencer confirmed that the reading frame of the amino acid upstream and downstream of HMG1 in the obtained plasmid DNA was not displaced, and that no amino acid substitution due to a PCR error occurred near the binding site. . The prepared plasmid is called pYHMG044.
イソペンテュル二リン酸△一イソメラーゼ遺伝子 IDI1 のクローニング 及び発現ベクターの作製  Cloning of Isopentyl Diphosphate Monoisomerase Gene IDI1 and Construction of Expression Vector
PCR法により、 S. cerevisiae cDNA ライブラリを鶬型にして、 S. cerevi siaeの IDI1 遺伝子約 0.9kbp 断片を増幅した。 PCRプライマーは、配列番号 2 7に示す塩基配列を持つプライマーと、 配列番号 2 8に示す塩基配列を持つブラ イマ一である。 PCR断片をァガロースゲル電気泳動によって精製した後、 ρΤ7Β1 ue-T へ T/A ライゲーシヨンによりクローニングした。 IDI1 は、 pT7Blue-T内 の lacZ と反対向きに揷入されていた。 クロ一ユングした断片の塩基配列決定を 行い、 SGD の配列と比較したところ、 PCRエラーは無かった。 作製したプラス ミ ド DNAを pT7IDIl と呼ぶ。 Using a PCR method, the S. cerevisiae cDNA library was converted into type III, and an approximately 0.9 kbp fragment of the IDI1 gene of S. cerevisiae was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 27 and a primer having the nucleotide sequence shown in SEQ ID NO: 28. After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into ρΤ7Β1 ue-T by T / A ligation. IDI1 is in pT7Blue-T LacZ was introduced in the opposite direction. When the base sequence of the cloned fragment was determined and compared with the SGD sequence, there was no PCR error. The prepared plasmid DNA is called pT7IDIl.
PT7IDI1 から BamHI-Sallによって切出される 0.9kbp断片を調製し、 pRS43 5GAP の BamHI-Sall 切断部位に揷入した。 IDI1 をサブクローン化したブラ スミ ドであって、 Ncol と BamHI認識部位マッピングにより計画通りに作製で きたプラスミ ドを、選抜した。 選抜したプラスミ ドを pRS435GAP-IDIl と呼ぶ。 ゲラニルゲラニルニリン酸合成酵素遺伝子 BTS1 のクローユングおよび発現 ベクターの作製  A 0.9 kbp fragment cut out from PT7IDI1 by BamHI-Sall was prepared and inserted into pRS435GAP at the BamHI-Sall cleavage site. A plasmid subcloned from IDI1, which was produced as planned by Ncol and BamHI recognition site mapping, was selected. The selected plasmid is called pRS435GAP-IDIl. Cloning of geranylgeranyl diphosphate synthase gene BTS1 and construction of expression vector
PCR法により、 ^ _ cerevisiae cDNA ライブラリを鎳型にして、 S. cerevis i の BTS1 遺伝子約 l . Okbp 断片を増幅した。 PCRプライマーは、 配列番号 2 9 に示す塩基配列を持つプライマーと、 配列番号 3 0に示す塩基配列を持つプライ マーである。 PCR断片をァガロースゲル電気泳動によって精製した後、 pT7Blue- T へ T/A ライゲーシヨンによりクローニングした。 クローユングした断片の塩基配 列決定を行い、 SGD の配列と比較したところ、 PCRエラーは無かった。 作製した プラスミ ド DNAを PT7BTS1と呼ぶ。  Using the PCR method, the ^ _cerevisiae cDNA library was converted into a type II, and an approximately l. Okbp fragment of the BTS1 gene of S. cerevisi was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 29 and a primer having the nucleotide sequence shown in SEQ ID NO: 30. After the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pT7Blue-T by T / A ligation. When the base sequence of the cleaved fragment was determined and compared with the SGD sequence, no PCR errors were found. The prepared plasmid DNA is called PT7BTS1.
PT7BTS1 から BamHI - Sai l によって切りされる 1. Okbp断片を調製し、 pYES2 の BamHI - Sai l 切断部位に挿入した。 作製したプラスミ ドを pYESGGPS と呼ぶ。  Cleavage from PT7BTS1 by BamHI-Sail 1. An Okbp fragment was prepared and inserted into pYES2 at the BamHI-Sail cleavage site. The prepared plasmid is called pYESGGPS.
次に、 pYESGGPS を制限酵素 BamHIと Mlul で切断し、 ァガロースゲル電気 泳動により 1. Okbp断片を精製した。 これを pRS435GAPと pRS445GAPの BamHI- Mlul 切断部位に揷入した。 これらのプラスミ ドを pRS435GAP- BTS1 、 PRS445GAP-BTS1 と呼ぶ。 Next, pYESGGPS was digested with restriction enzymes BamHI and Mlul, and the Okbp fragment was purified by agarose gel electrophoresis. This was inserted into the BamHI-Mlul cleavage site of pRS435GAP and pRS445GAP. These plasmids referred to as pRS43 5 GAP- BTS1, P RS445GAP- BTS1.
遺伝子 DPP1 のクローユングおよび発現ベクターの作製  Cloning of DPP1 gene and construction of expression vector
PCR法により、 ^ cerevi s iaeゲノム DNAを铸型にして、 ^ cerevis iaeの 遺伝子 DPP1 約 0. 9kbp 断片を増幅した。 PCRプライマーは、 配列番号 3 1に示 す塩基配列を持つプライマーと、 配列番号 3 2に示す塩基配列を持つプライマー である。 PCR断片をァガロースゲル電気泳動によって精製した後， pCR2. 1-T0P0 へ T/A ライゲーシヨンによりクローニングした。 クローニングした断片の塩基配 列決定を行い、 SGD の配列と比較したところ、 PCRエラーは無かった。 作成した プラスミ ド DNAを pCR-DPPl と呼ぶ。 The ^ cerevisiae genomic DNA was converted into type III by PCR, and an approximately 0.9 kbp fragment of the ^ cerevisiae gene DPP1 was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 31 and a primer having the nucleotide sequence shown in SEQ ID NO: 32. After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into pCR2.1-T0P0 by T / A ligation. When the base sequence of the cloned fragment was determined and compared with the SGD sequence, no PCR errors were found. Created The plasmid DNA is called pCR-DPPl.
pCR- DPP1から SacII- Xhol によって切出される 0. 9kbpの断片を調製し、 pRS4 34GAP, pRS436GAP の SacII- Xhol 切断部位に挿入した。 作製したプラスミ ドは p RS434GAP-DPP1 、 pRS436GAP- DPP1と呼ぶ。  A 0.9 kbp fragment cut out from pCR-DPP1 by SacII-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP and pRS436GAP. The plasmids thus prepared are called pRS434GAP-DPP1 and pRS436GAP-DPP1.
遺伝子 LPP1 のクローニングおよび pRS 発現ベクターの作製  Cloning of LPP1 gene and construction of pRS expression vector
PCR法により、 ^ _ cerevi siaeゲノム DNAを錄型にして、 S. cerevi siaeの 遺伝子 LPP1 約 0. 9kbp断片を増幅した。 PCRプライマーは、 配列番号 3 3に示す 塩基配列を持つプライマーと、 配列番号 3 4に示す塩基配列を持つプライマーで ある。 PCR断片をァガロースゲル電気泳動によって精製した後 pCR2. 1- T0P0へ T/A ライゲ一ションによりクローニングした。 クローユングした断片の塩基配列決定 を行い、 SGD の配列と比較したところ、 PCRエラ一は無かった。 作成したプラス ミ ド DNAを pCR - LPP1 と呼ぶ。  ^ _Cerevisiae genomic DNA was converted into type III by PCR, and an approximately 0.9 kbp fragment of the S. cerevisiae gene LPP1 was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 33 and a primer having the nucleotide sequence shown in SEQ ID NO: 34. The PCR fragment was purified by agarose gel electrophoresis and cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cleaved fragment was determined and compared with the SGD sequence, there were no PCR errors. The prepared plasmid DNA is called pCR-LPP1.
pCR - LPP1から SacI I- Xhol によって切出される 0. 9kbp 断片を調製し、 pRS4 34GAP、 pRS436GAPの SacII- Xhol切断部位に揷入した。 作製したプラスミ ドを pRS4 34GAP- LPP1、 pRS436GAP-LPPl と呼ぶ。  A 0.9 kbp fragment cut out from pCR-LPP1 by SacI I-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP and pRS436GAP. The plasmids thus prepared are called pRS434GAP-LPP1 and pRS436GAP-LPPl.
遺伝子 Dri42のクローニングおよび発現べクタ一の作製  Cloning of gene Dri42 and construction of expression vector
PCR法により、 ラッ ト norvegicus) の遺伝子 Dri42約 0. 9kbp 断片を 増幅した。 PCRプライマーは、 配列番号 3 5に示す塩基配列を持つプライマーと、 配列番号 3 6に示す塩基配列を持つプライマーである。 錶型 D N Aとしては、 ラ ッ ト肝臓由来の Pol yA+ RNA (CL0NTECH) から、 Marathon cDNA Ampl ication Kit (CL0NTECH) を用いてラッ ト肝臓 cDNA ライブラリを調製して用いた。 PCR 断片をァガロースゲル電気泳動によって精製した後、 pCR2. 1- T0P0へ T/Aライゲ ーションによりクローニングした。 クローニングした断片の塩基配列決定を行い、 GenBanKの配列と比較したところ、 PCRエラーは無かった。 作成したプラスミ ド DNAを pCR - Dri42と呼ぶ。 An approximately 0.9 kbp fragment of Dri42 gene of rat norvegicus was amplified by PCR. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 35 and a primer having the nucleotide sequence shown in SEQ ID NO: 36. A rat liver cDNA library was prepared from rat liver-derived PolyA + RNA (CL0NTECH) using a Marathon cDNA Amplification Kit (CL0NTECH) as type I DNA. After the PCR fragment was purified by agarose gel electrophoresis, it was cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of GenBanK, there was no PCR error. The resulting plasmid DNA is called pCR-Dri42.
CR-Dri42 から BamHI - Sail によって切出される 0. 9kbP 断片を調製し、 pR S434GAP, pRS436GAP の BaraHI- Sail切断部位に揷入した。 作製したプラスミ ドを、 pRS434GAP-Dri42 pRS436GAP-Dri42と呼ぶ。 遺伝子 PAP2 のクローニングおよび発現べクタ一の作製 A 0.9 kb P fragment cut out from CR-Dri42 by BamHI-Sail was prepared and inserted into the BaraHI-Sail cleavage site of pR S434GAP and pRS436GAP. The prepared plasmid is called pRS434GAP-Dri42 pRS436GAP-Dri42. Cloning of PAP2 gene and construction of expression vector
PCR法で、 norvegicusの遺伝子 PAP2 約 0. 9kbp断片を増幅した。 PCRプ ライマーは、 配列番号 3 7に示す塩基配列を持つプライマーと、 配列番号 3 8に 示す塩基配列を持つプライマーである。 鐯型 DNAとしては、 前記の PolyA+ RNA (CL0NTECH) から、 Marathon cDNA Ampl ication Kit (CLONTECH) を用いて、 ラ ット肝臓 cDNA ライブラリを調製して用いた。 PCR断片をァガロースゲル電気泳 動によって精製した後、 pCR2. 1- T0P0へ T/A ライゲーシヨンによりクローニング した。 クローユングした断片の塩基配列決定を行い GenBanKの配列と比較したと ころ、 PCRエラーは無かった。 作成したプラスミ ド DNAを pCR- PAP2 と呼ぶ。  By the PCR method, an approximately 0.9 kbp fragment of the norvegicus gene PAP2 was amplified. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 37 and a primer having the nucleotide sequence shown in SEQ ID NO: 38. As type I DNA, rat liver cDNA library was prepared from the above-mentioned PolyA + RNA (CL0NTECH) using Marathon cDNA Amplification Kit (CLONTECH). After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into pCR2.1-TOP0 by T / A ligation. When the nucleotide sequence of the cleaved fragment was determined and compared with the sequence of GenBanK, there was no PCR error. The prepared plasmid DNA is called pCR-PAP2.
pCR- PAP2から BamHI- EcoRIによって切出される 0. 9kbp 断片を調製し、 pRS4 34GAP, pRS436GAPの BamHI- EcoRI切断部位に挿入した。 作製したプラスミ ドを、 P S434GAP-PAP2 、 pRS436GAP- PAP2と呼ぶ。  A 0.9 kbp fragment cut out from pCR-PAP2 by BamHI-EcoRI was prepared and inserted into the BamHI-EcoRI cleavage site of pRS434GAP and pRS436GAP. The prepared plasmids are called PS434GAP-PAP2 and pRS436GAP-PAP2.
遺伝子 AtPAPl のクローユングおょぴ発現ベクターの作製  Construction of the expression vector for the gene AtPAPl
PCR法により、 thalianaの遺伝子 AtPAPl 約 0. 9kbp断片を増幅した。 PC Rプライマーは、 配列番号 3 9に示す塩基配列を持つプライマ一と、 配列番号 4 0 に示す塩基配列を持つプライマーである。 錶型 DNAとしては、 L thaliana cDNA Uni-ZAP XR Library (STRATAGENE) 40 μ L を 100° Cで 10分間加熱処理した後、 フエノール抽出、 クロ口ホルム抽出、 エタノール沈殿処理して ΙΟ μ ί の TEバッフ ァ一に溶解したものを用いた。 得られた PCR断片をァガロースゲル電気泳動によ つて精製した後、 pCR2. 1- T0P0へ T/Aライゲーシヨンによりクローニングした。 作成したプラスミ ド DNA を pCR- AtPAPl と呼ぶ。 クローユングした AtPAPl の塩基配列決定を行ったところ、 配列番号 1に示す通りであった。  An approximately 0.9 kbp fragment of the Thaliana gene AtPAPl was amplified by PCR. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 39 and a primer having the nucleotide sequence shown in SEQ ID NO: 40. As Type DNA DNA, heat-treat 40 μL of L-thaliana cDNA Uni-ZAP XR Library (STRATAGENE) at 100 ° C for 10 minutes, then extract with phenol, extract with black-mouth form, precipitate with ethanol, and perform 沈 殿 μί TE A solution dissolved in a buffer was used. The obtained PCR fragment was purified by agarose gel electrophoresis, and cloned into pCR2.1-TOP0 by T / A ligation. The generated plasmid DNA is called pCR-AtPAPl. The nucleotide sequence of the cleaved AtPAPl was determined and was as shown in SEQ ID NO: 1.
pCR- AtPAPlから BamHI - Xhol によって切出される 0. 9kbp 断片を調製し、 pR S434GAP, pRS436GAP の BamHI- Xhol 切断部位に挿入した。 作製したプラスミ ドを PRS434GAP - AtPAPl、 pRS436GAP- AtPAPl と呼ぶ。  A 0.9 kbp fragment cut out from pCR-AtPAPl by BamHI-Xhol was prepared and inserted into the BamHI-Xhol cleavage site of pRS434GAP and pRS436GAP. The prepared plasmids are called PRS434GAP-AtPAPl and pRS436GAP-AtPAPl.
ί»伝子 AtPAP2 のクローニングおよび発現ベクターの作製  ί »Cloning of AtPAP2 gene and construction of expression vector
PCR法により、 thal ianaの遺伝子 AtPAP2 約 0. 9kbp 断片を増幅した。 PCRプライマーは、 配列番号 4 1に示す塩基配列を持つプライマーと、 配列番号 4 2に示す塩基配列を持つプライマーである。 踌型 DNAとしては、 thal iana cD NA Uni-ZAP XR Library 40 μ L を 100° Cで 10分間加熱処理した後、 フエノール 抽出、 クロ口ホルム抽出、 エタノール沈殿処理して ΙΟ μ ί の TEバッファーに溶解 したものを用いた。 得られた PCR断片をァガロースゲル電気泳動によって精製し た後、 pCR2. 1-T0P0へ Τ/Α ライゲーシヨンによりクローニングした。 作成したプ ラスミ ド DNAを pCR - AtPAP2 と呼ぶ。 クロ一ニングした AtPAP2 の塩基配列決定 を行ったところ、 配列番号 2に示す通りであった。 An approximately 0.9 kbp fragment of the thaliana gene AtPAP2 was amplified by PCR. PCR primers include a primer having the nucleotide sequence shown in SEQ ID NO: 41 and a primer having SEQ ID NO: 4 This is a primer having the nucleotide sequence shown in 2. As type DNA DNA, heat-treat 40 μL of thaliana cDNA Uni-ZAP XR Library at 100 ° C for 10 minutes, then extract with phenol, extract with clonal form, precipitate with ethanol, and treat with ΙΟμί TE buffer. The dissolved one was used. After the obtained PCR fragment was purified by agarose gel electrophoresis, it was cloned into pCR2.1-T0P0 by Τ / Α ligation. The generated plasmid DNA is called pCR-AtPAP2. When the nucleotide sequence of the cloned AtPAP2 was determined, it was as shown in SEQ ID NO: 2.
PCR-AtPAP2 から BamHI- Xhol によって切出される 0. 9kbp 断片を調製し、 pRS434GAP、 pRS436GAPの BamHI-XhoI 切断部位に挿入した。 作製したプラスミ ド を、 pRS434GAP-AtPAP2 、 pRS436GAP-AtPAP2 と呼ぶ。 The 0. 9 kbp fragment cut from P CR-AtPAP2 by BamHI- Xhol was prepared and inserted pRS434GAP, the BamHI-XhoI cleavage site of PRS436GAP. The prepared plasmids are called pRS434GAP-AtPAP2 and pRS436GAP-AtPAP2.
組換え酵母の作製  Production of recombinant yeast
Frozen EZ yeast transformation kit (Zymo Research ) ¾r用レヽ S. cereviae の形質転換を行った。 形質転換には、 l w g のプラスミ ド DNA を用いた。 形質転 換を行った菌体を SD選択平板培地 〔1.7g/l Yeast Nitrogen Base without amin o acid (Difco) , 20g/l グルコース （Wako) , 0.77g/l Complete Supplement Mixture (Bio 101) 、 20g/l Agar (Wako) から選択マーカー遺伝子に対応する アミノ酸及び/又はヌクレオチドを除いたもの〕 に塗沫し、 生育したコロニーを さらに別の SD選択平板培地にス トリークすることにより組換え体を純化し、 以後 の実験に用いた。  Frozen EZ yeast transformation kit (Zymo Research) For transformation of S. cereviae. For transformation, lwg of plasmid DNA was used. The transformed cells were transferred to an SD selective plate medium (1.7 g / l Yeast Nitrogen Base without amino acid (Difco), 20 g / l glucose (Wako), 0.77 g / l Complete Supplement Mixture (Bio 101), 20 g). / l Agar (Wako) excluding amino acids and / or nucleotides corresponding to the selectable marker gene), and purify the recombinant by purifying the grown colonies on another SD selective plate medium. And used for subsequent experiments.
組換え酵母の培養  Culture of recombinant yeast
形質転換した ^ cereviaeは、 マーカー遺伝子に応じた SD選択培地 〔1.7g/l Yeast Nitrogen Base without ammo acid (Diico) , 20g/l glucose, 0.77g/l Complete Supplement Mixture (Bio 101) から選択マーカー遺伝子に対応する アミノ酸及び Z又はヌクレオチドを除いたもの、 pH 7〕 で 2日間、 30°Cで前培養 した。 次いで、 25 L の前培養液を 2. 5mL の YM培地 （0.3% Yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, pH 7 ) あるいは SG選択培地 （SD 選択培地のグルコース成分をガラク トースに置き換えたもの） に接種し、 30° C で 4日間 130r. p. m.の回転振盪培養で培養した。 ただし、 前培養液を SG選択培地 に加える前には、 菌体を生理食塩水で洗浄し、 グルコース成分の持ち込みがない ようにした。 また、 YPH499を宿主に用いたときには、 40μ g/mUこなるように aden ine hemisulfateを培地にカロ; tた。 The transformed ^ cereviae was selected from SD selection medium (1.7 g / l Yeast Nitrogen Base without ammo acid (Diico), 20 g / l glucose, 0.77 g / l Complete Supplement Mixture (Bio 101)) according to the marker gene. Pre-cultured at 30 ° C. for 2 days at pH 7, except for the amino acid and Z or nucleotide corresponding to. Then, 25 L of the preculture was added to 2.5 mL of YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, pH 7) or SG selective medium (Glucose component of SD selective medium (Replaced by toose)) and cultured at 30 ° C. for 4 days in a rotary shaking culture at 130 rpm. However, if the preculture is SG selective medium Before adding to the cells, the cells were washed with physiological saline so that glucose components were not introduced. When YPH499 was used as a host, adenine hemisulfate was added to the culture medium to a concentration of 40 μg / mU.
培養液からのプレニルアルコールの抽出  Extraction of prenyl alcohol from culture solution
上記組換え酵母の培養液から 50 Lを抜き取り、 30倍希釈液にして 0D6。。を測 定した。 残りの培養液に 2, 5mL のメタノ'ールを加えて混合した。 その後、 さらに 約 5mLのペンタンを加えてボルテックスミキサーで 1 5秒間激しく攪拌した後に 静置した。 そしてペンタン層を新しいガラス試験管にと り、 ドラフ ト中でペンタ ンを気化させて溶質成分を濃縮した後、 内部標準物質として 1.0mL/Lゥンデカノ ールを lOiiL 加え、 GC/MS 用サンプルとした。 Withdraw 50 L from the culture of the above recombinant yeast, make a 30-fold dilution, and add 0D 6 . . Was measured. 2.5 mL of methanol was added to the remaining culture and mixed. Thereafter, about 5 mL of pentane was further added, and the mixture was vigorously stirred with a vortex mixer for 15 seconds, and then allowed to stand. The pentane layer was transferred to a new glass test tube, pentane was vaporized in the draft, and the solute components were concentrated.Then, 1.0 mL / L pentadecanol was added as an internal standard substance and lOiiL was added. did.
GC/MS 解析  GC / MS analysis
ペンタン抽出画分を HP6890/5973 GC/MSシステム （Hewlett- Packard, Wilmin gton, DE ) を用いて分離、 同定、 定量した。 使用カラムは HP- 5MS (0.25mmX30 m 、 フィルム厚 0.25μηι ) であり、 分析条件は以下の通りである。  The pentane-extracted fraction was separated, identified, and quantified using an HP6890 / 5973 GC / MS system (Hewlett-Packard, Wilmington, DE). The column used was HP-5MS (0.25 mm × 30 m, film thickness 0.25 μηι), and the analysis conditions were as follows.
温度条件：注入口温度 250° C, 検出器温度 260° C Temperature conditions: inlet temperature 250 ° C, detector temperature 260 ° C
MSゾーン温度： MS Quad 150 ° C， MSソース 230 ° C， マススキャンレンジ 35 -200 。  MS zone temperature: MS Quad 150 ° C, MS source 230 ° C, mass scan range 35 -200.
注入条件： 自動注入モード， サンプル量 2ML , スプリ ッ ト比 1/20 ， キヤリ ァガス ；ヘリ ウム l.OmL/分， ソルベントディレイ 2分。 Injection conditions: automatic injection mode, sample volume 2 ML , split ratio 1/20, carrier gas; helium l.OmL / min, solvent delay 2 min.
昇温設定： 115° C 90秒一 250° Cまで昇温 （70° C/分） 一 250° C 2分一 300° Cまで昇温 （70° CZ分） 一 300° C 7 分。 ポス トタイム 0。 Heating setting: 115 ° C 90 seconds / 250 ° C (70 ° C / min) 1 250 ° C 2min-1 300 ° C (70 ° CZ) 1 300 ° C 7 min. Post time 0.
標準物質： 内部標準； 0.01 し 1一ゥンデ力ノール。 標準物質； （E)- nerolidoU Eisai) , (all-E)-farnesol (Sigma) , (all-E)-geranylgeraniol (Eisai) , squale ne (Tokyo Kasei Kogyo) 。 Standard material: internal standard; 0.01 to 1 pound of force. (E) -nerolidoU Eisai), (all-E) -farnesol (Sigma), (all-E) -geranylgeraniol (Eisai), squale ne (Tokyo Kasei Kogyo).
ホスファターゼ活性の発現  Expression of phosphatase activity
PRS436GAP-DPP1 、 RS436GAP-LPPl 、 pRS436GAP- Dri42、 pRS436GAP-PAP2 、 pRS436GAP - AtPAPl及ぴ pRS436GAP- AtPAP2 ベクターをそれぞれ ^_ cerevisiae A451 へ導入して形質転換した。 得られた組換え体を、 それぞれ 5mLの SD選択培 地で 0De。。 が約 1になるまで培養し、 遠心分離により茵体を集菌した。 菌体を生 理食塩水で 2度洗い、 300 の抽出バッファー （50mM Tris- HC1 (pH7. 5 ) 、 lOmM β -mercaptoethanol , 2ra EDTA、 Ira PMSF、 10 μ g/L aprotinin 、 10 μ g/L leupeptin 、 1 μ g/L pepstatin ) に懸濁した。 そして、 等量のガラスビーズ ( φ = 0. 45- 0. 5鹏 ) を加え、 ボルテックスミキサーにて 15 分間強く撹袢 （4 ° C ) して菌体を破砕した。 更に 300 z L の抽出バッファ一を加え、 遠心分離 （l, 000g、 5分） により未破砕の菌体を除去した。 その上清を粗酵素画分として、 酵素活性 測定に供した。 The PRS436GAP-DPP1, RS436GAP-LPPl, pRS436GAP-Dri42, pRS436GAP-PAP2, pRS436GAP-AtPAPl and pRS436GAP-AtPAP2 vectors were each introduced into ^ _cerevisiae A451 for transformation. Each of the obtained recombinants was subjected to 5 mL of SD selective culture. 0D e in the ground. . Was collected to about 1 and the cells were collected by centrifugation. The cells were washed twice with saline, and 300 extraction buffers (50 mM Tris-HC1 (pH 7.5), lOmM β-mercaptoethanol, 2ra EDTA, Ira PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin, 1 μg / L pepstatin). Then, an equal amount of glass beads (φ = 0.45-0.5 鹏) was added, and the cells were disrupted by vigorous stirring (4 ° C.) for 15 minutes using a vortex mixer. Further, 300 zL of extraction buffer was added, and the unbroken cells were removed by centrifugation (1,000 g, 5 minutes). The supernatant was used as a crude enzyme fraction for enzyme activity measurement.
GGPPホスファターゼ活性 （GGPPase 活性） の測定は、 5 <u M の GGPP (2. lkB qの [3H] GGPP を含む）を含む反応パッファ一（最終濃度 0. 1M citrate ( pH6. 0)、 5mM EDTA) に適当量の粗酵素画分を添加し、 30分間 37° Cで反応を行なった （合 計 100 μし ） 。 反応後、 アルコール K0H液 （90%エタノール'： 40%Κ0Η ( 1 : 1 ) )Measurements of GGPP phosphatase activity (GGPPase activity), 5 <u M of GGPP reaction puffer one containing (2. LKB containing [3 H] GGPP of q) (final concentration 0. 1M citrate (pH6. 0) , 5mM An appropriate amount of the crude enzyme fraction was added to EDTA), and the reaction was carried out at 37 ° C. for 30 minutes (total of 100 μl). After the reaction, alcohol K0H solution (90% ethanol ': 40% {0} (1: 1))
100 μ ί を添加して反応を停止した。 そして、 n-へキサン 500 し を加えた後、 1分間ボルテックスミキサーにて激しく撹拌して、 生成した [3H] GG0H をへキサン 層に抽出した。 このへキサン層 300 /i L を分取して、 液体シンチレ一ションカゥ ンタ一にて放射活性を測定した。 0〜500mU/mLのアルカリホスファターゼ （calf intestine, ベ一リンガー） を用いて検量線を作成し、 前記で求めた放射活性を酵 素活性値 （Unit) に換算した。 粗酵素画分中のタンパク質濃度は、 Bio- Rad Prot ein Assay (Bio-Rad) で BSA を標準タンパク質として測定し、 粗酵素液中の GGPPa se 比活性 （U/mg protein) を求めた。 野生株の GGPPase 比活性を 1とした場合の 相対活性を表 1に示す。 ホスファターゼ遺伝子導入株は野生株に比べて 2. 4 〜34. 8倍 GGPPase 比活性が増大したことから、 ホスファタ一ゼ遺伝子を導入することに より GGPPase 活性が発現することが確かめられた。 The reaction was stopped by adding 100 μ 100. After adding 500 ml of n-hexane, the mixture was vigorously stirred with a vortex mixer for 1 minute to extract the generated [ 3 H] GG0H into the hexane layer. 300 / i L of the hexane layer was fractionated, and the radioactivity was measured with a liquid scintillation counter. A calibration curve was prepared using 0 to 500 mU / mL alkaline phosphatase (calf intestine, Boehringer), and the radioactivity determined above was converted to an enzyme activity value (Unit). The protein concentration in the crude enzyme fraction was measured by using BSA as a standard protein by Bio-Rad Protein Assay (Bio-Rad), and the specific activity of GGPPase (U / mg protein) in the crude enzyme solution was determined. Table 1 shows the relative activity when the GGPPase specific activity of the wild strain is set to 1. The phosphatase gene-introduced strain had a 2.4 to 34.8-fold increase in GGPPase specific activity compared to the wild-type strain, confirming that the phosphatase gene was introduced to express GGPPase activity.
【表 1】  【table 1】
Figure imgf000037_0001
ホスファタ一ゼの基質特異性
Figure imgf000037_0001
Substrate specificity of phosphatases
「ホスファターゼ活性の発現」 の項で得られた形質転換体を 500mLの SD選択 培地で 0D6。。 が約 1になるまで培養し、 遠心分離により菌体を集菌した。 得られ た菌体ペレツ トカ ら、 Wu W. I. らの方法 （ （1998) J. Biol. Chem. 271 (4) , 1868-1876 ) を参考にして、 発現したホスファターゼの部分精製を行った。 部分 精製酵素を用いて、 ホスファターゼの基質特異性を以下の方法で調べた。 0D 6 transformants obtained in the section "Expression of phosphatase activity" in SD selection medium 500 mL. . Was cultured to about 1, and the cells were collected by centrifugation. The expressed phosphatase was partially purified with reference to the obtained bacterial pellets and Wu WI et al. ((1998) J. Biol. Chem. 271 (4), 1868-1876). Using the partially purified enzyme, the substrate specificity of phosphatase was examined by the following method.
5 μ GGPP (2. lkBqの [3H] GGPPを含む） および部分精製酵素を含む反応バッ ファー 〔最終濃度 0.1M citrate (pH6.0) 、 5mM EDTA 〕 に対して、 GGPP以外の追 加基質として ΙΟΟμΜ ATP、 100 グルコース 6 リン酸 （G6P) 、 100 μ Μ ピロリ ン酸ナトリ ウム （NaPP) 、 100 μΜ β - グリセ口リン酸 ( β一 GP) 、 100 μΜ ρ- ニトロフエ二ルリン酸 （ρΝΡΡ) 、 ΙΟΟμΜ Reaction buffer containing 5 μ GGPP (including 2.lkBq [ 3 H] GGPP) and partially purified enzyme [final concentration 0.1 M citrate (pH 6.0), 5 mM EDTA], additional substrate other than GGPP As ΙΟΟμΜ ATP, 100 glucose 6-phosphate (G6P), 100 μΜ sodium pyrophosphate (NaPP), 100 μΜ β-glycerol phosphate (β-GP), 100 μΜ ρ-nitrophenyl phosphoric acid (ρΝΡΡ) , ΙΟΟμΜ
MVP、 100 μ M MVPP、 100 μΜ IPP、 100 μ M DMAPP 、 100 M GPP 、 5μΜ FPP 又は 5μΜ GGPPのいずれかを添加し、 30分間 37° Cで反応を行なった。 そして 1 00 β ΐ アルコール K0H液を添加して反応を停止し、 へキサン 500<uL を加えた後、 1分間ボルテックスミキサーにて激しく撹拌し、 生成した [3H] GG0Hを抽出した。 このへキサン層 300/iL を分取して液体シンチレ一シヨンカウンタ一で放射活性 を測定することにより、 GGPPase 比活性を求めた。 [3H] GGPP以外の追加基質を 加えなかったとき （+none) の GGPPase比活性を 100%とした場合の相対活性を表 2にまとめた。 MVP, 100 μM MVPP, 100 μM IPP, 100 μM DMAPP, 100 M GPP, 5 μM FPP or 5 μM GGPP were added, and the reaction was carried out at 37 ° C. for 30 minutes. Then, 100 βΐ alcohol K0H solution was added to stop the reaction, 500 <uL of hexane was added, and the mixture was vigorously stirred for 1 minute with a vortex mixer to extract the generated [ 3 H] GG0H. The specific activity of GGPPase was determined by collecting 300 / iL of the hexane layer and measuring the radioactivity with a liquid scintillation counter. Table 2 summarizes the relative activities when the specific activity of GGPPase when no additional substrate other than [ 3 H] GGPP was added (+ none) was 100%.
【表 2】 GGPPase相対活性（%)  [Table 2] GGPPase relative activity (%)
追加基質 (¾度） DPP1 LPP1 PAP2 Dri42 AtPAPI AtPAP2 Additional substrate (¾ degree) DPP1 LPP1 PAP2 Dri42 AtPAPI AtPAP2
+ΠΟΠΘ 100 100 100 100 100 100+ ΠΟΠΘ 100 100 100 100 100 100
+ATP(100jUM) 100 100 100 100 100 100+ ATP (100jUM) 100 100 100 100 100 100
+G6P(100^M) 100 100 100 100 100 100+ G6P (100 ^ M) 100 100 100 100 100 100
+NaPP(100/ M) 100 100 100 100 100 100+ NaPP (100 / M) 100 100 100 100 100 100
+ /3-GP(100 iM) 100 100 100 100 100 100+ / 3-GP (100 iM) 100 100 100 100 100 100
■tpNPP(100// ) 100 100 100 100 , 100 100TpNPP (100 //) 100 100 100 100, 100 100
+MVP (卿 M) 97 99 96 . 98 99 97+ MVP (Sir M) 97 99 96. 98 99 97
+MVPP(100 iM) 96 98 96 97 95 97+ MVPP (100 iM) 96 98 96 97 95 97
+IPP(100jU ) 95 99 94 92 95 94+ IPP (100jU) 95 99 94 92 95 94
+DMAPP(100jUM) 96 92 96 91 90 92+ DMAPP (100jUM) 96 92 96 91 90 92
„+GPP(100 /M) 91 91 ,,92 ； 95 93 90„+ GPP (100 / M) 91 91 ,, 92; 95 93 90
+FPP(5)UM) 64 57 63 58 66 61+ FPP (5) UM) 64 57 63 58 66 61
+GGPP(5/iM) 52' 44 59 47 42 53 表 2の結果から、 プレニルニリン酸生合成経路に関係しないリン酸化合物 （A TP、 G6P、 NaPP、 — GP又は pNPP) を、 追加基質として GGPPの 20倍量加えて も、追加基質を加えなかつた場合と比較して GGPPに対するホスファターゼ活性は 90%以上保持していた。 即ち、 実験に供したホスファタ一ゼのプレニルニリン酸 生合成経路に関係しないリン酸化合物に対するホスファタ一ゼ活性は、 GGPPに対 する酵素活性に比べて低いことが確かめられた。 次に、 プレニルリン酸前駆体 （M VP , MVPP) 、 炭素数 5の IPP 又は DMAPP 、 あるいは炭素数 10の GPP を、 追加基質 として、 GGPPの 20倍量加えても追加基質を加えなかった場合と比較して GGPPに対 するホスファターゼ活性は 90%以上保持していた。 即ち、 実験に供したホスファ ターゼのプレニルリン酸前駆体及び炭素数 10以下のプレニルリン酸化合物に対す るホスファターゼ活性は、 GGPPに対するホスファターゼ活性に比べて低いことが 確かめられた。 一方、 炭素数 15 の cold FPPを、 追加基質として、 GGPPと等モル ( 5 μ Μ ) 加えた場合には、 基質競合により [ 3H] GGPP に対する見かけ上のホス ファターゼ活性が約 40〜60%減少した。 これは cold GGPP を、 追加基質として、 G GPPと等モル （ 5 μ Μ ) 加えた場合の見かけ上のホスファタ一ゼ活性の減少度合い と同程度であった。 即ち、 実験に供したホスファターゼの炭素数 15 の FPPに対す るホスファターゼ活性と炭素数 20 の GGPPに対する酵素活性は同等であること が確かめられた。 以上のことから、 実験に供したホスファターゼは次のような活 性を示すことが明らかとなった。 即ち、 炭素数 15以上のプレニルリン酸 （A ) を 基質とした酵素活性測定系において、 過剰量 （モル比で 20倍） のプレニルニリン 酸生合成経路に関係しないリン酸化合物又は炭素数 10以下のプレニルリン酸又は プレニルリン酸前駆体 （B ) を共存させても、 （A ) に対する酵素活性が （B ) を共存させなかった場合と比べて 90%以上の活性を示す。 + GGPP (5 / iM) 52 '44 59 47 42 53 From the results in Table 2, it was found that a phosphate compound (ATP, G6P, NaPP, —GP or pNPP) not involved in the prenyl diphosphate biosynthesis pathway was added as an additional substrate in an amount 20 times that of GGPP, but no additional substrate was added. Compared to the case, the phosphatase activity for GGPP was maintained at 90% or more. That is, it was confirmed that the phosphatase activity of the phosphatases used in the experiment for phosphate compounds not involved in the prenyl diphosphate biosynthesis pathway was lower than the enzyme activity for GGPP. Next, prenyl phosphate precursors (MVP, MVPP), IPP or DMAPP with 5 carbon atoms, or GPP with 10 carbon atoms were added as additional substrates, even when 20 times the amount of GGPP was added but no additional substrate was added. In comparison, phosphatase activity for GGPP was maintained at 90% or more. That is, it was confirmed that the phosphatase activity of the phosphatase used for the prenyl phosphate precursor and the prenyl phosphate compound having 10 or less carbon atoms was lower than the phosphatase activity for GGPP. On the other hand, when cold FPP with 15 carbon atoms was added as an additional substrate in an equimolar amount (5 μΜ) with GGPP, the apparent phosphatase activity on [ 3 H] GGPP was about 40-60% due to substrate competition. Diminished. This was comparable to the apparent decrease in phosphatase activity when cold GGPP was added as an additional substrate in an equimolar amount (5 μM) to GGPP. In other words, it was confirmed that the phosphatase used in the experiment had the same phosphatase activity with respect to FPP having 15 carbon atoms and the enzyme activity with respect to GGPP having 20 carbon atoms. From the above, it was clarified that the phosphatases used in the experiments exhibited the following activities. That is, in an enzyme activity measurement system using prenylphosphoric acid (A) having 15 or more carbon atoms as a substrate, an excess amount (20-fold by mole) of a phosphate compound unrelated to the prenylnylinic acid biosynthesis pathway or a prenylphosphine having 10 or less carbon atoms. Even when the acid or prenyl phosphate precursor (B) is present, the enzyme activity against (A) is 90% or more as compared with the case where (B) is not present.
酵母によるプレニルアルコールの生産  Production of prenyl alcohol by yeast
本発明に係るホスファターゼ遗伝子を用いて生体内でプレニルアルコールを 大量生産させるためには、 この遺伝子を適当なベクターあるいは D N A断片に組 んで宿主に導入し、 生体内で発現させればよい。  In order to mass-produce prenyl alcohol in vivo using the phosphatase gene of the present invention, the gene may be assembled into an appropriate vector or DNA fragment, introduced into a host, and expressed in vivo.
更に、 プレニルニリン酸生合成経路 （メバロン酸経路、 非メバロン酸経路を 含む） に関わる幾つかの酵素の遺伝子を用いてプレニルリン酸の供給能力を向上 む） に関わる幾つかの酵素の遺伝子を用いてプレニルリ ン酸の供給能力を向上さ せたもとで上記ホスファターゼ遺伝子を導入すれば、 より高いプレニルアルコ一 ル生産性を期待できる。 例えば、 ヒ ドロキシメチルダルタリノレ CoA (HMG-CoA ) 還元酵素は、 メバロン酸経路の中で律速酵素であることが知られている （Dimster — Denk, D. et al (1994) Mol. Biol . Cel l. 5, 655—665. ) 。 又、 イ ソペンテュル 二リン酸イソメラーゼ （IPP イソメラ一ゼ） 遺伝子の発現を増強した！^ co l iで カロテノィ ドの生産量が増加することが報告されている （三沢ら、 特開平 8-24286 1) 。 GGPP合成酵素遺伝子 （例えば、 ^_ cerevi ae BTS1 ) は、 GG0Hの前駆体であ る GGPPを合成する酵素である。 かかる GGPP合成酵素遺伝子の発現の増強により、 G GPP生産能力の強化を期待できる。 よって、 これらの遺伝子の導入又は発現強化と、 本発明に係るホスファターゼ遺伝子の導入又は発現強化とを組合わせることによ り、 より高いプレニルアルコール生産性の実現を期待できる。 Furthermore, the prenyl diphosphate biosynthesis pathway (mevalonate pathway, non-mevalonate pathway The above-mentioned phosphatase gene is introduced under the condition that the supply capacity of prenyl phosphoric acid is improved by using the genes of several enzymes related to If so, higher prenyl alcohol productivity can be expected. For example, hydroxymethyldaltarinole CoA (HMG-CoA) reductase is known to be the rate-limiting enzyme in the mevalonate pathway (Dimster — Denk, D. et al (1994) Mol. Biol Cel l. 5, 655-665.). Also, the expression of the isopenthl diphosphate isomerase (IPP isomerase) gene was enhanced! It has been reported that carotenoid production increases in ^ coli (Misawa et al., JP-A-8-24286-1). The GGPP synthase gene (eg, ^ _cereviae BTS1) is an enzyme that synthesizes GGPP, a precursor of GG0H. By enhancing the expression of the GGPP synthase gene, enhancement of GGPP production capacity can be expected. Therefore, by combining the introduction or enhancement of these genes and the introduction or enhancement of the phosphatase gene according to the present invention, higher prenyl alcohol productivity can be expected.
ホスファタ一ゼ遺伝子を導入した W303 - 1B によるプレニルアルコールの生産 PRS435GAP-BTS1 を^ _ cerevi siae W303-1Bに導入し、 得られた組換え体に 対して更に、 本発明に係るホスファタ一ゼ遺伝子を組込んだ発現べクタ一 pRS436G AP-DPP1 , pRS436GAP-LPPl 、 pRS436GAP- Dri42、 pRS436GAP-PAP2、 pRS436GAP- AtPAPl又は pRS436GAP- AtPAP2 を導入した。 各組換え体及び宿主の GGOH生産性を 測定した結果を表 3に示す。  Production of prenyl alcohol by W303-1B into which the phosphatase gene has been introduced PRS435GAP-BTS1 was introduced into ^ _cerevisiae W303-1B, and the phosphatase gene according to the present invention was further transformed with respect to the obtained recombinant. The incorporated expression vectors pRS436GAP-DPP1, pRS436GAP-LPP1, pRS436GAP-Dri42, pRS436GAP-PAP2, pRS436GAP-AtPAPl or pRS436GAP-AtPAP2 were introduced. Table 3 shows the results of measuring the GGOH productivity of each recombinant and host.
【表 3】  [Table 3]
GG0H生産  GG0H production
導入した発現べクタ- S  Introduced expression vector-S
N.D.  N.D.
PRS435GAP-BTS1 0.2  PRS435GAP-BTS1 0.2
pRS435GAP-BTS1, pRS436GAP-DPP1 1.7  pRS435GAP-BTS1, pRS436GAP-DPP1 1.7
p S435GAP-BTS1 , P S436GAP-LPP1 3.0 p S435GAP-BTS1, P S436GAP-LPP1 3.0
p S435GAP-BTS1 , p S436GAP-Dri42 2.1  p S435GAP-BTS1, p S436GAP-Dri42 2.1
PRS435GAP-BTS1 , pRS436GAP-PAP2 1.0  PRS435GAP-BTS1, pRS436GAP-PAP2 1.0
PRS435GAP-BTS1 , pRS436GAP-AtPAP1 1.2  PRS435GAP-BTS1, pRS436GAP-AtPAP1 1.2
P S435GAP-BTS1 , pRS436GAP-AtPAP2 2.8  P S435GAP-BTS1, pRS436GAP-AtPAP2 2.8
W303-1B では GGOHの生産は検出されなかったが、 GGPP合成酵素遺伝子を導入 することにより GGOH 力'； 0. 2mg/L生産されるようになった。 これは、 GGPP合成酵 活性上昇により GGPP が生産され、 この GGPP が酵母菌体内在性の脱リン酸化活 性により GG0H に変換されたことを示唆している。 更に、 この株に本発明に係る ホスファタ一ゼ遗伝子を導入することにより、 GGPP合成酵素遺伝子のみを導入し た場合に比べて、 5. 7〜17倍に GG0H の生産性が向上した。 即ち、 GGPP合成酵素 遺伝子の導入によりある程度の GG0H 生産性向上が認められるが、 GG0Hを更に効 率的に大量生産させるためには、 本発明に係るホスファターゼ遺伝子の導入が重 要であることが判った。 GGOH production was not detected in W303-1B, but GGPP synthase gene was introduced. As a result, GGOH power '; 0.2 mg / L was produced. This suggests that GGPP was produced by increasing the activity of the GGPP synthase and this GGPP was converted to GG0H by the intrinsic dephosphorylation activity of the yeast. Furthermore, by introducing the phosphatase gene of the present invention into this strain, the productivity of GG0H was improved 5.7 to 17 times as compared with the case where only the GGPP synthase gene was introduced. That is, although the introduction of the GGPP synthase gene improves the GG0H productivity to some extent, it has been found that the introduction of the phosphatase gene according to the present invention is important in order to mass-produce GG0H more efficiently. Was.
ホスファタ一ゼ遺伝子を導入した YPH499 によるプレニルアルコールの生産 PRS434TEF - HMG1、 pRS445GAP - BTS1又は RS435GAP-IDI l を、 cerevis iae YPH499 に導入した。 又、 別途に、 pRS434TEF-HMGl 及び pRS445GAP- BTS1 の 2種 のプラスミ ドを YPH499 に導入した。 上記 2種のプラスミ ドを導入した組換え体 に対して、 更に本発明のホスファタ一ゼ遺伝子を組込んだ発現ベクター pRS436GA P-DPP1 、 p S436GAP-LPPl 、 S436GAP-Dri42 、 pRS436GAP-PAP2 、 PRS436GAP -AtPAPl 又は pRS436GAP- AtPAP2 の各プラスミ ドを導入した。 各組換え体及び宿 主の F0H 、 GG0H生産性を測定した結果を表 4に示す。 Production of prenyl alcohol by YPH499 into which phosphatase gene was introduced. PRS434TEF-HMG1, pRS445GAP-BTS1 or RS435GAP-IDIl was introduced into cerevisiae YPH499. Separately, two plasmids, pRS434TEF-HMGl and pRS445GAP-BTS1, were introduced into YPH499. Against recombinant was introduced above two plasmids, further expression vector pRS436GA P-DPP1 incorporating phosphatase Ichize gene of the present invention, p S436GAP-LPPl, S436GAP- Dri42, pRS436GAP-PAP2, P RS436GAP Each plasmid of -AtPAPl or pRS436GAP-AtPAP2 was introduced. Table 4 shows the results of measuring the F0H and GG0H productivity of each recombinant and host.
【表 4】  [Table 4]
GGOH生産量 導入した発現ベクター  GGOH production amount Expression vector introduced
(m /ϋ  (m / ϋ
- N.D.  -N.D.
pRS435GAP-IDI1 N.D.  pRS435GAP-IDI1 N.D.
PRS434TEF-HMG1 0.05 PRS434TEF-HMG1 0.05
P S445GAP-BTS1 0.20P S445GAP-BTS1 0.20
PRS434TEF-HMG1. p S445GAP-BTS1 0.40 PRS434TEF-HMG1.p S445GAP-BTS1 0.40
PRS434TEF-H G1 , pRS445GAP-BTS1 , pRS436GAP-DPP1 1.41  PRS434TEF-H G1, pRS445GAP-BTS1, pRS436GAP-DPP1 1.41
PRS434TEF-HMG1 , pRS445GAP- BTS1, pRS436GAP-LPP1 2.37  PRS434TEF-HMG1, pRS445GAP-BTS1, pRS436GAP-LPP1 2.37
P S434TEF-H G1 , p S445GAP-BTS1, p S436GAP-Dri42 0.73 pRS434TEF-HMG1 , pRS445GAP-BTS1 , pRS436GAP-PAP2 0.79  P S434TEF-H G1, p S445GAP-BTS1, p S436GAP-Dri42 0.73 pRS434TEF-HMG1, pRS445GAP-BTS1, pRS436GAP-PAP2 0.79
PRS434TEF-H G1 , pRS445GAP-BTS1 , PRS436GAP-AtPAP1 2.26 PRS434TEF-H G1, pRS445GAP-BTS1 , P RS436GAP-AtPAP1 2.26
DRS434TEF-HMG1 , pRS445GAP-BTS1, pRS436GAP-AtPAP2 0.51  DRS434TEF-HMG1, pRS445GAP-BTS1, pRS436GAP-AtPAP2 0.51
YPH499 では GGOH は検出されなかった。 IPP -△イソメラーゼ遺伝子のみの 導入では GG0Hは検出されなかったが、 HMG- CoA 還元酵素遺伝子のみの導入によつ て GG0Hの生産量がある程度増大し （ GQ0H 0. 05mg/L) 、 GGPP合成酵素遺伝子の導 入によっても GGOH の生産量がある程度増大した （GG0H 0, 2mg/L) 。 GGOH was not detected in YPH499. GG0H was not detected when only the IPP- △ isomerase gene was introduced, but GG0H production was increased to some extent (GQ0H 0.05 mg / L) by introducing only the HMG-CoA reductase gene. Gene transfer GGOH production also increased to some extent (GG0H 0, 2 mg / L).
HMG-CoA 還元酵素遺伝子と GGPP 合成酵素遺伝子の 2種の遣伝子を導入する ことによりプレニルアルコール生産性向上の相乗効果が認められた。 但しその場 合に比較して、 上記の 2種の遺伝子に加えて更に本発明のホスファターゼ遺伝子 を導入することにより、 1. 3倍〜 5. 9倍に GGOH 生産性が向上した。  The synergistic effect of improving prenyl alcohol productivity was confirmed by introducing two genes, the HMG-CoA reductase gene and the GGPP synthase gene. However, in comparison with that case, by further introducing the phosphatase gene of the present invention in addition to the above two genes, the GGOH productivity was improved 1.3 to 5.9 times.
ホスファタ一ゼ遺伝子を導入した A451によるプレニルアルコールの生産 A451 の AUR1 遺伝子が AUR1- C 遺伝子に置換 ¾異した株を AURGG101 (AUR 1 : : AUR1- C) と呼ぶ。 AURGG101 に pYHMG044 を導入し （ 15-2 と呼ぶ） 、 15 - 2に 更に pRS435GAP- BTS 1 を導入し、 pRS435GAP- BTS 1/15- 2を得た。 pRS435GAP- BTS 1 /15— 2に pRS434GAP - DPP l , pRS434GAP-LPPl , pRS434GAP-Dri42 , PRS434GAP-PAP2, PRS436GAP-AtPAP l 又は pRS434GAP- AtPAP2 を導入した。 各組換え体及び宿主の プレニルアルコール生産性を測定した結果を表 5に示す。 Production of prenyl alcohol by A451 into which phosphatase gene has been introduced The AUR1 gene of A451 has been replaced with AUR1-C gene. The different strain is called AURGG101 (AUR1 :: AUR1-C). PYHMG044 was introduced into AURGG101 (referred to as 15-2), and pRS435GAP-BTS1 was further introduced into 15-2 to obtain pRS435GAP-BTS 1 / 15-2. pRS435GAP- BTS 1 / 15- 2 to pRS434GAP - DPP l, pRS434GAP-LPPl , was introduced pRS434GAP-Dri42, P RS434GAP-PAP2 , PRS436GAP-AtPAP l or pRS434GAP- AtPAP2. Table 5 shows the results of measuring the prenyl alcohol productivity of each recombinant and host.
【表 5】  [Table 5]
FOH生産： £ GGOH生逢量 宿 導入した発現べクタ- a w 42· 74 FOH production: £ GGOH production amount Introduced expression vector-a w 42 · 74
A451 N.D. A451 N.D.
15-2* pRS435GAP-BTS1 0.415-2 * pRS435GAP-BTS1 0.4
15-2 pRS435GAP-BTS1 , PRS434GAP-DPP1 10.915-2 pRS435GAP-BTS1, PRS434GAP-DPP1 10.9
15-2 pRS435GAP-BTS1. PRS434GAP-LPP1 4.815-2 pRS435GAP-BTS1. PRS434GAP-LPP1 4.8
15-2 pRS435GAP-BTS1 , P S434GAP-DH42 4.315-2 pRS435GAP-BTS1, P S434GAP-DH42 4.3
15-2 pRS435GAP-BTS1 , PRS434GAP-PAP2 1 1.615-2 pRS435GAP-BTS1, PRS434GAP-PAP2 1 1.6
15-2 pRS435GAP-BTS1 , p S434GAP-AtPAP1 3.315-2 pRS435GAP-BTS1, p S434GAP-AtPAP1 3.3
15-2 j)RS435GAP-BTS1 , pRS434GAP-AtPAP2 7.0 15-2 j) RS435GAP-BTS1, pRS434GAP-AtPAP2 7.0
A451 ではプレニルアルコールの生産は検出されなかった。 HMG- CoA 還元酵 素遺伝子と GGPP合成酵素遺伝子の 2種の遺伝子を導入した場合、 FOH 、 GG0Hがあ る程度生産されるようになった （FOH 1. 4mg/L ， GG0H 0. 4mg/L) 。 更にこの株に 本発明に係るホスファタ—ゼ遺伝子を導入すると、 上記 2種の遺伝子を導入した 場合に比較して F0H で 2. 6〜20倍、 GG0Hで 8. 8倍〜 31倍に生産性が向上した。 No prenyl alcohol production was detected in A451. When two types of genes, the HMG-CoA reductase gene and the GGPP synthase gene, were introduced, FOH and GG0H were produced to some extent (FOH 1.4 mg / L, GG0H 0.4 mg / L ). Furthermore, when the phosphatase gene according to the present invention was introduced into this strain, the productivity was increased by 2.6 to 20 times with F0H and 8.8 to 31 times with GG0H as compared with the case where the above two genes were introduced. Improved.
この結果は、 HMG- CoA 還元酵素遺伝子や GGPP 合成酵素遺伝子が導入されて いる場合、 F0Hや GG0Hの生産の増量に対しては、 ホスファターゼ遺伝子の導入が 決定的であることを示している。  This result indicates that when the HMG-CoA reductase gene or GGPP synthase gene has been introduced, the introduction of the phosphatase gene is crucial for increasing the production of F0H and GG0H.
GGPP合成酵素遺伝子 BTS1とプレニルリン酸特異的ホスファターゼ遺伝子 DPP1 の連結 GGPP synthase gene BTS1 and prenyl phosphate specific phosphatase gene DPP1 Concatenation of
Nikawaらの方法（Nikawa, J. and Ka abata, M. (1998) Nucleic Acids Res. 26. 860- 861. )を参考にして、 PCR法により、 プレニルリン酸特異的ホスファタ ゼ遺伝子 DPP1と GGPP合成酵素遺伝子 BTS1の連結遺伝子 （以後、 「DPGGJ と呼ぶ） を作製した。  Based on the method of Nikawa et al. (Nikawa, J. and Ka abata, M. (1998) Nucleic Acids Res. 26. 860-861.), The prenyl phosphate-specific phosphatase genes DPP1 and GGPP synthase were determined by PCR. A gene linked to the gene BTS1 (hereinafter referred to as "DPGGJ") was prepared.
1 st PCRの条件を以下に示す。 1XK0D buffer (T0Y0B0) ， 0.2mM dNTPs , ImM MgCl2 ， 2U KOD DNA polymerase (T0Y0B0) ， 20pmolプライマー 1 , 20pmolプィ マー 2 , 60ng YPH499 ゲノム DNA を含む 40μ ί の反応液を調製し、 次のように I s t PCRを行った。 The conditions for 1st PCR are shown below. Prepare a 40μί reaction solution containing 1XK0D buffer (T0Y0B0), 0.2mM dNTPs, ImM MgCl 2 , 2U KOD DNA polymerase (T0Y0B0), 20pmol primer 1, 20pmol primer 2, 60ng YPH499 genomic DNA as follows. Ist PCR was performed.
98° C— 15秒， 44° C- 2秒， 74° C— 30秒； 25サイクル  98 ° C—15 seconds, 44 ° C-2 seconds, 74 ° C—30 seconds; 25 cycles
プライマ一 1 として配列番号 4 3に示す塩基配列を持つ S - DPP1-1、 プライマ 一 2として配列番号 4 4に示す塩基配列を持つ DPP- 2 を用いたときの PCR産物を #1とした。 また、 プライマー 1として配列番号 4 5に示す塩基配列を持つ BTS- 3 、 プライマー 2として配列番号 4 6に示す塩基配列を持つ X - BTS - 4 を用いたときの PCR産物を #2とした。 得られた PCR産物をァガロースゲル電気泳動により精製し た。 次に、 Annealing and extension 反応を以下の条件で行った。 1XK0D buffer, 0.2mM dNTPs , I mM MgCl2, 0.25U KOD DNA polymerase, 0.5μ L PCR 産物 #1， 0.5 μ ΐ PCR 産物 #2を含む 10/iL の反応液を調製し、 次のように行った。 The PCR product when S-DPP1-1 having the nucleotide sequence shown in SEQ ID NO: 43 as the primer 1 and DPP-2 having the nucleotide sequence shown in SEQ ID NO: 44 as the primer 1 was used as # 1. The PCR product obtained when BTS-3 having the nucleotide sequence shown in SEQ ID NO: 45 as primer 1 and X-BTS-4 having the nucleotide sequence shown in SEQ ID NO: 46 as primer 2 was used was # 2. The obtained PCR product was purified by agarose gel electrophoresis. Next, Annealing and extension reaction was performed under the following conditions. 1XK0D buffer, 0.2mM dNTPs, I mM MgCl 2, prepared 0.25U KOD DNA polymerase, 0.5μ L PCR product # 1, a reaction solution of 10 / iL containing 0.5 μ ΐ PCR product # 2, carried out as follows Was.
98° C— 15秒， 54° C— 2 秒， 74° C— 30秒； 5サイクル 98 ° C—15 seconds, 54 ° C—2 seconds, 74 ° C—30 seconds; 5 cycles
得られた PCR産物の一部についてァガロースゲル電気泳動を行い、 PCR産物 # 1および #2の連結遺伝子 （約 1.9kb ) が合成されていることを確認した。  A part of the obtained PCR product was subjected to agarose gel electrophoresis, and it was confirmed that the ligation gene (about 1.9 kb) of PCR products # 1 and # 2 was synthesized.
続いて 2nd PCRを以下の条件で行った。 1XK0D buffer (T0Y0B0) , 0.2mM dNT Ps , 1 mM MgCl2, 0.5U KOD DNA polymerase (T0Y0B0) , 20pmol S- DPP - 1, 20ol X - BTS- 4， 2μ Ι Annealing and extension 反応液を含む lO L の反応液を調製 し、 次のように 2nd PCR反応を行った。 Subsequently, 2nd PCR was performed under the following conditions. 1XK0D buffer (T0Y0B0), 0.2mM dNT Ps, 1 mM MgCl 2, 0.5U KOD DNA polymerase (T0Y0B0), 20pmol S- DPP - 1, 20ol X - BTS- 4, lO includes a 2μ Ι Annealing and extension reaction L The reaction solution was prepared, and 2nd PCR reaction was performed as follows.
98° C— 15秒ヽ 58° C— 2 秒， 74° C一 30秒； 25サイクル 98 ° C—15 seconds ヽ 58 ° C—2 seconds, 74 ° C—30 seconds; 25 cycles
得られた PCR産物をァガロースゲル電気泳動により精製した （これを SX - DPGG と呼ぶ） 。 SX- DPGG を制限酵素 SacII および Xholで消化した後、 pRS434GAP の S acl l- Xhol切断部位に挿入し、 pRS434GAP- DPGGを得た。 pRS434GAP- DPGG の構造遺 伝子部分と、 制限酵素サイ ト周辺との DNAシークェンシングを行い、 設計通りの 塩基配列を持っていることを確認した。 以上の方法により、 DPP1と BTS1の結合領 域にリンカ一配列 （5' ggtggtggttct 3' ) を導入したホスファターゼ連結遺伝子 D PGGの発現ベクターを作製した。 The resulting PCR product was purified by agarose gel electrophoresis (this is called SX-DPGG). After digesting SX-DPGG with restriction enzymes SacII and Xhol, pRS434GAP Insertion into the acl-Xhol cleavage site yielded pRS434GAP-DPGG. DNA sequencing between the structural gene portion of pRS434GAP-DPGG and the vicinity of the restriction enzyme site confirmed that the nucleotide sequence was as designed. By the above method, an expression vector for the phosphatase-linked gene DPGG in which a linker sequence (5 ′ ggtggtggttct 3 ′) was introduced into the binding region between DPP1 and BTS1 was prepared.
フアルネシル二リン酸 （ FPP) 合成酵素遺伝子 ERG20とプレニルリン酸特異 的ホスファターゼ遺伝子 DPP1の連結  Ligation of the funaresyl diphosphate (FPP) synthase gene ERG20 and the prenyl phosphate specific phosphatase gene DPP1
前記 BTS1と DPP1の連結の場合と同様にして、 FPP 合成酵素遺伝子 ERG20 (Gen Bank access ion No. NC001142, 105006. . 106064) とプレニルリン酸特異的ホスフ ァターゼ遺伝子 DPP1を連結した遺伝子 （DPF と呼ぶ） を作製した。  A gene obtained by ligating the FPP synthase gene ERG20 (Gen Bank access ion No. NC001142, 105006.. Was prepared.
プライマー 1として配列番号 4 7に示す塩基配列を持つ S - DPP13A、 プライマ — 2として DPP - 2 を用いて 1 st PCRを行い、 PCR産物 #3を得た。 又、 プライマー 1として配列番号 4 8に示す塩基配列を持つ DPF3、 プライマー 2として配列番号 4 9に示す塩基配列を持つ X- FPS4を用いて 1 st PCRを行い、 PCR産物 #4を得た。 これらの PCR産物をァガロースゲル電気泳動により精製した。  1st PCR was performed using S-DPP13A having the nucleotide sequence of SEQ ID NO: 47 as primer 1 and DPP-2 as primer-2 to obtain PCR product # 3. In addition, 1st PCR was performed using DPF3 having the nucleotide sequence shown in SEQ ID NO: 48 as primer 1, and X-FPS4 having the nucleotide sequence shown in SEQ ID NO: 49 as primer 2, to obtain a PCR product # 4. These PCR products were purified by agarose gel electrophoresis.
PCR産物 #3および #4を用いて Anneal ing and extens ion 反応を行い、 得られ た PCR産物の一部についてァガロースゲル電気泳動を行い、 PCR産物 #3および #4 の連結遺伝子 （約 1. 9kb) が合成されていることを確認した。 Anneal ing and ext ens ion 反応液、 S- DPP13Aおよび X- FPS4を用いて 2 nd PCRを行い、 得られた PCR産 物をァガロースゲル電気泳動により精製した （これを SX- DPFと呼ぶ） 。 SX- DPFを 制限酵素 SacI I および Xholで消化した後、 pRS434GAP および pRS435GAP の SacII - X hoi切断部位に揷入し、 それぞれ PRS434GAP- DPF、 pRS435GAP-DPF を得た。 pRS434 GAP - DPF、 pRS435GAP - DPF の構造遺伝子部分と制限酵素サイ ト周辺との DNAシー クェンシングを行い、 設計通りの塩基配列を持っていることを確認した。 以上の 方法により、 DPP1と ERG20 の結合領域にリンカ一配列 （5' ggtggtggttct 3' ) を 導入したホスファターゼ連結遺伝子 DPF の発現ベクターを作製した。 Annealing and extension reactions were performed using PCR products # 3 and # 4, and agarose gel electrophoresis was performed on a portion of the obtained PCR products. The linked genes of PCR products # 3 and # 4 (about 1.9 kb) Was confirmed to be synthesized. 2nd PCR was performed using Annealing and extension reaction solution, S-DPP13A and X-FPS4, and the resulting PCR product was purified by agarose gel electrophoresis (this is called SX-DPF). After digesting SX-DPF with restriction enzymes SacII and Xhol, it was inserted into the SacII-Xhoi cleavage site of pRS434GAP and pRS435GAP to obtain PRS434GAP-DPF and pRS435GAP-DPF, respectively. DNA sequencing was performed between the structural gene portion of pRS434 GAP-DPF and pRS435GAP-DPF and the vicinity of the restriction enzyme site, and it was confirmed that the nucleotide sequence was as designed. By the above method, an expression vector for a phosphatase-linked gene DPF in which a linker sequence ( 5 ′ ggtggtggttct 3 ′) was introduced into the binding region between DPP1 and ERG20 was prepared.
ホスファターゼ連結遺伝子 DPF および DPGGを導入した YPH499の GGPPase 活性 の発現 DPF を発現した場合は DPP1がコードするホスファターゼと ERG20 がコードす るフアルネシルニリン酸合成酵素との融合酵素が発現され、 DPGGを発現した場合 は DPP1がコードするホスファタ一ゼと BTS1がコードするゲラニルゲラ-ルニリン 酸合成酵素との融合酵素が発現されると考えられる。 そこで、 連結遺伝子 DPF お よび DPGGを発現した場合でも GGPPase 活性が発現されるかどうかを調べた。 Expression of GGPPase activity of YPH499 transfected with phosphatase-linked genes DPF and DPGG When DPF is expressed, a fusion enzyme of the phosphatase encoded by DPP1 and the pharmacophorase synthase encoded by ERG20 is expressed, and when DPGG is expressed, the phosphatase encoded by DPP1 and BTS1 encode It is considered that a fusion enzyme with geranylgera-lunirate synthase is expressed. Therefore, it was examined whether GGPPase activity was expressed even when the linked genes DPF and DPGG were expressed.
PRS434GAP-DPP 1 , pRS434GAP-DPF 及び pRS434GAP-DPGGをそれぞれ ^ cerevi s i ae YPH499 へ導入して形質転換を行い、 得られた組換え体の GGPPase 比活性を、 前記 「ホスファターゼ活性の発現」 の項で記載した方法と同様にして測定した。 その結果を表 6に示す。  PRS434GAP-DPP 1, pRS434GAP-DPF and pRS434GAP-DPGG were each introduced into ^ cerevisiae YPH499 for transformation, and the GGPPase specific activity of the obtained recombinant was determined in the section of `` Expression of phosphatase activity ''. The measurement was performed in the same manner as described above. Table 6 shows the results.
【表 6】  [Table 6]
GGPPase比活性 fo l d  GGPPase specific activity fo l d
宿主 導入した発現ベクター  Host Introduced expression vector
(U/mg)  (U / mg)
YPH499 0. 9 1. 0 YPH499 0.9.1 1.0
YPH499 PRS4346AP-DPP1 8. 9 9. 9YPH499 PRS4346AP-DPP1 8.9 9. 9
YPH499 P S4346AP-DPF 3. 9 4. 3YPH499 P S4346AP-DPF 3.9.4.3
YPH499 P S434GAP-DP66 4. 0 4. 4 YPH499 P S434GAP-DP66 4.0 4.4
DPP 1を単独で発現させた株の GGPPase 比活性は野生株の約 10倍であった。一 方、 DPF 及び DPGGを発現させた株の GGPPase 比活性は野生株の約 4倍であった。 即ち、 DPF 及び DPGGを発現させた株においては、 DPP 1を単独で発現させた場合に 比べると GGPPase 活性の発現量は少ないものの、 野生株に比べると有意に GGPPase 比活性が増大した。 この結果から、 連結遺伝子 DPF および DPGGを発現した場合で も GGPPase 活性が発現されることが明らかとなった。 The GGPPase specific activity of the strain expressing DPP1 alone was about 10 times that of the wild-type strain. On the other hand, the GGPPase specific activity of the strain expressing DPF and DPGG was about 4 times that of the wild type. That is, in the strain expressing DPF and DPGG, the GGPPase activity was significantly lower than that in the case where DPP1 was expressed alone, but the GGPPase specific activity was significantly increased as compared with the wild-type strain. These results revealed that GGPPase activity was expressed even when the linked genes DPF and DPGG were expressed.
ホスファタ一ゼ連結遺伝子 DPF および DPGGを導入した A451によるプレニルァ ノレコーノレ;  A451 with phosphatase-linked genes DPF and DPGG introduced
HMG- CoA還元酵素遺伝子 HMG1の発現べクタ一である PRS434GAP- HMG1を A451に 導入し （これを AH1と呼ぶ） 、 AH1 に更に PRS435GAP- DPF を導入した。 こうして得 られた組換え体、 及び A451、 AH1 を YM培地で培養し、 F0H 生産性を測定した。 そ の結果を表 7に示す。 HMG- CoA reductase gene P RS434GAP- HMG1 expression is base Kuta one HMG1 were introduced into A451 (referred to as AH1), was introduced more PRS435GAP- DPF to AH1. The recombinant thus obtained, and A451 and AH1 were cultured in a YM medium, and the F0H productivity was measured. Table 7 shows the results.
【表 7】 ' FOH [Table 7] '' FOH
宿主 S入した発現ベクター  Expression vector containing host S
(mg/L)  (mg / L)
A451 N. D.  A451 N. D.
AH1 0. 3  AH1 0.3
AH1 PRS435GAP-0PF 3. 4  AH1 PRS435GAP-0PF 3.4
A451では FOH の生産は検出されなかった。 HMG1を発現した AH1 では F0H があ る程度生産されるようになった （ F0H 0. 3mg/L) 。 AH1 にホスファタ一ゼ連結遺 伝子 DPF を導入すると、 AH1 に比べて F0H の生産性が 11. 3倍に向上した （ F0H 3. 4mg/L) 。 この結果から、 ホスファターゼ遺伝子とプレニルニリン酸生合成系路に 関与する酵素の遺伝子 （特にプレニルニリン酸合成酵素遺伝子） とを連結した連 結遺伝子の導入が、 プレニルアルコールの生産性向上に有効であることが分かつ た。 No FOH production was detected for A451. AH1 expressing HMG1 produced a certain amount of F0H (F0H 0.3 mg / L). Introduction of phosphatase-linked gene DPF into AH1 increased the productivity of F0H by 11.3 times compared to AH1 (F0H 3.4 mg / L). These results indicate that the introduction of a phosphatase gene and a gene linked to an enzyme involved in the prenyl diphosphate biosynthetic pathway (particularly the prenyl diphosphate synthase gene) is effective in improving prenyl alcohol productivity. I was divided.
ホスファタ一ゼ連結遺伝子 DPF および DPGGを導入した AURGG101によるプレニ ルアルコール生産  Aurgg101 with phosphatase-linked genes DPF and DPGG for prenyl alcohol production
前記した 15 2 に対して、 それぞれ PRS434GAP- DPFおよび PRS434GAP- DPGGを 導入した。 又、 別途に 15-2 に対して PRS435GAP- BTS 1と PRS434GAP- DPP 1の 2種の プラスミ ドを導入した。 各組換え体および宿主を SG選択培地で培養し、 プレニル アルコール生産性を測定した結果を.表 8に示す。  PRS434GAP-DPF and PRS434GAP-DPGG were respectively introduced into 152 described above. In addition, two types of plasmids, PRS435GAP-BTS1 and PRS434GAP-DPP1, were separately introduced into 15-2. Table 8 shows the results of measuring the prenyl alcohol productivity by culturing each recombinant and host in an SG selective medium.
【表 8】  [Table 8]
F0H GG0H  F0H GG0H
宿主 導入した発現ベクター  Host Introduced expression vector
(mg/L) (mg/L) (mg / L) (mg / L)
■ A451 N. D. N. D. ■ A451 N.D.N.D.
AUR6Q101 0. 01 0. 02  AUR6Q101 0.01 0.02
15-2 8. 5 2. 2 15-2 PRS434GAP-DPF 66. 7 5. 7 15-2 PRS435GAP-BTS1 , PRS434GAP-DPP1 28. 8 13. 7 15-2 PRS434GAP-DPGG 68. 7 75. 7  15-2 8.5 2.2 15-2 PRS434GAP-DPF 66.7 5.7 15-2 PRS435GAP-BTS1, PRS434GAP-DPP1 28.8 13.7 15-2 PRS434GAP-DPGG 68.7 75.7
*N. D. = not detected  * N.D. = not detected
A451ではプレニルアルコールの生産は検出されず、 AURGG101のプレニルアル コール生産量は微量であった。 AURGG101に HMG044遺伝子を導入した 15- 2では、 F0H, GGOH がある程度生産されるようになった （F0H 8. 5mg/L， GG0H 2. 2mg/L )。 1 5-2にホスファターゼ連結遺伝子 DPF を導入すると、 15- 2に比べて F0H では 7. 8 倍、 660^1では2. 6 倍に生産性が向上した （F0H 66. 7mg/L, GGOH 5. 7mg/L ) 。 次に、 15 - 2に GGPP合成酵素遺伝子 BTS1とプレニルリン酸特異的ホスファタ一ゼ遗伝子 DP PIの 2種の遺伝子を別々のプラスミ ド上に組み込んで導入したところ、 15 - 2と比 ベて FOH, GG0H の生産性が向上した （F0H 28. 8mg/L, GGOH 13. 7mg/L) 。 一方、 1 5 - 2に連結遺伝子 DPGGを導入すると、 BTS1と DPP1を別々のプラスミ ド上に組み込ん で導入した場合と比べて、 F0H では 2. 4倍、 GG0Hでは 5. 5倍に生産性が向上したNo production of prenyl alcohol was detected with A451, and the amount of prenyl alcohol produced by AURGG101 was very small. In AURGG101, the HMG044 gene was introduced 15-2, F0H, GGOH has been produced to some extent (F0H 8.5 mg / L, GG0H 2.2 mg / L). When the phosphatase-linked gene DPF was introduced into 15-2, the productivity was improved 7.8-fold in F0H and 2.6-fold in 660 ^ 1 compared to 15-2 (F0H 66.7 mg / L, GGOH 5 7mg / L). Next, two genes, the GGPP synthase gene BTS1 and the prenylphosphate-specific phosphatase gene DPPI, were incorporated into separate plasmids at 15-2 and introduced. The productivity of FOH and GG0H was improved (F0H 28.8 mg / L, GGOH 13.7 mg / L). On the other hand, when the linked gene DPGG was introduced into 15-2, the productivity was 2.4 times higher in F0H and 5.5 times higher in GG0H than when BTS1 and DPP1 were incorporated into separate plasmids. Improved
(F0H 68. 7mg/L, GGOH 75. 5 mg/L) 。 (F0H 68.7 mg / L, GGOH 75.5 mg / L).
この結果から、 ホスファターゼ遺伝子とプレニルニリン酸生合成系路に関与 する酵素の遺伝子 （特にプレニルニリン酸合成酵素遺伝子） とを連結した連結遺 伝子の導入が、 プレニルアルコールの生産性向上に非常に有効であることが分か つた  These results indicate that the introduction of a connecting gene that links the phosphatase gene to a gene of an enzyme involved in the prenyl diphosphate biosynthetic pathway (particularly the prenyl diphosphate synthase gene) is extremely effective in improving prenyl alcohol productivity. I knew something
ジャー培養  Jar culture
15 - 2に連結遺伝子 DPGGを導入した株 （PRS434GAP- DPGG/15- 2) を 5 L容ジャ —フアーメンタ （MSJ- U2W；丸菱バイオェンジ） で培養して、 その生産性を測定し、 連結遺伝子 DPGGを導入することによる生産性向上への効果を更に確認することと した。 培地は、 50 g ガラク トース （ナカライ） 、 100 g パク トペプトン （DIFC 0) 、 50 g 酵母エキス （DIFC0) 、 50ml 大豆油 （ナカライ） 、 5ml アデカノー ル LG109、 水道水 5 1 を混合して作製し、 ジャーフアーメンタで滅菌 （121°C、 20分） して培養に用いた。  The strain (PRS434GAP-DPGG / 15-2) in which the connecting gene DPGG was introduced into 15-2 was cultured in a 5 L capacity fermenter (MSJ-U2W; Marubishi Biohenge), and its productivity was measured. It was decided to further confirm the effect of introducing DPGG on productivity improvement. The medium was prepared by mixing 50 g of galactose (Nacalai), 100 g of paptopeptone (DIFC0), 50 g of yeast extract (DIFC0), 50 ml of soybean oil (Nacalai), 5 ml of Adecanol LG109, and 51 tap water. The cells were sterilized with a jar armament (121 ° C, 20 minutes) and used for culture.
ジャー培養条件は次の通りである。 植菌量 2%、 温度 33°C、 通気量 1VVM、 か くはん速度 300rpm、 ガラク トース ' フィード ' レート =3. 4g/L、 pH7. 0 (4N NaOHで 制御） 。 経時的に培養液を採取してプレニルアルコール生産量を定量した結果を 図 2に示す。  The jar culture conditions are as follows. Inoculation rate 2%, temperature 33 ° C, aeration rate 1 VVM, stirring speed 300 rpm, galactose 'feed' rate = 3.4 g / L, pH 7.0 (controlled with 4N NaOH). Figure 2 shows the results of quantifying the amount of prenyl alcohol produced by collecting the culture solution over time.
培養 134時間目に FOHおよび GGOHの生産量は極大に達し、 それぞれ 0.38g / L、 0.24 g / Lであった。  At 134 hours of culture, the production of FOH and GGOH reached a maximum, 0.38 g / L and 0.24 g / L, respectively.
酸性ホスファタ一ゼ遺伝子 PH03およぴァルカリ性ホスファタ一ゼ潰伝子 PH08 のクローユングおょぴ発現ベクターの作製 Acid phosphatase gene PH03 and alkaline phosphatase gene PH08 Of expression vector
リン酸エステル化合物に対する加水分解活性において基質特異性のない （非 特異的な） ホスファタ一ゼをコ一ドする遺伝子のクロ一エング及び発現ベクター の作製を、 以下の方法で行った。 非特異的ホスファターゼとして ^ cerevisiae の酸性ホスファターゼ遺伝子 PH03 (GenBank accession No. NC001134, complemen t (427657. . 429060) ) 及ぴアルカリ性ホスファターゼ遺伝子 PH08 (GenBank access ion No. NC001136, complement (1418537. . 1420237) ) を用いた。  Cloning of a gene encoding a phosphatase encoding (non-specific) phosphatase having no substrate specificity in the activity of hydrolyzing a phosphate ester compound and production of an expression vector were performed by the following methods. ^ Cerevisiae acid phosphatase gene PH03 (GenBank accession No. NC001134, complement (427657.. 429060)) and alkaline phosphatase gene PH08 (GenBank access ion No. NC001136, complement (1418537.. 1420237)) as non-specific phosphatases Was used.
PCR法により、 ^ cerevisiae YPH499 ゲノム DNA を铸型として、 S. cerev isiaeの遺伝子 PH03 約 1. 4kbp断片を増幅した。 PCRプライマーは、 配列番号 5 0 に示す塩基配列を持つプライマーと、 配列番号 5 1に示す塩基配列を持つプライ マーである。 PCR 断片をァガロースゲル電気泳動によって精製した後、 pCR2. 1- T 0P0へ T/A ライゲーシヨンによりクローニングした。 クローユングした断片の塩基 配列決定を行い、 SGD の配列と比較したところ、 PCRエラーは無かった。 作成し たプラスミ ド DNA を pCR - PH03と呼ぶ。 pCR - PH03から SacII - Xholによって切出され る 1. 4kbp 断片を調製し、 pRS434GAPの SacII- Xhol切断部位に揷入した。 作製し たプラスミ ドを pRS434GAP - 1¾03と呼ぶ。  Using the genomic DNA of ^ cerevisiae YPH499 genomic DNA as a 約 type, an approximately 1.4 kbp fragment of the gene PH03 of S. cerevisiae was amplified by PCR. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 50 and a primer having the nucleotide sequence shown in SEQ ID NO: 51. After purifying the PCR fragment by agarose gel electrophoresis, it was cloned into pCR2.1-TOP0 by T / A ligation. When the cleaved fragment was sequenced and compared to the SGD sequence, there were no PCR errors. The resulting plasmid DNA is called pCR-PH03. A 1.4 kbp fragment cut out from pCR-PH03 by SacII-Xhol was prepared and inserted into the SacII-Xhol cleavage site of pRS434GAP. The prepared plasmid is called pRS434GAP-1¾03.
PCR法により、 ^ cerevisiae YPH499 ゲノム DNA を鐯型として、 ^_ cerev isiaeの遺伝子 PH08 約 1. 7kbP断片を増幅した。 PCRプライマーは、 配列番号 5 2 に示す塩基配列を持つプライマーと、 配列番号 5 3に示す塩基配列を持つプライ マーである。 PCR 断片をァガロースゲル電気泳動により精製した後 pCR2. 1- T0P0 へ T/A ライゲーシヨンによりクローニングした。 クローユングした断片の塩基配 列決定を行い、 SGD の配列と比較したところ、 PCRエラーは無かった。 作成した プラスミ ド DNAを pCR- PH08と呼ぶ。 pCR - PH08から SacII - Smal によって切出され る 1. 7kbp断片を調製し、 pRS434GAPの SacII- Smal切断部位に挿入した。 作製した プラスミ ドを pRS434GAP- PH08と呼ぶ。 Using the ^ cerevisiae YPH499 genomic DNA as a 鐯 type, an approximately 1.7 kb P fragment of the ^ _ cerevisiae gene PH08 was amplified by PCR. The PCR primers are a primer having the nucleotide sequence shown in SEQ ID NO: 52 and a primer having the nucleotide sequence shown in SEQ ID NO: 53. The PCR fragment was purified by agarose gel electrophoresis and cloned into pCR2.1-TOP0 by T / A ligation. When the base sequence of the cleaved fragment was determined and compared with the SGD sequence, no PCR errors were found. The prepared plasmid DNA is called pCR-PH08. A 1.7 kbp fragment cut out from pCR-PH08 by SacII-Smal was prepared and inserted into the SacII-Smal cleavage site of pRS434GAP. The resulting plasmid is called pRS434GAP-PH08.
非特異的ホスファターゼ活性の測定  Measurement of non-specific phosphatase activity
酸性ホスファターゼ活性は Ferminanらの方法 （E. Ferminan, et al. (1997) Microbiol. 143, 2615-2625) を参考にして以下のようにして測定した。 pRS434GA P- PH03を前記 15-2 に導入し、 得られた組換え体および宿主を 5mLの SD選択培地 で 1日間培養し、 遠心分離により菌体を集菌した。 菌体を生理食塩水で 2度洗い、 300 μ I の抽出ノ ッファ一 (lOmM citrate (pH4.3)、 10mM β -mercaptoethanol, 1 mM PMSF 、 10 μ g/L aprotinin 、 10 μ g/L leupeptin 、 1 μ g/L pepstatin ) に懸濁した。 そして、 等量のガラスビーズ （φ =0.45— 0.5mm) を加え、 ボルテ ックスミキサーにて 15分間強く攪拌 （ 4° C) して菌体を破砕した。 更に 300 し の抽出バッファーを加え、 遠心分離 （18，000Xg 、 15分、 4° C) した後、 上清 を分取して粗酵素画分とした。 10mM citrate ( pH4.3) で適当に希釈した粗酵 素画分 100 し に、 100 の ρ—ニトロフエニルリン酸 （ρΝΡΡ) 溶液 （ 10mM ρ NPP、 200mMcitrate (PH4.3) ) を添加し、 30° Cで 15分間反応を行った。 50μ L の 1M NaOH水溶液を添加して反応を停止し、 405nmの吸光度を測定した。 0〜 0.03U/mL の酸性ホスファターゼ （Roche Diagnostics ) を用いて検量線を作成し、 前記で求めた 405nmの吸光度を酵素活性値 （Unit) に換算した。 粗酵素画分中の タンパク質濃度は、 Bio- Rad Protein Assay で、 BSA を標準タンパク質として測 定し、 粗酵素画分中の酸性ホスファターゼ比活性 （U/mg protein) を求めた。 ま た、 ここで得られた粗酵素画分の GGPPase 比活性は、 反応バッファ一として 120m M citrate (PH4.3) を用いて、 前記 「ホスファターゼ活性の発現」 の項で記載した 酵素活性測定法と同様の方法で測定した。 Acid phosphatase activity was measured as follows with reference to the method of Ferminan et al. (E. Ferminan, et al. (1997) Microbiol. 143, 2615-2625). pRS434GA P-PH03 was introduced into the above 15-2, and the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. Wash the cells twice with saline, and add 300 μl of extraction buffer (10 mM citrate (pH 4.3), 10 mM β-mercaptoethanol, 1 mM PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin. , 1 μg / L pepstatin). Then, an equal amount of glass beads (φ = 0.45-0.5 mm) was added, and the cells were disrupted by vigorous stirring (4 ° C) for 15 minutes using a vortex mixer. An additional 300 extraction buffers were added, centrifuged (18,000 Xg, 15 minutes, 4 ° C), and the supernatant was collected to obtain a crude enzyme fraction. To the crude enzyme Motoga fraction 100, who appropriately diluted with 10mM citrate (pH4.3), it was added 100 of ρ- nitrophenyl phosphate (ρΝΡΡ) solution (10mM ρ NPP, 200mMcitrate (P H4.3)) The reaction was performed at 30 ° C. for 15 minutes. The reaction was stopped by adding 50 μL of a 1 M aqueous NaOH solution, and the absorbance at 405 nm was measured. A calibration curve was prepared using 0 to 0.03 U / mL acid phosphatase (Roche Diagnostics), and the absorbance at 405 nm determined above was converted to an enzyme activity value (Unit). The protein concentration in the crude enzyme fraction was determined by Bio-Rad Protein Assay using BSA as a standard protein, and the specific activity of the acid phosphatase (U / mg protein) in the crude enzyme fraction was determined. Also, where the resulting GGPPase specific activity of the crude enzyme fraction, with 120m M citrate (P H4.3) as a reaction buffer one, the enzyme activity measured as described in the paragraph of the "expression of phosphatase activity" It measured by the same method as the method.
アルカリ性ホスファタ一ゼ活性は Dhamija らの方法 （S. S. Dhamija, et al. Alkaline phosphatase activity was determined by the method of Dhamija et al. (S. S. Dhamija, et al.
(1987) Current Genetics, 11, 67-473) を参考にして以下のようにして測定し た。 PRS434GAP- PH08を前記 15-2 に導入し、 得られた組換え体および宿主を 5mL の SD選択培地で 1 日間培養し、 遠心分離により菌体を集菌した。 菌体を生理食塩 水で 2度洗い、 300 μ L の抽出バッファー （lOOmM Tris · HC1 (pH8.5)、 ImM MgCl2 、 lOmM β -mercaptoethanolN ImM PMSF、 10 μ g/L aprotinin 、 10 μ g/L leupeptin 、 1 μ g/L pepstatin ) に懸濁した。 そして、 等量のガラスビーズ（φ = 0.45-0.5mm ) を加え、 ボルテックスミキサーにて 15分間強く攪拌 （4° C) して菌体を破枠した。 更に 300 L の抽出バッファーを加え、 遠心分離 （18, 000 Xg 、 15分、 4° C) により可溶性画分 （上清） を分取して粗酵素画分とした。 1 OOm Tris - HC1 (pH8. 5)で適当に希釈した粗酵素画分 lOO L に、 100 M L の p—ニ トロフエ二ルリ ン酸 （pNPP) 溶液 （10mM pNPP、 lOOmM Tris · HC1 (pH8, 5) ) を添加し、 30° Cで 15分間反応を行った。 50 L の 1M NaOH水溶液を添加し て反応を停止し、 405nmの吸光度を測定した。 0〜0. 3U/mL のアルカリ性ホスフ ァターゼ （calf intestine, Roche Diagnostics) を用いて検量線を作成し、 前記 で求めた 405nm の吸光度を酵素活性値 （Unit) に換算した。 アル力リ性ホスファ ターゼ比活性 （U/mg protein) は酸性ホスファターゼの場合と同様にして求めた。 又、 ここで得られた粗酵素画分の GGPPase 比活性は、 反応バッファ一として 60mM(1987) Current Genetics, 11, 67-473). PRS434GAP-PH08 was introduced into the above 15-2, and the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. Wash the cells twice with physiological saline, and add 300 μL of extraction buffer (100 mM TrisHC1 (pH 8.5), ImM MgCl 2 , 10 mM β-mercaptoethanol N ImM PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin, 1 μg / L pepstatin). Then, an equal amount of glass beads (φ = 0.45-0.5 mm) was added, and vigorously stirred (4 ° C.) for 15 minutes with a vortex mixer to break the cells. Further, 300 L of extraction buffer was added, and the soluble fraction (supernatant) was collected by centrifugation (18,000 Xg, 15 minutes, 4 ° C) to obtain a crude enzyme fraction. 1 OOm Tris-100 mL of p-nitrotrophyl phosphoric acid (pNPP) solution (10 mM pNPP, lOOmM Tris HC1 (pH 8.5)) was added to 100 mL of p-nitrotrophyl phosphoric acid (pNPP) in the crude enzyme fraction lOO L appropriately diluted with HC1 (pH 8.5). ) Was added and reacted at 30 ° C. for 15 minutes. The reaction was stopped by adding 50 L of a 1 M aqueous NaOH solution, and the absorbance at 405 nm was measured. A calibration curve was prepared using alkaline phosphatase (calf intestine, Roche Diagnostics) at 0 to 0.3 U / mL, and the absorbance at 405 nm obtained in the above was converted to an enzyme activity value (Unit). Specific phosphatase specific activity (U / mg protein) was determined in the same manner as for acid phosphatase. The GGPPase specific activity of the crude enzyme fraction obtained here was 60 mM as the reaction buffer.
Tris - HC1 (pH8. 5)、 Im MgCl 2 を用いて、 前記 「ホスファターゼ活性の発現」 の項で記載した酵素活性測定法と同様の方法で測定した。 Using Tris-HC1 (pH 8.5) and Im MgCl 2 , the measurement was carried out in the same manner as the enzyme activity measurement method described in the section “Expression of phosphatase activity”.
以上の方法で測定した非特異的ホスファタ一ゼ比活性 （酸性ホスファターゼ比 活性おょぴアルカリ性ホスファターゼ比活性） を以下 pNPPase 比活性と呼ぶ。  The non-specific phosphatase specific activity (acid phosphatase specific activity or alkaline phosphatase specific activity) measured by the above method is hereinafter referred to as pNPPase specific activity.
非特異的ホスファタ一ゼ遺伝子を導入した 15 - 2のホスファターゼ活性の発現 とプレニルアルコールの生産  Expression of 15-2 phosphatase activity and production of prenyl alcohol by introducing non-specific phosphatase gene
酸性ホスファタ一ゼ遺伝子 PH03及ぴアル力リ性ホスファターゼ遺伝子 PH08を それぞれ組込んだ発現ベクターを導入した 15-2 の pNPPase 比活性、 GGPPase 比 活性及ぴプレニルアルコール生産性 （SG選択培地で培養） を測定した。 その結果 を表 9に示す。 表 9では、 宿主を 1とした相対値で表示している。 表中の GGPPase 比活性の項で、 +PNPP の列は、 前記 「ホスファタ一ゼの基質特異性」 の項で記載 したのと同様に、 GGPP以外の追加基質として ΙΟΟ μ Μ の pNPPを添加した場合の GGP Pase 比活性値を示し、 - pNPP の列は、 pNPPを添加せずに GGPPase 比活性を測定し た値を示す。  The pNPPase specific activity, GGPPase specific activity, and prenyl alcohol productivity (cultured in SG selective medium) of 15-2 were introduced with expression vectors incorporating the acid phosphatase gene PH03 and the alkaline phosphatase gene PH08, respectively. It was measured. Table 9 shows the results. In Table 9, relative values are shown with the host as 1. In the GGPPase specific activity section of the table, in the + PNPP column, ΙΟΟμΜ of pNPP was added as an additional substrate other than GGPP in the same manner as described in the section `` Substrate specificity of phosphatases ''. The column of -pNPP shows the value of the GGPPase specific activity measured without adding pNPP.
【表 9】  [Table 9]
GGPPase比活性 プしニルアルコール pNPPase比活性 GGPPase specific activity Puccinyl alcohol pNPPase specific activity
宿主 導入した発現ベクター (U/m l ) 生産性 （相対値）  Host Expression vector (U / ml) Productivity (relative value)
(U/rn l )  (U / rn l)
- pNPP +pNPP FOH 660H -pNPP + pNPP FOH 660H
15-2 0. 02 0. 11 0 1 . 0 1. 015-2 0.02 0.11 0 1 .0 1.0
15-2 PRS434GAP-PH03 0. 47 0. 37 0 0. 8 0. 315-2 PRS434GAP-PH03 0.47 0.37 0 0.8 0.3
15-2 PRS4346AP-PH08 0. 60 0. 68 0 - 1. 0 0. 8 酸性ホスファターゼ遺伝子又はアル力リ性ホスファターゼ遺伝子の導入によ り、 pNPPase 比活性は宿主に比べて 28〜； 35倍に増大した。 GGPPase 比活性は、 追 加基質として pNPPを加えない場合は宿主に比べて 3〜 6倍に増大した。 しかし、 追加基質として pNPPを加えると [ 3H] GGPPに対する見かけ上のホスファターゼ活性 はゼロとなった。 このことから、 酸性ホスファターゼ遺伝子およびアルカ リ性ホ スファターゼ遺伝子の導入により発現されたホスファターゼは、 GGPPと pNPPのい ずれとも反応する基質特異性の低いホスファターゼであることが示された。 この 結果は、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファターゼ遺伝 子を導入した場合と、 対照的である （表 2参照） 。 一方、 プレニルアルコールの 生産性は、 酸性ホスファターゼ遺伝子又はアル力リ性ホスファターゼ遺伝子を導 入しても向上しなかった。 以上の結果から、 非特異的ホスファターゼ遺伝子を宿 主に導入して、 pNPPase 活性又は GGPPase 活性を上昇させても、 プレニルアルコ ールの生産性の向上への効果は無いことが分かった。 15-2 PRS4346AP-PH08 0.60 0.68 0-1.0.0 0.8 By the introduction of the acid phosphatase gene or the alkaline phosphatase gene, the specific activity of pNPPase was increased 28- to 35-fold compared to the host. The specific activity of GGPPase was increased 3 to 6 times compared to the host when pNPP was not added as an additional substrate. However, the addition of pNPP as an additional substrate resulted in zero apparent phosphatase activity for [ 3 H] GGPP. This indicated that the phosphatase expressed by the introduction of the acid phosphatase gene and the alkaline phosphatase gene was a phosphatase with low substrate specificity that reacted with both GGPP and pNPP. This result is in contrast to the case where a phosphatase gene having substrate specificity was introduced into prenyl phosphate having 15 or more carbon atoms (see Table 2). On the other hand, the productivity of prenyl alcohol was not improved by introducing the acid phosphatase gene or the alkaline phosphatase gene. From the above results, it was found that introduction of a non-specific phosphatase gene into a host to increase pNPPase activity or GGPPase activity had no effect on improving prenyl alcohol productivity.
実施例の評価  Evaluation of Examples
以上の実施例は、 次のように評価される。  The above embodiment is evaluated as follows.
即ち、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼを コードする 6種類の遺伝子を、 それぞれ F0H および GG0H産生酵母に導入すること により、 6種類いずれの遺伝子を導入した場合においても、 GGPPase 比活性が上 昇した。 更に、 6種類いずれの遺伝子を導入した場合においても、 F0H および GG0 Hの生産性が実際に数倍〜数十倍に向上することを示した。  That is, by introducing six kinds of genes encoding phosphatases having substrate specificity to prenyl phosphate having 15 or more carbon atoms into F0H and GG0H-producing yeasts, respectively, even when any of the six kinds of genes are introduced, GGPPase specific activity increased. Furthermore, it was shown that the productivity of F0H and GG0H actually increased several to several tens of times when any of the six genes was introduced.
本願発明者らは、 F0H および GG0Hの生産性を向上させることのできるホスフ ァターゼのアミノ酸配列には、 第 1図に示すホスファタ一ゼモチーフが含まれて いることを見出した。 このホスファターゼモチーフは、 炭素数 I 5以上のプレニル リン酸に対する基質特異性に関係があると推測している。 The present inventors have found that the amino acid sequence of a phosphatase capable of improving the productivity of F0H and GG0H contains the phosphatase motif shown in FIG. We speculate that this phosphatase motif is related to the substrate specificity for prenyl phosphates having at least 5 carbon atoms.
更に、 炭素数 15以上のプレニルリ ン酸に基質特異性のあるホスファターゼを コードする遺伝子とプレニルニリン酸生合成系路に関与する酵素をコードする遺 伝子を連結してなる遺伝子を、 F0H および GG0H 産生酵母に導入することにより、 F0H および GG0Hの生産性が更に向上した。 上記連結遺伝子を導入した場合、 連結 せずに別々のプラスミ ド上に組み込んで導入した場合と比べて F0H および GG0Hの 生産性が、 数倍に向上することも示した。 Furthermore, a gene encoding a phosphatase having a substrate specificity of prenyl phosphinate having at least 15 carbon atoms and a gene encoding an enzyme involved in a prenyl diphosphate biosynthetic pathway was linked to F0H and GG0H production. By introducing into yeast, the productivity of F0H and GG0H was further improved. When the above linked gene is introduced, It was also shown that the productivity of F0H and GG0H was improved several times compared to the case where they were incorporated into separate plasmids without introduction.
一方、 リン酸エステル化合物に対する加水分解活性において基質特異性のな い （非特異的な） ホスファターゼをコードする遺伝子 （PH03， PH08) を、 F0H お ょぴ GG0H産生酵母に導入した場合、 GGPPase 比活性は上昇したが、 F0H および GG0 Hの生産性は向上しなかった。 非特異的ホスファターゼは、 基質特異性がないため に菌体中のあらゆるリン酸エステル化合物と反応すると考えられる。 そのため、 菌体中のプレニルリン酸以外のリン酸ヱステル化合物 （例えば菌体中に多量に存 在すると考えられる ATP 等） とも反応してしまう。 そして、 実質的にプレニルリ ン酸と反応してプレニルアルコールを産生できる非特異的ホスファタ一ゼの数は 非常に限られてしまうため、 プレニルアルコールの生産性が向上しなかったと推 定される。 On the other hand, if we name substrate specificities in the hydrolysis activity with respect to phosphoric acid ester compounds a gene encoding a (non-specific) phosphatase (PH0 3, PH08), was introduced into F0H Contact Yopi GG0H producing yeast, GGPPase ratio Activity increased, but F0H and GG0H productivity did not increase. Non-specific phosphatases are considered to react with any phosphate compound in the cells due to lack of substrate specificity. For this reason, it also reacts with phosphate esters other than prenyl phosphate in the cells (for example, ATP which is considered to be present in large amounts in the cells). It is presumed that the productivity of prenyl alcohol did not improve because the number of non-specific phosphatases capable of producing prenyl alcohol by substantially reacting with prenyl phosphinate was very limited.
又、 これらの非特異的なホスファタ一ゼのァミノ酸配列を詳細に検討した結 果、 第 1図に示したようなホスファターゼモチーフは含まれていないことが明ら かとなつた。 このことは、 上記ホスファターゼモチーフを含み炭素数 15以上のプ レニルリン酸に基質特異性のあるホスファターゼ遺伝子の導入又は発現強化が、 プレニルアルコールの生産性の向上に対して決定的であることを示している。  Further, as a result of detailed examination of the amino acid sequences of these non-specific phosphatases, it was found that the phosphatase motif as shown in FIG. 1 was not contained. This indicates that the introduction or enhanced expression of a phosphatase gene containing the above phosphatase motif and having substrate specificity to prenyl phosphate having 15 or more carbon atoms is crucial for improving prenyl alcohol productivity. I have.
産業上の利用分野 Industrial applications
以上のように、 本発明は、 プレニルアルコール、 特に生物学的に活性な全ト ランス型で、 しかも炭素数 15以上であるプレニルアルコールの効率的な大量生産 を可能とする。  As described above, the present invention enables efficient mass production of prenyl alcohols, particularly, prenyl alcohols having a biologically active all-trans form and having 15 or more carbon atoms.

Claims

請 求 の 範 囲 The scope of the claims
1 . 配列番号 1〜配列番号 6のいずれかに示すアミノ酸配列を有し、 炭素数 15以 上のプレニルリン酸に基質特異性のあるホスファターゼ活性を示すポリべプチド。1. A polypeptide having an amino acid sequence represented by any one of SEQ ID NOS: 1 to 6 and having a phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
2. 配列番号 1〜配列番号 6のいずれかに示すアミノ酸配列における 5個以下のァ ミノ酸が置換， 欠失又は付加されたアミノ酸配列を有し、 炭素数 15以上のプレニ ルリン酸に基質特異性のあるホスファターゼ活性を示すポリぺプチド。 2. The amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6 has an amino acid sequence in which 5 or less amino acids have been substituted, deleted or added, and has a substrate specificity for prenyl phosphoric acid having 15 or more carbon atoms. A polypeptide exhibiting potent phosphatase activity.
3. 以下 （1 ) 〜 ( 3 ) 式の内の少なくとも一つのホスファターゼモチーフを含み、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファターゼ活性を示すポ リぺプチド。  3. A polypeptide comprising at least one phosphatase motif of the following formulas (1) to (3) and exhibiting phosphatase activity having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
( 1 ) KXXXGXXRP (式中、 X は任意のアミノ酸残基から選ばれることを表す） (1) KXXXGXXRP (where X represents an amino acid residue)
( 2 ) 1XXSGH (式中、 1 はアミノ酸残基 S 又は T から選ばれ、 X は任意のァミノ 酸残基から選ばれることを表す） (2) 1XXSGH (where 1 is selected from amino acid residues S or T, and X is selected from any amino acid residues)
( 3 ) SR2XDXXHXXXDVXXGXXX3 (式中、 2 はアミノ酸残基 V 又は T から選ばれ、 3 はアミノ酸残基 G又は A から選ばれ、 X は任意のアミノ酸残基から選ばれること を表す） 。  (3) SR2XDXXHXXXDVXXGXXX3 (wherein, 2 is selected from amino acid residues V or T, 3 is selected from amino acid residues G or A, and X is selected from any amino acid residues).
4 . 前記 （1 ) 〜 （ 3 ) 式で示されるホスファターゼモチーフにおいて、 種類が 特定されている任意の 1以上のアミノ酸残基が同類置換を受けている請求の範囲 3項に記載のポリぺプチド。  4. The polypeptide according to claim 3, wherein in the phosphatase motifs represented by the formulas (1) to (3), any one or more amino acid residues whose type is specified have been conservatively substituted. .
5. 前記プレニルリン酸がブアルネシルリン酸、 ゲラニルゲラニルリン酸、 ゲラニ ルフアルネシルリン酸、 へキサプレニルリン酸、 ヘプタプレニルリン酸、 ォクタ プレニルリン酸、 ノナプレニルリン酸、 デカプレニ/レリン酸、 ゥンデカプレニノレ リン酸又はドデカブレニルリン酸の 1種又は 2種以上である請求の範囲 1項ない し 4項のいずれかに記載のポリペプチド。  5. The prenyl phosphate is buarnesyl phosphate, geranylgeranyl phosphate, geranilfalnesyl phosphate, hexaprenyl phosphate, heptaprenyl phosphate, octaprenyl phosphate, nonaprenyl phosphate, decapreni / rephosphoric acid, pendecapreninoleic acid Or the polypeptide according to any one of claims 1 to 4, which is one or more of dodecabrenyl phosphate.
6. 配列番号 7〜配列番号 1 2のいずれかに示す塩基配列を有し、 炭素数 15以上の プレニルリン酸に基質特異性のあるホスファタ一ゼをコ一ドするポリヌクレオチ ド、。 6. A polynucleotide having a base sequence represented by any one of SEQ ID NOs: 7 to 12, and encoding a phosphatase having substrate specificity to prenyl phosphate having 15 or more carbon atoms.
7. 配列番号 7〜配列番号 1 2のいずれかに示す塩基配列又はこれと相補的な塩基 配列に対してストリンジェントな条件下でハイブリダィズする塩基配列を有し、 炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼをコ一ドする ポリヌクレオチド。 7. It has a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence shown in any one of SEQ ID NOs: 7 to 12 or a nucleotide sequence complementary thereto, and is capable of producing a prenyl phosphate having 15 or more carbon atoms. A polynucleotide encoding a phosphatase with substrate specificity.
8. 前記プレニルリン酸がフアルネシルリン酸、 ゲラ -ルゲラニルリン酸、 ゲラニ ノレフアルネシルリン酸、 へキサプレニルリン酸、 ヘプタプレニルリン酸、 ォクタ プレニルリン酸、 ノナプレニルリン酸、 デカプレニルリン酸、 ゥンデカプレニル リン酸又はドデカブレニルリン酸の 1種又は 2種以上である請求の範囲 6項又は 7 項に記載のポリヌクレオチド。  8. The prenyl phosphoric acid is selected from the group consisting of phthalnesyl phosphate, gera-rugeranyl phosphate, gerani norlefarnesyl phosphate, hexaprenyl phosphate, heptaprenyl phosphate, octaprenyl phosphate, nonaprenyl phosphate, decaprenyl phosphate, pendecaprenyl phosphate, and dodecaphosphate. 8. The polynucleotide according to claim 6, which is one or more of brenyl phosphate.
9. 請求の範囲 1項〜 5項に記載のいずれかのポリべプチドをコ一ドするポリヌク レオチド。  9. A polynucleotide encoding any of the polypeptides according to claims 1 to 5.
10.請求の範囲 6項〜 9項に記載のいずれかのポリヌクレオチドを含む組換え核酸。 10. A recombinant nucleic acid comprising the polynucleotide according to any one of claims 6 to 9.
11. 請求の範囲 10項に記載のいずれかの組換え核酸が導入され又は発現を強化さ れている組換え体。 11. A recombinant into which any of the recombinant nucleic acids according to claim 10 has been introduced or whose expression has been enhanced.
1 2. 前記組換え体の宿主細胞が、真菌 (Eumycetes ) ， 子嚢菌類 (Ascomycetes )， 単細胞真核生物のいずれかである請求の範囲 11項に記載の組換え体。  12. The recombinant according to claim 11, wherein the host cell of the recombinant is any of fungi (Eumycetes), ascomycetes (Ascomycetes), and unicellular eukaryotes.
1 3. 前記組換え体の宿主細胞が酵母である請求の範囲 11項に記載の組換え体。 13. The recombinant according to claim 11, wherein the host cell of the recombinant is yeast.
1 4. 前記酵母がサッカロミセス属 (Saccharomycetes ) の酵母である請求の範囲 1 3項に記載の組換え体。 14. The recombinant according to claim 13, wherein the yeast is a yeast belonging to the genus Saccharomycetes.
1 5. 前記酵母がサッカロミセス 'セレピシェ （Saccharomyces cerevisiae) YPH4 99株， YPH500株， A451株， W303-1A株， W303-1B株又はこれらに由来する株であ る請求の範囲 1 3項に記載の組換え体。  15. The method according to claim 13, wherein the yeast is Saccharomyces cerevisiae YPH499, YPH500, A451, W303-1A, W303-1B, or a strain derived therefrom. Recombinant.
1 6. ホスファターゼ遺伝子を導入又は発現強化した宿主細胞を培養し、 その培養 物からプレニルアルコールを採取するプレニルアルコールの製造方法。  1 6. A method for producing prenyl alcohol, comprising culturing host cells into which a phosphatase gene has been introduced or having enhanced expression, and collecting prenyl alcohol from the culture.
1 7. ホスファターゼ遺伝子とプレニルニリン酸生合成経路に関与する酵素の遺伝 子とを導入又は発現強化した宿主細胞を培養し、 その培養物からプレニルアルコ ールを採取するプレニルアルコールの製造方法。  1 7. A method for producing prenyl alcohol, comprising culturing host cells into which a phosphatase gene and a gene of an enzyme involved in the prenyl diphosphate biosynthetic pathway have been introduced or having enhanced expression, and collecting prenyl alcohol from the culture.
1 8. 前記ホスファターゼ遺伝子とプレニルニリン酸生合成経路に関与する酵素の 遺伝子とが連結遺伝子として導入又は発現強化されている請求の範囲 1 7項に記 載のプレニルアルコールの製造方法。 1 8. The phosphatase gene and the enzyme involved in the prenyl diphosphate biosynthesis pathway 18. The method for producing prenyl alcohol according to claim 17, wherein the gene and the gene are introduced as a linked gene or expression is enhanced.
1 9. 前記プレニルニリン酸生合成経路に関与する酵素の遺伝子が以下の （4 ) 及 び Z又は （5 ) の遺伝子である請求の範囲 1 7項に記載のプレニルアルコールの製 造方法。  19. The method for producing prenyl alcohol according to claim 17, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway is the following gene of (4) and / or Z or (5).
( 4 ) フアルネシル二リン酸合成酵素遺伝子及ぴゲラ二ルゲラ二ルニリン酸合成 酵素遺伝子から選ばれる少なくとも 1の遺伝子。  (4) At least one gene selected from the pharmacogene synthase gene and the gerneryl geraniline diphosphate synthase gene.
( 5 ) ァセチル CoA合成酵素遺伝子、 ァセチル CoA —ァセチルトランスフェラー ゼ遺伝子、 ヒ ドロキシメチルダルタリル CoA合成酵素遺伝子、 ヒ ドロキシメチル グルタリル CoA還元酵素遺伝子、 メバロン酸キナーゼ遺伝子、 メバロン酸リン酸 キナーゼ遺伝子、 メバロン酸二リン酸デカルポキシラーゼ遺伝子、 イソペンテ二 ルニリン酸ィソメラーゼ遺伝子、 デォキシキシルロースリン酸リダク トイソメラ ーゼ遺伝子、 デォキシキシルロースリン酸合成酵素遺伝子、 MEP シチジリルトラ ンスフヱラーゼ遺伝子、 CDP- MEキナーゼ遺伝子及び MECDP 合成酵素遺伝子から選 ばれる少なくとも 1の遺伝子。  (5) Acetyl-CoA synthase gene, acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalon Acid diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, doxyxylulose phosphate reductoisomerase gene, doxyxylulose phosphate synthase gene, MEP citidylyltransferase gene, CDP-ME kinase gene and MECDP At least one gene selected from a synthase gene.
2 0 . 前記プレニルニリン酸生合成経路に関与する酵素の遺伝子が以下の （4 ) 及び 又は （5 ) の遺伝子である請求の範囲 18項に記載のプレニルアルコールの 製造方法。  20. The method for producing prenyl alcohol according to claim 18, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway is the gene of the following (4) and / or (5).
( 4 ) フアルネシル二リン酸合成酵素遺伝子及びゲラニルゲラ二ルニリン酸合成 酵素遺伝子から選ばれる少なくとも 1の遺伝子。  (4) at least one gene selected from a pharmacogene synthase gene and a geranylgeraniline diphosphate synthase gene;
( 5 ) ァセチル CoA合成酵素遺伝子、 ァセチル CoA —ァセチルトランスフェラー ゼ遺伝子、 ヒ ドロキシメチルダルタリル CoA 合成酵素遺伝子、 ヒ ドロキシメチル グルタリル Co A還元酵素遺伝子、 メバロン酸キナーゼ遺伝子、 メバロン酸リン酸 キナーゼ遺伝子、 メバロン酸二リン酸デカルボキシラーゼ遺伝子、 イソペンテ二 ルニリン酸ィソメラ一ゼ遺伝子、 デォキシキシルロースリン酸リダク トイソメラ ーゼ遺伝子、 デォキシキシルロースリン酸合成酵素遺伝子、 MEP シチジリルトラ ンスフェラーゼ遺伝子、 CDP- MEキナーゼ遺伝子及ぴ MECDP 合成酵素遺伝子から選 ばれる少なくとも 1の遺伝子。 (5) acetyl-CoA synthase gene, acetyl-CoA-acetyltransferase gene, hydroxymethyldaltharyl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, Mevalonate diphosphate decarboxylase gene, isopentenyl diphosphate isomerase gene, dexoxylulose phosphate reductoisomerase gene, dexoxylulose phosphate synthase gene, MEP citidylyltransferase gene, CDP-ME kinase At least one gene selected from genes and MECDP synthase genes.
2 1 . 前記ホスファターゼ遺伝子が、 リン酸エステル化合物に対する加水分解活性 において炭素数 15以上のプレニルリン酸に基質特異性のあるホスファタ一ゼをコ 一ドする遺伝子である請求の範囲 1 6項〜 2 0項のいずれかに記載のプレニルァ ルコールの製造方法。 21. The phosphatase gene according to claim 16, wherein the phosphatase gene encodes a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms in hydrolyzing activity for a phosphate compound. The method for producing prenyl alcohol according to any one of the above items.
2 2 . 前記プレニルリン酸がブアルネシルリン酸、 ゲラエルゲラニルリン酸、 ゲラ ニルブアルネシルリン酸、 へキサプレニルリン酸、 ヘプタプレニルリン酸、 オタ タプレニルリ ン酸、 ノナプレニルリン酸、 デカプレニルリ ン酸、 ゥンデカプレニ ルリン酸又はドデカブレニルリ ン酸の 1種又は 2種以上である請求項 2 1に記載 のプレニルアルコールの製造方法。  22. The prenyl phosphate is buarnesyl phosphate, geraergeranyl phosphate, geranyl buarnesyl phosphate, hexaprenyl phosphate, heptaprenyl phosphate, otataprenyl phosphate, nonaprenyl phosphate, decaprenyl phosphate, pendecaprenyl phosphate. 22. The method for producing a prenyl alcohol according to claim 21, wherein the method is one or two or more of dodecabrenyl phosphoric acid.
2 3 . 前記ホスファターゼ遺伝子が、 請求の範囲 6項〜 9項に記載のいずれかのポリ ヌクレオチドである請求の範囲 1 6項〜 2 0項のいずれかに記載のプレニルアル コールの製造方法。  23. The method for producing prenyl alcohol according to any one of claims 16 to 20, wherein the phosphatase gene is any one of the polynucleotides according to claims 6 to 9.
24 . 前記ホスファターゼ遺伝子がリン酸エステル化合物に対する加水分解活性に おいて炭素数 15以上のプレニルリン酸に基質特異性のあるホスファターゼ以外の ホスファタ一ゼをコ一ドする遺伝子である請求の範囲 1 6項〜 2 0項のいずれか に記載のプレニルアルコールの製造方法。  24. The method according to claim 16, wherein the phosphatase gene encodes a phosphatase other than a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms in hydrolyzing activity for a phosphate compound. Item 20. The method for producing prenyl alcohol according to any one of Items 20 to 20.
2 5 . 前記宿主細胞が、 請求の範囲 1 2項〜 1 5項に記載のいずれかの宿主細胞で ある請求の範囲 1 6項〜 2 0項のいずれかに記載のプレニルアルコールの製造方法。  25. The method for producing prenyl alcohol according to any one of claims 16 to 20, wherein the host cell is any one of the host cells according to claims 12 to 15.
2 6 . 前記プレ-ルアルコールが炭素数 15以上のものである請求の範囲 1 6項〜 2 0項のいずれかに記載のプレニルアルコールの製造方法。 26. The method for producing prenyl alcohol according to any one of claims 16 to 20, wherein the prenyl alcohol has 15 or more carbon atoms.
2 7 . 前記プレニルアルコールがフアルネソ—ル、 ゲラニルゲラ二オール、 ゲラニ ルファノレネシソ一ノレ、 へキサプレニルアルコール、 ヘプタプレニルアルコール、 ォクタプレニノレアノレコール、 ノナブレニルアルコーノレ、 デカプレニルァノレコール、 ゥンデカプレニルアルコール又はドデカプレニルアルコールの 1種又は 2種以上 である請求の範囲 2 6項に記載のプレ-ルアルコールの製造方法。  27. The prenyl alcohol may be fuarnesol, geranylgeranol, gerani ruphanolenesicole, hexaprenyl alcohol, heptaprenyl alcohol, octapreninolenolecole, nonabrenyl alcoholonole, decaprenilanolecole, phenol. 27. The method for producing a phenol alcohol according to claim 26, which is one or more of decaprenyl alcohol and dodecaprenyl alcohol.
2 8 . 前記プレニルアルコールが全トランス型である請求の範囲 1 6項〜 2 0項の いずれかに記載のプレニルアルコールの製造方法。 28. The method for producing prenyl alcohol according to any one of claims 16 to 20, wherein the prenyl alcohol is of an all-trans type.
2 9 . 前記プレニルアルコールが全トランス型である請求の範囲 2 6項に記載のプ レニルアルコールの製造方法。 29. The process according to claim 26, wherein the prenyl alcohol is of an all-trans type. Method for producing renyl alcohol.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150191746A1 (en) * 2008-09-15 2015-07-09 Danisco Us Inc. Conversion of prenyl derivatives to isoprene
CN114934094A (en) * 2022-06-27 2022-08-23 南京大学 Short-chain isopentenyl transferase activity detection method

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53127889A (en) * 1977-04-08 1978-11-08 Kuraray Co Ltd Preparation of all-trans polyprenyl alcohol
JPS56151492A (en) * 1980-04-23 1981-11-24 Nisshin Flour Milling Co Ltd Production of all-trans polyprenyl alcohol
WO1998020138A1 (en) * 1996-11-05 1998-05-14 Toyota Jidosha Kabushiki Kaisha Mutated prenyl diphosphate synthases
WO2000005385A1 (en) * 1998-07-24 2000-02-03 Calgene Llc Plant phosphatidic acid phosphatases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53127889A (en) * 1977-04-08 1978-11-08 Kuraray Co Ltd Preparation of all-trans polyprenyl alcohol
JPS56151492A (en) * 1980-04-23 1981-11-24 Nisshin Flour Milling Co Ltd Production of all-trans polyprenyl alcohol
WO1998020138A1 (en) * 1996-11-05 1998-05-14 Toyota Jidosha Kabushiki Kaisha Mutated prenyl diphosphate synthases
WO2000005385A1 (en) * 1998-07-24 2000-02-03 Calgene Llc Plant phosphatidic acid phosphatases

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXANDER FAULKNER ET AL.: "The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in saccharomyces cerevisiae", J. BIOL. CHEM., vol. 274, no. 21, 1999, pages 14831 - 14837, XP002908896 *
DANIELA BARILA ET AL.: "The Dri42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein", J. BIOL. CHEM., vol. 271, no. 47, 1996, pages 29928 - 29936, XP002908895 *
DAVID A. TOKE ET AL.: "Isolation and characterization of saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase", J. BIOL. CHEM., vol. 273, no. 6, 1998, pages 3278 - 3284, XP002908892 *
DAVID A. TOKE ET AL.: "Isolation and characterization of saccharomyces cerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase", J. BIOL. CHEM., vol. 273, no. 23, 1998, pages 14331 - 14338, XP002908893 *
DAVID N. BRINDLEY ET AL.: "Minireview: mammalian lipid phosphate phosphohydrolases", J. BIOL. CHEM., vol. 273, no. 38, 1998, pages 24281 - 24284, XP002908894 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150191746A1 (en) * 2008-09-15 2015-07-09 Danisco Us Inc. Conversion of prenyl derivatives to isoprene
CN114934094A (en) * 2022-06-27 2022-08-23 南京大学 Short-chain isopentenyl transferase activity detection method

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