WO2003000902A1 - Groupe d'aphidicoline-biosynthases - Google Patents

Groupe d'aphidicoline-biosynthases Download PDF

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Publication number
WO2003000902A1
WO2003000902A1 PCT/JP2002/004381 JP0204381W WO03000902A1 WO 2003000902 A1 WO2003000902 A1 WO 2003000902A1 JP 0204381 W JP0204381 W JP 0204381W WO 03000902 A1 WO03000902 A1 WO 03000902A1
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Prior art keywords
seq
dna
amino acid
acid sequence
protein
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PCT/JP2002/004381
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English (en)
Japanese (ja)
Inventor
Hideaki Oikawa
Takeshi Sassa
Tomonobu Toyomasu
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Japan Science And Technology Agency
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Priority to JP2003507285A priority Critical patent/JPWO2003000902A1/ja
Publication of WO2003000902A1 publication Critical patent/WO2003000902A1/fr

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    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01029—Geranylgeranyl diphosphate synthase (2.5.1.29)

Definitions

  • the present invention relates to a gene encoding each enzyme necessary for the biosynthesis of DNA polymerase specific inhibitor aphidicolin, a protein encoded by the gene, and the like.
  • Aphidicolin (aphidicoline) is a specific inhibitor of DNA polymerase ⁇ , which plays a major role in eukaryotic replication. Because the inhibition is reversible, it is an essential drug in the field of cell physiology, which is concerned with the cell cycle, for example, in studies of synchronized culture systems. However, since this compound is produced only by special filamentous fungi and can only be produced by companies that own the strain, it is currently supplied as a more expensive reagent than certain reagent companies. There are five or more other filamentous fungi that produce the compound.However, the increase in production cannot be relied on empirical breeding. Absent.
  • a group of compounds called diterpenes such as aphidicolin, whose complex carbon skeleton is made from geranylgeranilurinic acid (GGDP) by a cyclizing enzyme.
  • GGDP geranylgeranilurinic acid
  • the present invention relates to a gene encoding the following protein (a) or (b):
  • Amino acid sequence A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a), and having an aphidicolan-16) 3-ol synthase activity.
  • the present invention further provides, in a second aspect, a DNA containing the following DNA (a) or (b): Pertaining to the gene:
  • the present invention further relates, as a fourth aspect, to a gene comprising the DNA of the following (a) or (b):
  • the present invention further relates, as a sixth aspect, to a gene containing DNA of the following (a) or (b):
  • VANaSil SH ⁇ I3 ⁇ 43a9a ⁇ IA3 ⁇ 4iiaAnnHSlIiaVHSSViiI3 ⁇ 43 ⁇ I3VH33H0HAlSaVaXI viOd3 ⁇ 4ovNa ⁇ iaa ⁇ iv3 ⁇ 4vA3 ⁇ 4iiiad & aJVA33b (mssab TM vai ⁇ ip3 ⁇ 4viHX3Adit) X3a smd3 Aiiaiiiaid
  • the present invention further relates, as a tenth aspect, to a gene comprising the DNA of the following (a) or (b):
  • the present invention also relates to an aphidicolin biosynthesis gene cluster containing two or more DNAs and / or genes selected from the group consisting of any of the above-mentioned DNAs and genes.
  • the present invention further relates to afaidicolan-16j3-ol synthase, geranylgeranyl diphosphate synthase, cytochrome P450 hydroxylase (P450-1), transporter protein (TP ) And cytochrome P450 hydroxylase (P450-2).
  • P450-1 cytochrome P450 hydroxylase
  • TP transporter protein
  • P450-2 cytochrome P450 hydroxylase
  • the affidicolan- 16] 3-ol synthase product deduced from the gene of the present invention is a protein consisting of 944 amino acids shown in SEQ ID NO: 1 (estimated molecular weight 1065580). .
  • the term "affidicolan-16 / 3-ol synthase” includes proteins having aphidikolan-16 ⁇ -ol synthase activity.
  • the term “activity” refers to an activity capable of substantially cyclizing a substrate, geranylgeraernilic acid, in an enzyme reaction system shown below, that is, an activity from the substrate in such a reaction system. It refers to an activity capable of synthesizing a significant amount of aphidicolan- 16 / 3-ol.
  • the aphidikolan-16 / 3-ol synthase of the present invention is divided into two domains having different functions from the N-terminal side and the C-terminal side, and the former is DX DD and the latter is DE XXE as a substrate recognition site.
  • the former is DX DD and the latter is DE XXE as a substrate recognition site.
  • the former shows the cyclization to the intermediate sya-coparillinylate, which is not released from geranylgeranilurinic acid, and the latter shows the cyclization of affidicolan- 16-ol (aphidicolan- It is expected to catalyze the cyclization to 16 j8-o 1).
  • deletion or substitution of one or several amino acids in the gene encoding the enzyme having the amino acid sequence shown in SEQ ID NO: 1 of the present invention may cause cyclization activity using geranylgeraniline diphosphate as a substrate.
  • it occurs at no more than 40 amino acid residues from the N-terminal and / or C-terminal of the amino acid sequence.
  • the reaction system for measuring the cyclization activity of the enzyme of the present invention using geranylgeranyl diphosphate as a substrate or the synthesis activity of aphidicolan-16-ol (aphidicolan-l6 ⁇ -ol) is, for example, as follows. Can be done with
  • partially purified enzyme (985 ⁇ l, enzyme concentration 10-1000 ng / iQl), 1 M magnesium chloride (5 / xl, final concentration 5 mM), geranylgeranyl diphosphate (10 / zl, lmg / mK, manufactured by Sigma) 30
  • the reaction can be performed at ° C for 12 hours. Thereafter, 1 ml of hexane is added to the reaction solution, and after extraction, the solvent is distilled off under a stream of nitrogen. Hexane 1001 is added to this and dissolved, and 11 is subjected to GC-MS analysis.
  • the GC-MS analysis is carried out using a Thermo Quest GCQ instrument and a calibration ram DB-1 (J & W Scientific, 0.25 mm ID, 30 m length) under the following conditions.
  • Splitless mode initial temperature 100 ° C (holding time 2 minutes), temperature rise setting 5 ° C / min, final temperature 280 (holding time 2 minutes), helium gas flow lm1 / min.
  • Mass measurement conditions scan 1 second / scan, source temperature 200, transfer line temperature 275 ° C, full scan mode 40—450.
  • Enzyme reaction product retention time 26 minutes 30 seconds.
  • geranylgeranyl diphosphate synthase includes a protein having a geranylgeranyl diphosphate synthase activity
  • geranilgeranyl diphosphate synthase activity refers to the substrate dimethylaryl It means an activity capable of synthesizing a significant amount of geranylgeranyl diphosphate from diphosphate or farnesyl diphosphate and isopentenyl diphosphate.
  • cytochrome P450 hydroxylase (P450-1)” and “cytochrome P450 hydroxylase (P450-2)” are cytochrome P450 hydroxylases.
  • the term "cytochrome P450 hydroxylase activity" which includes a protein having an activity, refers to an activity capable of introducing a hydroxyl group at the 3-, 17-, and 18-positions herein.
  • transport overnight protein () includes a protein having a transporter (TP) activity
  • transporter (TP) activity refers to the term It means an activity that can be excreted outside the body.
  • the gene or DNA encoding Affidicolan- 16) 3-ol synthase was cloned by a method such as RT-PCR using filamentous fungal mRNA as a template, and its nucleotide sequence was determined.
  • filamentous fungi include Phoma betae, Cephalosporium aphidicola (Vertici 11 ium 1 ecanii), Nigrospora sphaer ica (Nigrospora oryza e), Herziella en tomophi 11a and the like.
  • the filamentous fungus Phoma betae is a researcher of Nippon Sugar Beet Sugar Co., Ltd. (Registration number: PH-27) and American Type Culture Co., Ltd. (ATCC) (Registration number: 6550). 4, 246 3 5).
  • a database is prepared based on the nucleotide sequence obtained as a result of chromosome walking based on the sequence of the thus obtained afidicolin-l6-ol synthase (affidicolin biosynthetic intermediate synthase (ACS) gene).
  • ACS affidicolin biosynthetic intermediate synthase
  • a homology search was performed for the GGD P synthase gene upstream of the ACS gene and for the hydroxylase (P450-1), resistance (transport Yuichi), and hydroxylase (P450-2) downstream. We succeeded in finding each gene.
  • a specific gene or DNA can be hybridized in a buffer well known to those skilled in the art under stringent conditions under various conditions such as appropriate temperature and salt concentration.
  • DNA that hybridizes with the gene or DNA of the present invention under such stringent conditions and encodes a protein having each enzyme activity of the present invention include, for example, 70% homology with the gene. As described above, DNA having preferably 90% or more can be mentioned.
  • the gene, DNA, or afadicolin biosynthesis gene cluster of the present invention may contain various sequences known to those skilled in the art of gene recombination, such as regulatory elements such as promoters and enhancers, restriction enzyme sites, and selection markers. (Marker-enzyme, etc.)
  • regulatory elements such as promoters and enhancers, restriction enzyme sites, and selection markers.
  • selection markers Marker-enzyme, etc.
  • a DNA molecule to which a gene or the like is appropriately linked is also within the scope of the present invention.
  • an expression vehicle such as the gene, DNA, aphidicolin biosynthesis gene cluster of the present invention or a recombinant vector containing the above DNA molecule; Transformants such as cells can be easily prepared by means known in the art.
  • the enzymes and affidicolin of the present invention can be easily produced by culturing the above-mentioned expression vehicle, transformant, and the like, and then performing operations such as recovery and purification using known means in the art. be able to.
  • cDNA of the aphidicolin biosynthesis intermediate synthase gene was expressed in Escherichia coli as a fusion protein with daryuthion-1S-transferase, and the actual biosynthesis intermediate, aphidikolan-1 6 j3-ol (aphidi colan- 16/3 -ol).
  • a degenerate primer (1: 5'-GCITA (T / C) GA (T / C) ACIGCITGGGT-3 '(SEQ ID NO: 3); 2: 5 '-(A / G) AAIGCCATIGCIGT (A / G) TC (A / G) -TC-3' (SEQ ID NO: 4)) and mRNA extracted from the fungus! 01 ⁇ iae RT-PCR was performed using as a template. The full-length cDNA was analyzed using the 3′-RACE and 5′-RACE methods based on the amplified DNA fragment of about 1.1 kb.
  • Transformants were pre-cultured in a LB medium (2.5 ml) supplemented with 0.1% glucose and Ampicillin (final concentration 100 g / ml) at 200 rpm for 30 hours at 30 ° C. went. This was transferred to a 2 YT medium (500 ml) supplemented with Ampici 11 in (final concentration 100 g / m 1), and cultured at 30 ° C. for 3 hours at 200 rpm with stirring. After the culture solution was left on ice for 30 minutes, IPTG was added to a final concentration of 0.5 mM, and culturing was performed at 20 ° C. for 20 hours at 180 ° C. with stirring.
  • Genomic DNA extracted from the cells of the aphidicolin-producing bacterium W / o / Ba6ae was digested with eight types of restriction enzymes, and then a commercially available kit adapter (GTAATACGACTCACTATAGG GCACGCGTGGTCGACGGCCCGGGCTGGT) was ligated.
  • a commercially available kit adapter GTAATACGACTCACTATAGG GCACGCGTGGTCGACGGCCCGGGCTGGT
  • primer 1 5 'side 1.CCAACAGGGAATGTAGCG CAGAGGTTGCGA / 2. GTCCAGCGATCGTGTTCAGAACTCCGTCG, 3) based on the nucleotide sequence of the aphidikolan-16-ol synthase gene obtained in 'Side 1.
  • CAAGCTATCGCAGTC AGCTCTACCGTTGGG / 2.GTCGCATGTACAACGACATCGGCTCGTGG) was prepared, and PCR was performed under the same conditions as in Example 1 with an adapter primer (1. GTAATACGACTCACTATAGGGC / 2. ACTATAGGGCACGCGTGGT).
  • the upstream and downstream sequences of the ACS gene were analyzed by repeating this procedure, and a sequence of a gene cluster consisting of 15 kb in total length was obtained. Then the analyzed distribution The following primers were designed from the column, and using the iRNA of the same bacterium as a template, the full length of each cDNA was obtained using the 3'-RACE and 5'-RACE methods under the same conditions as in Example 1. Was analyzed. As a result, the sequences of GGDP synthase (343aa), P450-1 (486aa), transporter (564aa), and P450-2 (541aa) were clarified.
  • genes encoding all the enzymes required for aphidicolin biosynthesis have been found.Thus, by expressing these biosynthetic genes by heterologous microorganisms with established culture methods, mass production of afadicolin is possible. become. In addition, the present invention opens the door to the large supply of this useful agent.

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  • Chemical & Material Sciences (AREA)
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Abstract

Cette invention a pour but de trouver des gènes nécessaires à la production de l'aphidicoline, qui est un agent inhibiteur spécifique de l'ADN-polymérase α jouant un rôle important dans la réplication des eucaryotes, et d'exprimer ces biosynthases dans un micro-organisme étranger, dont le procédé de culture a été mis au point pour permettre la production d'aphidicoline en grande quantité. Cette invention concerne des gènes codant une synthase GGDP, une cyclase et des enzymes catalysant l'oxydation des trois phases qui sont nécessaires à la biosynthèse d'une aphidicoline inhibitrice spécifique de l'ADN-polymérase α; un gène de la tolérance contre la toxicité de l'aphidicoline; des protéines codées par ces gènes et l'aphidicoline; et des procédés de production de ceux-ci.
PCT/JP2002/004381 2001-06-21 2002-05-02 Groupe d'aphidicoline-biosynthases WO2003000902A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58220690A (ja) * 1982-06-14 1983-12-22 Asahi Chem Ind Co Ltd アフイデイコリンおよび／またはその誘導体の製造法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58220690A (ja) * 1982-06-14 1983-12-22 Asahi Chem Ind Co Ltd アフイデイコリンおよび／またはその誘導体の製造法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Alessandra CARATTOLIT et al., "The Neurospora crassa Carotenoid Biosynthetic Gene (Albino 3) Reveals Highly Conserved Regions among Prenyltransferases" The Journal of Biological Chemistry. 1991, Vol. 266, No. 9, pages 5854 to 5859 *
Hideaki OIKAWA et al., "Biosynthesis of Diterpenoid Aphidicolin: Isolation of Intermediates from P-450 Inhibitor Treated Mycelia of Phoma betae" Tetrahedron. 1999, Vol. 55, No. 24, pages 7541 to 7554 *
Hideaki OIKAWA et al., "Cloning and Functional Expression of cDNA Encoding Aphidicolan-16 Beta-ol Synthase: A Key Enzyme Responsible for Formation of an Unusual Dieterpene Skeleton in Biosynthesis of Aphidicolin" J. Am. Chem. Soc. 2001, Vol. 123, No. 21, pages 5154 to 5155 *
Hideaki OIKAWA et al., "Diversity of diterpene hydrocarbons in fungus Phoma betae" Tetrahedron Letters. March 2001, Vol. 42, No. 12, pages 2329 to 2332 *
Mark J. ACKLAND et al., "Studies in Terpenoid Biosynthesis. Part 35. Biosynthetic Sequences leading to the Diterpenoid Aphidicolin in Cephalosporium aphidicola" J. Chem. Soc. Perkin Trans. I. 1988, No. 6, pages 1477 to 1480 *
Tomonobu TOYOMASA et al., "Cloning of a Full-length cDNA Encoding ent-Kaurene Synthase from Gibberella fujikuroi: Functional Analysis of a Bifunctional Diterpene Cyclase" Biosci. Biotechnol. Biochem. 2000, Vol. 64, No. 3, pages 660 to 664 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US9909130B2 (en) 2005-03-18 2018-03-06 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi
US9297031B2 (en) 2006-09-28 2016-03-29 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi

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