US20190376093A1 - Method for producing trans-polyisoprenoid, vector, transgenic plant, method for producing pneumatic tire and method for producing rubber product - Google Patents

Method for producing trans-polyisoprenoid, vector, transgenic plant, method for producing pneumatic tire and method for producing rubber product Download PDF

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US20190376093A1
US20190376093A1 US16/466,193 US201716466193A US2019376093A1 US 20190376093 A1 US20190376093 A1 US 20190376093A1 US 201716466193 A US201716466193 A US 201716466193A US 2019376093 A1 US2019376093 A1 US 2019376093A1
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trans
producing
tpt
polyisoprenoid
rubber
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Yuko Sakurai
Haruhiko Yamaguchi
Yukino Inoue
Kazuhisa Fushihara
Seiji Takahashi
Satoshi Yamashita
Toru Nakayama
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Tohoku University NUC
Sumitomo Rubber Industries Ltd
Kanazawa University NUC
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Tohoku University NUC
Sumitomo Rubber Industries Ltd
Kanazawa University NUC
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Assigned to KANAZAWA UNIVERSITY, TOHOKU UNIVERSITY, SUMITOMO RUBBER INDUSTRIES, LTD. reassignment KANAZAWA UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUSHIHARA, KAZUHISA, INOUE, YUKINO, SAKURAI, YUKO, YAMAGUCHI, HARUHIKO, NAKAYAMA, TORU, TAKAHASHI, SEIJI, YAMASHITA, SATOSHI
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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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60C—VEHICLE TYRES; TYRE INFLATION; TYRE CHANGING; CONNECTING VALVES TO INFLATABLE ELASTIC BODIES IN GENERAL; DEVICES OR ARRANGEMENTS RELATED TO TYRES
    • B60C1/00—Tyres characterised by the chemical composition or the physical arrangement or mixture of the composition
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29D—PRODUCING PARTICULAR ARTICLES FROM PLASTICS OR FROM SUBSTANCES IN A PLASTIC STATE
    • B29D30/00—Producing pneumatic or solid tyres or parts thereof
    • B29D30/06—Pneumatic tyres or parts thereof (e.g. produced by casting, moulding, compression moulding, injection moulding, centrifugal casting)
    • B29D30/0601—Vulcanising tyres; Vulcanising presses for tyres

Definitions

  • the present invention relates to a method for producing a trans-polyisoprenoid, a vector, a transgenic plant, a method for producing a pneumatic tire, and a method for producing a rubber product.
  • natural rubber for use in industrial rubber products is produced by cultivating rubber-producing plants, such as para rubber tree ( Hevea brasiliensis ) of the family Euphorbiaceae or Indian rubber tree ( Ficus elastica ) of the family Moraceae.
  • rubber-producing plants such as para rubber tree ( Hevea brasiliensis ) of the family Euphorbiaceae or Indian rubber tree ( Ficus elastica ) of the family Moraceae.
  • Such natural rubber is a polyisoprenoid (cis-natural rubber) in which isoprene units are linked in a cis configuration.
  • trans-polyisoprenoids trans rubber
  • Trans rubber can be extracted from the seeds or pericarp tissue of Eucommia ulmoides .
  • Trans rubber can also be chemically synthesized.
  • Such trans rubber has different characteristics from cis-natural rubber and has been used in crack-resistant golf balls or dental materials used to fill cavities in teeth.
  • the trans-polyisoprenoid extracted and purified from Eucommia ulmoides is a polyisoprenoid having a weight average molecular weight of about 1.8 ⁇ 10 6 in which at least 99% of the units of the straight chain are trans-linked, and has been used as eucommia lastomer.
  • Eucommia ulmoides which is used as a healthy food or herbal medicine, is industrially used to extract and purify a trans-polyisoprenoid, this may potentially compete with use as a food material.
  • the chemically synthesized trans-polyisoprenoids do not have a trans content of 100% but contain about 1.2 to 4% of cis bonds. They also have a molecular weight of about 250,000, and it is very difficult to synthesize a trans-polyisoprenoid having an ultra-high molecular weight of 1,000,000 or higher. Furthermore, their chemical synthesis requires a supply of raw materials, including petroleum-derived materials, which is hardly an eco-friendly (environmentally friendly) procurement process.
  • Trans rubber has a trans-1,4-polyisoprene structure that is biosynthesized by addition polymerization of isopentenyl diphosphate (IPP) with a starting substrate such as dimethylallyl diphosphate (DMAPP) or farnesyl diphosphate (FPP), and the nature of this structure suggests that a trans-prenyltransferase (tPT) may be involved in trans rubber biosynthesis.
  • IPP isopentenyl diphosphate
  • DMAPP dimethylallyl diphosphate
  • FPP farnesyl diphosphate
  • Patent Literature 1 describes that trans-1,4-polyisoprene can be efficiently produced by transforming a plant with an expression vector containing a gene coding for a long-chain trans-prenyl diphosphate synthase (trans-prenyltranspherase) to produce a plant containing an increased amount of trans-1,4-polyisoprene, and cultivating the plant.
  • trans-prenyltranspherase trans-prenyl diphosphate synthase
  • trans-polyisoprenoids trans rubber
  • one possible approach to solving these problems is to stabilize and enhance the activity of tPT in trans rubber biosynthesis in order to increase trans rubber production.
  • the present invention aims to solve the problems and provide a method for producing a trans-polyisoprenoid which can increase trans rubber production in vitro.
  • the present invention also aims to solve the above problems and provide a vector that can be introduced into a plant using genetic transformation techniques to enhance trans-polyisoprenoid production. Further objects are to provide a transgenic plant into which the vector has been introduced and to provide a method for enhancing production of a trans-isoprenoid or trans-polyisoprenoid in a plant by introducing the vector into the plant.
  • the present invention relates to a method for producing a trans-polyisoprenoid, the method including binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro.
  • This invention is hereinafter called the first aspect of the present invention and also referred to as the first invention.
  • the trans-prenyltransferase (tPT) family protein contains, at positions 183 to 187 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions,
  • X 1 and X 2 are the same as or different from each other and each represent any amino acid residue, or the following amino acid sequence (A2):
  • X 1 , X 2 , X 3 , and X 4 are the same as or different from each other and each represent any amino acid residue
  • trans-prenyltransferase (tPT) family protein contains, at positions 310 to 314 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions,
  • X 11 and X 12 are the same as or different from each other and each represent any amino acid residue.
  • the gene coding for a trans-prenyltransferase (tPT) family protein is derived from a plant.
  • the gene coding for a trans-prenyltransferase (tPT) family protein is derived from a rubber-producing plant.
  • the gene coding for a trans-prenyltransferase (tPT) family protein is derived from Hevea brasiliensis.
  • the binding includes performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a trans-prenyltransferase (tPT) family protein to bind the tPT family protein to the rubber particles.
  • tPT trans-prenyltransferase
  • the cell-free protein synthesis solution contains a germ extract.
  • the germ extract is derived from wheat.
  • the rubber particles are present in the cell-free protein synthesis solution at a concentration of 5 to 50 g/L.
  • the first invention is also directed to a method for producing a pneumatic tire, the method including: kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention with an additive to obtain a kneaded mixture; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • the first invention is also directed to a method for producing a rubber product, the method including: kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention with an additive to obtain a kneaded mixture; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • the present invention also relates to a vector, including: a promoter having a promoter activity that drives laticifer-specific gene expression; and a gene coding for a trans-prenyltransferase (tPT) family protein functionally linked to the promoter.
  • a promoter having a promoter activity that drives laticifer-specific gene expression
  • tPT trans-prenyltransferase family protein functionally linked to the promoter.
  • This invention is hereinafter called the second aspect of the present invention and also referred to as the second invention.
  • the promoter having a promoter activity that drives laticifer-specific gene expression is at least one selected from the group consisting of a promoter of a gene coding for rubber elongation factor (REF), a promoter of a gene coding for small rubber particle protein (SRPP), a promoter of a gene coding for Hevein 2.1 (HEV2.1), and a promoter of a gene coding for MYC1 transcription factor (MYC1).
  • REF rubber elongation factor
  • SRPP small rubber particle protein
  • HEV2.1 Hevein 2.1
  • MYC1 transcription factor MYC1 transcription factor
  • the second invention is also directed to a transgenic plant into which any one of the above-described vectors has been introduced.
  • the second invention is also directed to a method for enhancing trans-isoprenoid production in a plant by introducing any one of the above-described vectors into the plant.
  • the second invention is also directed to a method for enhancing trans-polyisoprenoid production in a plant by introducing any one of the above-described vectors into the plant.
  • the second invention is also directed to a method for producing a pneumatic tire, the method including: kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing any one of the above-described vectors into a plant; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • the second invention is also directed to a method for producing a rubber product, the method including: kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing any one of the above-described vectors into a plant; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • the method for producing a trans-polyisoprenoid of the first invention includes binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro.
  • tPT trans-prenyltransferase
  • the method for producing a pneumatic tire of the first invention includes kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention with an additive to obtain a kneaded mixture; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • This method which produces a pneumatic tire from a trans-polyisoprenoid obtained by a highly efficient polyisoprenoid production method, it is possible to use plant resources effectively to produce environmentally friendly pneumatic tires.
  • the method for producing a rubber product of the first invention includes kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention with an additive to obtain a kneaded mixture; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • the vector of the second invention includes a promoter having a promoter activity that drives laticifer-specific gene expression and a gene coding for a trans-prenyltransferase (tPT) family protein functionally linked to the promoter.
  • tPT trans-prenyltransferase
  • the method for producing a pneumatic tire of the second invention includes kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector of the second invention into a plant; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • the method for producing a rubber product of the second invention includes kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector of the second invention into a plant; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • FIG. 1 is a presumptive diagram illustrating rubber synthesis by tPT on a rubber particle.
  • FIG. 2 is a schematic diagram illustrating part of a trans-polyisoprenoid biosynthesis pathway.
  • FIG. 3 is an outline diagram illustrating the dialysis process in Example.
  • FIG. 4 illustrates a graph of the measured molecular weight distributions of the very long chain polyisoprenoids synthesized in Example 1 and Comparative Example 2.
  • FIG. 5 is an outline diagram illustrating the results of multiple sequence alignment of tPT family proteins derived from various organisms.
  • the first invention and the second invention are also referred to collectively as the present invention.
  • the first invention will be described first, and the second invention will be described later.
  • the method for producing a trans-polyisoprenoid of the first invention includes binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro.
  • tPT trans-prenyltransferase
  • FIG. 1 schematically illustrates an exemplary synthesis of trans rubber within a rubber particle by polymerization of an isopentenyl diphosphate (IPP) substrate by a tPT family protein depicted as tPT.
  • IPP isopentenyl diphosphate
  • trans-polyisoprenoid trans rubber
  • the production method of the first invention may include any other step as long as it involves the above binding step, and each step may be performed once or repeated multiple times.
  • the amount of the tPT family protein to be bound to the rubber particles is not particularly limited in the first invention.
  • binding a tPT family protein to rubber particles means that, for example, the tPT family protein is fully or partially incorporated into the rubber particles or inserted into the membrane structure of the rubber particles. It is not limited to these embodiments and also includes embodiments in which, for example, the tPT family protein is localized on the surface or inside of the rubber particles. Moreover, the concept of binding to rubber particles also includes embodiments in which the tPT family protein forms a complex with another protein bound to the rubber particles to exist in the form of the complex on the rubber particles.
  • CPT cis-prenyltranspherase
  • tPT family proteins are not present on rubber particles in vivo, particularly in rubber-producing plants capable of producing cis-natural rubber, a person skilled in the art has no motivation to bind a tPT family protein to rubber particles. If a skilled person were to consider biding a tPT family protein to rubber particles, the skilled person, who knows the above fact, could not predict at all that the binding of a tPT family protein to rubber particles would lead to rubber synthesis.
  • the origin of the rubber particles is not particularly limited.
  • the rubber particles may be derived from the latex of a rubber-producing plant such as Hevea brasiliensis, Taraxacum kok - saghyz, Parthenium argentatum, Sonchus oleraceus , or Ficus elastica.
  • the particle size of the rubber particles is also not particularly limited. Rubber particles having a predetermined particle size may be sorted out and used, or a mixture of rubber particles having different particle sizes may be used. When rubber particles having a predetermined particle size are sorted out and used, the rubber particles may be either small rubber particles (SRP) having a small particle size or large rubber particles (LRP) having a large particle size.
  • SRP small rubber particles
  • LRP large rubber particles
  • centrifugation In order to sort out the rubber particles having a predetermined particle size, commonly used methods may be used, including, for example, methods which involve centrifugation, preferably multistage centrifugation.
  • a specific method includes centrifugation at 500-1500 ⁇ g, centrifugation at 1700-2500 ⁇ g, centrifugation at 7000-9000 ⁇ g, centrifugation at 15000-25000 ⁇ g, and centrifugation at 40000-60000 ⁇ g, carried out in that order.
  • the duration of each centrifugation treatment is preferably 20 minutes or longer, more preferably 30 minutes or longer, still more preferably 40 minutes or longer, but is preferably 120 minutes or shorter, more preferably 90 minutes or shorter.
  • the temperature for each centrifugation treatment is preferably 0 to 10° C., more preferably 2 to 8° C., particularly preferably 4° C.
  • a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein is bound to rubber particles in vitro.
  • the origin of the gene coding for a trans-prenyltransferase (tPT) family protein is not particularly limited.
  • the gene may be derived from a microorganism, an animal, or a plant, preferably a plant, more preferably a rubber-producing plant, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium .
  • it is further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the plant is not particularly limited, and examples include Hevea species such as Hevea brasiliensis; Sonchus species such as Sonchus oleraceus, Sonchus asper , and Sonchus brachyotus; Solidago species such as Solidago altissima, Solidago virgaurea subsp. asiatica, Solidago virgaurea subsp. leipcarpa, Solidago virgaurea subsp. leipcarpa f. paludosa, Solidago virgaurea subsp. gigantea , and Solidago gigantea Ait. var.
  • Hevea species such as Hevea brasiliensis
  • Sonchus species such as Sonchus oleraceus, Sonchus asper , and Sonchus brachyotus
  • Solidago species such as Solidago altissima, Solidago virgaurea subsp. asiatica, Solidago virgaurea subsp. lei
  • Ficus species such as Ficus carica, Ficus elastica, Ficus pumila L., Ficus erecta Thumb., Ficus ampelas Burm. f., Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarp
  • Ficus benghalensis Parthenium species such as Parthenium argentatum, Parthenium hysterophorus , and Ambrosia artemisiifolia ( Parthenium hysterophorus ); lettuce ( Lactuca sativa ); Ficus benghalensis; Arabidopsis thaliana ; and Eucommia ulmoides.
  • trans-prenyltransferase (tPT) family protein refers to an enzyme that catalyzes a reaction of trans-chain elongation of an isoprenoid compound.
  • trans-polyisoprenoids are biosynthesized via trans-polyisoprenoid biosynthesis pathways as shown in FIG. 2 , in which tPT family proteins are considered to be enzymes that catalyze the reactions enclosed by the dotted frame in FIG. 2 .
  • the tPT family proteins are characterized by having an amino acid sequence contained in the trans-IPPS HT domain (NCBI Accession No. cd00685).
  • isoprenoid compound refers to a compound containing an isoprene unit (C 5 H 8 ).
  • trans-isoprenoid refers to a compound including an isoprenoid compound in which isoprene units are trans-linked (in particular, the content of trans bonds is preferably at least 90%, more preferably at least 95%, still more preferably at least 97% of the total bonds), and examples include trans-polyisoprenoids (trans rubber) such as farnesyl diphosphate, geranylgeranyl diphosphate, hexaprenyl diphosphate, heptaprenyl diphosphate, and trans-1,4-polyisoprene.
  • FIG. 5 is an outline diagram illustrating the results of multiple sequence alignment of tPT family proteins derived from various organisms.
  • literature such as Andrew H.-J. Wang et al., Eur. J. Biochem. 269, pp. 3339-3354 (2002)
  • box A corresponding to positions 183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2
  • box B corresponding to positions 310 to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 in FIG. 5 are parts of highly conserved regions of tPT family proteins derived from various organisms.
  • the term “conserved region” refers to a site having a similar sequence (structure) which is presumed to have a similar protein function.
  • SEQ ID NO:2 an amino acid sequence at positions corresponding to positions 183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2
  • amino acid sequence at positions corresponding to positions 310 to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 are conserved as specific motifs, and proteins having these motifs at the respective positions have the functions of tPT family proteins.
  • the multiple sequence alignment can be carried out as described later in EXAMPLES.
  • the trans-prenyltransferase (tPT) family protein preferably contains, at positions 183 to 187 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions, the following amino acid sequence (A1):
  • X 1 and X 2 are the same as or different from each other and each represent any amino acid residue, or the following amino acid sequence (A2):
  • X 1 , X 2 , X 3 , and X 4 are the same as or different from each other and each represent any amino acid residue
  • trans-prenyltransferase (tPT) family protein contains, at positions 310 to 314 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions, the following amino acid sequence (B):
  • X 11 and X 12 are the same as or different from each other and each represent any amino acid residue.
  • the tPT family protein having such a sequence is considered to have the functions of tPT family proteins, including the function as an enzyme that catalyzes a reaction of trans-chain elongation of an isoprenoid compound. By binding this tPT family protein to rubber particles, it is possible to synthesize trans rubber in the rubber particles.
  • the tPT family protein preferably contains, at positions 183 to 187 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions, the following amino acid sequence (A1):
  • X 1 and X 2 are the same as or different from each other and each represent any amino acid residue, or the following amino acid sequence (A2):
  • X 1 , X 2 , X 3 , and X 4 are the same as or different from each other and each represent any amino acid residue. More preferably, in the amino acid sequences (A1) and (A2), X 1 denotes M, I, or V, and X 2 denotes M, I, or L.
  • the tPT family protein contains, at positions 310 to 314 in the amino acid sequence of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding positions, the following amino acid sequence (B):
  • X 11 and X 12 are the same as or different from each other and each represent any amino acid residue. More preferably, in the amino acid sequence (B), X 11 denotes Y, M, I, or V, and X 12 denotes L.
  • the conserved region corresponding to positions 183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 corresponds for example to:
  • the conserved region corresponding to positions 310 to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO: 2 corresponds for example to:
  • tPT family protein examples include: tPT derived from yeast, such as FPPS (Erg20p [ Saccharomyces cerevisiae R103]) and TPT (Coq1p [ Saccharomyces cerevisiae YJM1342]); tPT derived from Eucommia ulmoides , such as EuFPPS (farnesyl pyrophosphate synthetase [ Eucommia ulmoides ]); tPT derived from Hevea brasiliensis , such as HbFPPS (farnesyl diphosphate synthase [ Hevea brasiliensis ]) and HbSDS (solanesyl diphosphate synthase [ Hevea brasiliensis ]); tPT derived from Arabidopsis thaliana , such as AtSDS1 (solanesyl diphosphate synthase 1 [ Arabidopsis thaliana ]); tPT derived from human, such as
  • trans rubber can be synthesized in rubber particles by binding any tPT family protein, regardless of the origin, type, and other factors of the protein, to the rubber particles.
  • trans rubber can be synthesized in rubber particles by using any tPT family protein, for example, regardless of whether the gene coding for the tPT family protein is derived from a rubber-producing plant or any other organism or whether it is naturally involved in rubber synthesis.
  • the tPT family protein used in the present invention desirably has a transmembrane domain on the N-terminal side to have a higher affinity for rubber particles.
  • a transmembrane domain may be artificially fused to the N-terminal side of the tPT family protein.
  • the transmembrane domain to be fused may have any amino acid sequence, desirably an amino acid sequence of the transmembrane domain of a protein inherently bound to rubber particles in nature.
  • tPT family protein examples include the following protein [1]:
  • proteins having one or more amino acid substitutions, deletions, insertions, or additions relative to the original amino acid sequence can have the inherent function.
  • tPT family protein is the following protein [2]:
  • the protein preferably has an amino acid sequence containing one or more, more preferably 1 to 83, still more preferably 1 to 62, further preferably 1 to 41, particularly preferably 1 to 20, most preferably 1 to 8, yet most preferably 1 to 4 amino acid substitutions, deletions, insertions, and/or additions relative to the amino acid sequence represented by SEQ ID NO:2.
  • conservative substitutions are preferred. Specific examples include substitutions within each of the following groups in the parentheses: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine), and (phenylalanine, tyrosine).
  • tPT family protein is the following protein [3]:
  • sequence identity to the amino acid sequence represented by SEQ ID NO:2 is preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, particularly preferably at least 98%, most preferably at least 99%.
  • sequence identity between amino acid sequences or nucleotide sequences may be determined using the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] developed by Karlin and Altschul or FASTA [Methods Enzymol., 183, 63 (1990)].
  • Whether it is a protein having the above enzyme activity or not may be determined by conventional techniques, such as by expressing a target protein in a transformant produced by introducing a gene coding for the target protein into Escherichia coli or other host organisms, and analyzing the presence or absence of the function of the target protein by the corresponding activity measuring method.
  • the gene coding for the tPT family protein is not particularly limited as long as it codes for the tPT family protein to express and produce the tPT family protein.
  • Specific examples of the gene include the following DNAs [1] and [2]:
  • hybridize means a process in which a DNA hybridizes to a DNA having a specific nucleotide sequence or a part of the DNA.
  • the DNA having a specific nucleotide sequence or part of the DNA may have a nucleotide sequence long enough to be usable as a probe in Northern or Southern blot analysis or as an oligonucleotide primer in polymerase chain reaction (PCR) analysis.
  • the DNA to be used as a probe may have a length of at least 100 bases, preferably at least 200 bases, more preferably at least 500 bases although it may be a DNA of at least 10 bases, preferably at least 15 bases.
  • the stringent conditions may include, for example, an overnight incubation at 42° C. of a DNA-immobilized filter and a DNA probe in a solution containing 50% formamide, 5 ⁇ SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/L denatured salmon sperm DNA, followed by washing the filter for example in a 0.2 ⁇ SSC solution at approximately 65° C. Less stringent conditions may also be used. Changes in stringency may be accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lower stringency), salt concentrations or temperature.
  • low stringent conditions include an overnight incubation at 37° C. in a solution containing 6 ⁇ SSCE (20 ⁇ SSCE: 3 mol/L sodium chloride, 0.2 mol/L sodium dihydrogen phosphate, 0.02 mol/L EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 ⁇ g/L denatured salmon sperm DNA, followed by washing in a 1 ⁇ SSC solution containing 0.1% SDS at 50° C.
  • washes performed following hybridization may be done at higher salt concentrations (e.g. 5 ⁇ SSC) in the above-mentioned low stringent conditions.
  • Variations in the above conditions may be accomplished through the inclusion or substitution of blocking reagents used to suppress background in hybridization experiments.
  • the inclusion of blocking reagents may require modification of the hybridization conditions for compatibility.
  • the DNA capable of hybridization under stringent conditions as described above may have a nucleotide sequence with at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, particularly preferably at least 99% sequence identity to the nucleotide sequence represented by SEQ ID NO:1 as calculated using a program such as BLAST or FASTA with the parameters mentioned above.
  • DNA which hybridizes to the aforementioned DNA under stringent conditions is a DNA coding for a protein having a predetermined enzyme activity or not may be determined by conventional techniques, such as by expressing a target protein in a transformant produced by introducing a gene coding for the target protein into Escherichia coli or other host organisms, and analyzing the presence or absence of the function of the target protein by the corresponding activity measuring method.
  • the RACE method (rapid amplification of cDNA ends method) refers to a method in which, when the nucleotide sequence of a cDNA is partially known, PCR is performed based on the nucleotide sequence data of such a known region to clone an unknown region extending to the cDNA terminal. This method is capable of cloning full-length cDNA by PCR without preparing a cDNA library.
  • the degenerate primers may each preferably be prepared from a plant-derived sequence having a highly similar sequence part to the target protein.
  • the full-length nucleotide sequence or amino acid sequence can be identified by designing a primer containing a start codon and a primer containing a stop codon using the known nucleotide sequence, followed by performing RT-PCR using a synthesized cDNA as a template.
  • additional proteins may further be bound to the rubber particles as long as the protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein is bound to the rubber particles in vitro.
  • tPT trans-prenyltransferase
  • the origin of the additional proteins is not particularly limited, but preferably the additional proteins are derived from any of the plants mentioned above, more preferably rubber-producing plants, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium . In particular, they are further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the additional proteins are not limited and may each be any protein, but in view of rubber synthesis activity of the rubber particles, they are each preferably a protein that inherently exists on rubber particles in a rubber-producing plant.
  • the protein that exists on rubber particles may be a protein bound to a large part of the membrane surface of rubber particles, or a protein inserted into and bound to the membrane of rubber particles, or a protein that forms a complex with another protein bound to the membrane to exist on the membrane surface.
  • Examples of the protein that inherently exists on rubber particles in a rubber-producing plant include Nogo-B receptor (NgBR), rubber elongation factor (REF), small rubber particle protein (SRPP), ⁇ -1,3-glucanase, and Hevein.
  • NgBR Nogo-B receptor
  • REF rubber elongation factor
  • SRPP small rubber particle protein
  • ⁇ -1,3-glucanase ⁇ -1,3-glucanase
  • Hevein Hevein.
  • the binding step may be carried out by any method that can bind a tPT family protein to rubber particles in vitro, such as, for example, by performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a tPT family protein to bind the tPT family protein to the rubber particles.
  • the binding step preferably includes performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a tPT family protein to bind the tPT family protein to the rubber particles, among other methods.
  • rubber particles bound to a tPT family protein by performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for the tPT family protein (more specifically, using a mixture of rubber particles with a cell-free protein synthesis solution containing an mRNA coding for the tPT family protein).
  • liposomes are artificially produced as lipid bilayer membranes formed of phospholipids, glyceroglycolipids, cholesterol, or other components, no protein is bound to the surface of the produced liposomes.
  • rubber particles collected from the latex of rubber-producing plants are also coated with a lipid membrane, the membrane of the rubber particles is a naturally derived membrane in which proteins that have been synthesized in the plants are already bound to the surface of the membrane. In view of this, it is expected to be more difficult to bind an additional protein to rubber particles that are already bound to and coated with proteins than to bind it to liposomes not bound to any protein. There is also concern that the proteins already bound to rubber particles could inhibit cell-free protein synthesis.
  • the protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a tPT family protein is namely the synthesis of a tPT family protein by cell-free protein synthesis, and the synthesized tPT family protein maintains its biological function (native state).
  • the synthesized tPT family protein in its native state can be bound to the rubber particles.
  • each tPT family protein synthesized by the protein synthesis is fully or partially incorporated into the rubber particles or inserted into the membrane structure of the rubber particles. It is not limited to these embodiments and also includes, for example, embodiments in which the protein is localized on the surface or inside of the rubber particles. Moreover, the concept of binding to rubber particles also includes embodiments in which the protein forms a complex with another protein bound to the rubber particles as described above to exist in the form of the complex on the rubber particles.
  • Each mRNA coding for a tPT family protein serves as a translation template that can be translated to synthesize the tPT family protein.
  • the origin of the mRNA coding for a tPT family protein is not particularly limited, and the mRNA may be derived from a microorganism, an animal, or a plant, preferably a plant, more preferably any of the plants mentioned above, still more preferably a rubber-producing plant, further preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium .
  • it is especially preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , most preferably from Hevea brasiliensis.
  • the mRNA coding for a tPT family protein may be prepared by any method as long as the prepared mRNA serves as a translation template that can be translated to synthesize the tPT family protein.
  • the mRNA may be prepared by extracting total RNA from the latex of a rubber-producing plant by, for example, the hot phenol method, synthesizing cDNA from the total RNA, obtaining a DNA fragment of a gene coding for a tPT family protein using primers prepared based on the nucleotide sequence data of the gene coding for a tPT family protein, and performing an ordinary in vitro transcription reaction of the DNA fragment.
  • the cell-free protein synthesis solution contains the mRNA coding for a tPT family protein, it may contain mRNAs coding for additional proteins.
  • the mRNAs coding for additional proteins may be ones that can be translated to express the additional proteins.
  • the additional proteins may be as described above.
  • cell-free synthesis of a tPT family protein is preferably performed in the presence of rubber particles.
  • the cell-free protein synthesis may be carried out using the cell-free protein synthesis solution in a similar manner to conventional methods.
  • the cell-free protein synthesis system used may be a common cell-free protein synthesis means, such as rapid translation system RTS500 (Roche Diagnostics); or wheat germ extracts prepared in accordance with Proc. Natl. Acad. Sci. USA, 97:559-564 (2000), JP 2000-236896 A, JP 2002-125693 A, and JP 2002-204689 A, or cell-free protein synthesis systems using the wheat germ extracts (JP 2002-204689 A, Proc. Natl. Acad. Sci. USA, 99:14652-14657 (2002)). Systems using germ extracts are preferred among these.
  • the cell-free protein synthesis solution contains a germ extract.
  • the source of the germ extract is not particularly limited. From the standpoint of translation efficiency, it is preferred to use a plant-derived germ extract for cell-free protein synthesis of a plant protein. It is particularly preferred to use a wheat-derived germ extract. Thus, in another suitable embodiment of the first invention, the germ extract is derived from wheat.
  • the method for preparing the germ extract is not particularly limited, and may be carried out conventionally, as described in, for example, JP 2005-218357 A.
  • the cell-free protein synthesis solution preferably further contains a cyclic nucleoside monophosphate derivative or a salt thereof (hereinafter, also referred to simply as “activity enhancer”). Protein synthesis activity can be further enhanced by the inclusion of the activity enhancer.
  • the cyclic nucleoside monophosphate derivative or salt thereof is not particularly limited as long as it can enhance cell-free protein synthesis activity.
  • Examples include adenosine-3′,5′-cyclic monophosphoric acid and its salts; adenosine-3′,5′-cyclic monophosphorothioic acid (Sp-isomer) and its salts; adenosine-3′,5′-cyclic monophosphorothioic acid (Rp-isomer) and its salts; guanosine-3′,5′-cyclic monophosphoric acid and its salts; guanosine-3′,5′-cyclic monophosphorothioic acid (Sp-isomer) and its salts; guanosine-3′,5′-cyclic monophosphorothioic acid (Rp-isomer) and its salts; 8-bromoadenosine-3′,5′-cyclic monophosphoric acid (bromo-cAMP) and its salts
  • the base that forms a salt with the cyclic nucleoside monophosphate derivative is not particularly limited as long as it is biochemically acceptable and forms a salt with the derivative.
  • Preferred are, for example, alkali metal atoms such as sodium or potassium, and organic bases such as tris-hydroxyaminomethane, among others.
  • adenosine-3′,5′-cyclic monophosphoric acid or adenosine-3′,5′-cyclic monophosphate sodium salt is particularly preferred.
  • These activity enhancers may be used alone or in combinations of two or more.
  • the activity enhancer may be added to the cell-free protein synthesis solution in advance. If the activity enhancer is unstable in the solution, it is preferably added during the protein synthesis reaction performed in the presence of both the cell-free protein synthesis solution and rubber particles.
  • the amount of the activity enhancer added is not particularly limited as long as the activity enhancer is at a concentration that can activate (increase) the protein synthesis reaction in the cell-free protein synthesis solution.
  • the final concentration in the reaction system may usually be at least 0.1 millimoles/liter.
  • the lower limit of the concentration is preferably 0.2 millimoles/liter, more preferably 0.4 millimoles/liter, particularly preferably 0.8 millimoles/liter, while the upper limit of the concentration is preferably 24 millimoles/liter, more preferably 6.4 millimoles/liter, particularly preferably 3.2 millimoles/liter.
  • the temperature of the cell-free protein synthesis solution to which the activity enhancer is added is not particularly limited, but is preferably 0 to 30° C., more preferably 10 to 26° C.
  • the cell-free protein synthesis solution also contains ATP, GTP, creatine phosphate, creatine kinase, L-amino acids, potassium ions, magnesium ions, and other components required for protein synthesis, and optionally an activity enhancer.
  • ATP ATP
  • GTP GTP
  • creatine phosphate creatine phosphate
  • creatine kinase L-amino acids
  • potassium ions potassium ions
  • magnesium ions and other components required for protein synthesis
  • an activity enhancer can serve as a cell-free protein synthesis reaction system.
  • the germ extract prepared as described in JP 2005-218357 A contains tRNA in an amount necessary for protein synthesis reaction, addition of separately prepared tRNA is not required when the germ extract prepared as above is used in the cell-free protein synthesis solution. In other words, tRNA may be added to the cell-free protein synthesis solution, if necessary.
  • the binding step in the first invention preferably includes performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a tPT family protein. Specifically, this can be accomplished by adding rubber particles to the cell-free protein synthesis solution at a suitable point either before or after protein synthesis, preferably before protein synthesis.
  • the rubber particles are preferably present in the cell-free protein synthesis solution at a concentration of 5 to 50 g/L. In other words, 5 to 50 g of rubber particles are preferably present in 1 L of the cell-free protein synthesis solution. If the concentration of rubber particles present in the cell-free protein synthesis solution is less than 5 g/L, a rubber layer may not be formed by separation treatment (e.g., ultracentrifugation) for collecting the rubber particles bound to the synthesized tPT family protein, and therefore it may be difficult to collect the rubber particles bound to the synthesized tPT family protein.
  • separation treatment e.g., ultracentrifugation
  • the concentration of rubber particles present in the cell-free protein synthesis solution exceeds 50 g/L, the rubber particles may coagulate, so that the synthesized tPT family protein may fail to bind well to the rubber particles.
  • the concentration of rubber particles is more preferably 10 to 40 g/L, still more preferably 15 to 35 g/L, particularly preferably 15 to 30 g/L.
  • additional rubber particles may be appropriately added as the reaction progresses.
  • the cell-free protein synthesis solution and rubber particles are preferably present together during the period when the cell-free protein synthesis system is active, such as 3 to 48 hours, preferably 3 to 30 hours, more preferably 3 to 24 hours after the addition of rubber particles to the cell-free protein synthesis solution.
  • the rubber particles do not have to be subjected to any treatment, e.g., pretreatment, before use in the binding step in the first invention, preferably before being combined with the cell-free protein synthesis solution.
  • proteins may be removed from the rubber particles with a surfactant beforehand to increase the proportion of the tPT family protein desired to be bound by the method of the first invention, among the proteins present on the rubber particles.
  • the rubber particles used in the first invention are washed with a surfactant before use in the binding step in the first invention, preferably before being combined with the cell-free protein synthesis solution.
  • the surfactant is not particularly limited, and examples include nonionic surfactants and amphoteric surfactants.
  • Nonionic or amphoteric surfactants are suitable because they have only a little denaturing effect on the proteins on the membrane, and amphoteric surfactants are especially suitable.
  • the surfactant is an amphoteric surfactant.
  • surfactants may be used alone or in combinations of two or more.
  • nonionic surfactants examples include polyoxyalkylene ether nonionic surfactants, polyoxyalkylene ester nonionic surfactants, polyhydric alcohol fatty acid ester nonionic surfactants, sugar fatty acid ester nonionic surfactants, alkyl polyglycoside nonionic surfactants, and polyoxyalkylene polyglucoside nonionic surfactants; and polyoxyalkylene alkylamines and alkyl alkanolamides.
  • Polyoxyalkylene ether or polyhydric alcohol fatty acid ester nonionic surfactants are preferred among these.
  • polyoxyalkylene ether nonionic surfactants examples include polyoxyalkylene alkyl ethers, polyoxyalkylene alkylphenyl ethers, polyoxyalkylene polyol alkyl ethers, and polyoxyalkylene mono-, di- or tristyryl phenyl ethers. Among these, polyoxyalkylene alkylphenyl ethers are suitable.
  • the “polyol” is preferably a C2-C12 polyhydric alcohol, such as ethylene glycol, propylene glycol, glycerin, sorbitol, glucose, sucrose, pentaerythritol, or sorbitan.
  • polyoxyalkylene ester nonionic surfactants examples include polyoxyalkylene fatty acid esters and polyoxyalkylene alkyl rosin acid esters.
  • polyhydric alcohol fatty acid ester nonionic surfactants include fatty acid esters of C2-C12 polyhydric alcohols and fatty acid esters of polyoxyalkylene polyhydric alcohols. More specific examples include sorbitol fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, and pentaerythritol fatty acid esters, as well as polyalkylene oxide adducts of the foregoing such as polyoxyalkylene sorbitan fatty acid esters and polyoxyalkylene glycerin fatty acid esters. Among these, sorbitan fatty acid esters are suitable.
  • sugar fatty acid ester nonionic surfactants include fatty acid esters of sucrose, glucose, maltose, fructose, and polysaccharides, as well as polyalkylene oxide adducts of the foregoing.
  • alkyl polyglycoside nonionic surfactants include those having, for example, glucose, maltose, fructose, or sucrose as the glycoside, such as alkyl glucosides, alkyl polyglucosides, polyoxyalkylene alkyl glucosides, and polyoxyalkylene alkyl polyglucosides, as well as fatty acid esters of the foregoing.
  • Polyalkylene oxide adducts of any of the foregoing may also be used.
  • alkyl groups in these nonionic surfactants include C4-C30 linear or branched, saturated or unsaturated alkyl groups.
  • the polyoxyalkylene groups may have C2-C4 alkylene groups, and may have about 1 to 50 moles of added ethylene oxide, for example.
  • fatty acids include C4-C30 linear or branched, saturated or unsaturated fatty acids.
  • nonionic surfactants polyoxyethyleneethylene (10) octylphenyl ether (Triton X-100) or sorbitan monolaurate (Span 20) is particularly preferred for their ability to moderately remove membrane-associated proteins while keeping the membrane of rubber particles stable and, further, having only a little denaturing effect on the proteins.
  • amphoteric surfactants examples include zwitterionic surfactants such as quaternary ammonium salt group/sulfonate group (—SO 3 H) surfactants, water-soluble quaternary ammonium salt group/phosphate group surfactants, water-insoluble quaternary ammonium salt group/phosphate group surfactants, and quaternary ammonium salt group/carboxyl group surfactants.
  • the acid group in each of these zwitterionic surfactants may be a salt.
  • such a zwitterionic surfactant preferably has both positive and negative charges in a molecule.
  • the acid dissociation constant (pKa) of the acid group is preferably 5 or less, more preferably 4 or less, still more preferably 3 or less.
  • amphoteric surfactants include ammonium sulfobetaines such as 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate (CHAPSO), 3-[(3-cholamidopropyl)-dimethylamino]-propanesulfonate (CHAPS), N,N-bis(3-D-gluconamidopropyl)-cholamide, n-octadecyl-N,N′-dimethyl-3-amino-1-propanesulfonate, n-decyl-N,N′-dimethyl-3-amino-1-propanesulfonate, n-dodecyl-N,N′-dimethyl-3-amino-1-propanesulfonate, n-tetradecyl-N,N′-dimethyl-3-amino-1-propanesulfonate (ZwittergentTM
  • the concentration of the surfactant for the treatment is preferably within three times the critical micelle concentration (CMC) of the surfactant used.
  • CMC critical micelle concentration
  • the membrane stability of the rubber particles may be reduced if they are treated with the surfactant at a concentration exceeding three times the critical micelle concentration.
  • the concentration is more preferably within 2.5 times, still more preferably within 2.0 times the CMC.
  • the lower limit of the concentration is preferably at least 0.05 times, more preferably at least 0.1 times, still more preferably at least 0.3 times the CMC.
  • Examples of protein synthesis protein synthesis reaction systems or apparatuses for protein synthesis that can be used in the cell-free protein synthesis include a batch method (Pratt, J. M. et al., Transcription and Translation, Hames, 179-209, B. D. & Higgins, S. J., eds, IRL Press, Oxford (1984)), a continuous cell-free protein synthesis system in which amino acids, energy sources, and other components are supplied continuously to the reaction system (Spirin, A. S.
  • Another method may be to supply template RNA, amino acids, energy sources, and other components, if necessary, to the protein synthesis reaction system, and discharge the synthesis product or decomposition product as required.
  • the dialysis method is preferred.
  • the reason for this is as follows.
  • the overlay method has the advantage of easy operation, but unfortunately rubber particles disperse in the reaction solution and thus are difficult to efficiently bind to the synthesized tPT family protein.
  • the dialysis method since the amino acids used as raw materials of the tPT family protein to be synthesized can pass through the dialysis membrane while rubber particles cannot pass therethrough, it is possible to prevent dispersal of rubber particles and thus to efficiently bind the synthesized tPT family protein to rubber particles.
  • the dialysis method refers to a method in which protein synthesis is carried out using the reaction solution for the cell-free protein synthesis as an internal dialysis solution, and an apparatus in which the internal dialysis solution is separated from an external dialysis solution by a dialysis membrane capable of mass transfer.
  • a translation template is added to the synthesis reaction solution excluding the translation template, optionally after pre-incubation for an appropriate amount of time, and then the solution is put in an appropriate dialysis container as the internal reaction solution.
  • dialysis container examples include containers with a dialysis membrane attached to the bottom (e.g., Dialysis Cup 12,000 available from Daiichi Kagaku) and dialysis tubes (e.g., 12,000 available from Sanko Junyaku Co., Ltd.).
  • the dialysis membrane used may have a molecular weight cutoff of 10,000 daltons or more, preferably about 12,000 daltons.
  • the external dialysis solution used may be a buffer containing amino acids. Dialysis efficiency can be increased by replacing the external dialysis solution with a fresh one when the reaction speed declines.
  • the reaction temperature and time are selected appropriately according to the protein synthesis system used. For example, in the case of a system using a wheat-derived germ extract, the reaction may be carried out usually at 10 to 40° C., preferably 18 to 30° C., more preferably 20 to 26° C., for 10 minutes to 48 hours, preferably for 10 minutes to 30 hours, more preferably for 10 minutes to 24 hours.
  • the mRNA coding for a tPT family protein contained in the cell-free protein synthesis solution is easily broken down, the mRNA may be additionally added as appropriate during the protein synthesis reaction to make the protein synthesis more efficient.
  • the mRNA coding for a tPT family protein is additionally added during the protein synthesis reaction.
  • the addition time, the number of additions, the addition amount, and other conditions of the mRNA are not particularly limited, and may be selected appropriately.
  • the step of collecting the rubber particles may optionally be performed after the step of binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro.
  • tPT trans-prenyltransferase
  • the rubber particle collection step may be carried out by any method that can collect the rubber particles. It may be carried out by conventional methods for collecting rubber particles. Specific examples include methods using centrifugation. When the rubber particles are collected by the centrifugation methods, the centrifugal force, centrifugation time, and centrifugation temperature may be selected appropriately so as to be able to collect the rubber particles. For example, the centrifugal force during the centrifugation is preferably 15000 ⁇ g or more, more preferably 20000 ⁇ g or more, still more preferably 25000 ⁇ g or more.
  • the upper limit of the centrifugal force is preferably 50000 ⁇ g or less, more preferably 45000 ⁇ g or less.
  • the centrifugation time is preferably at least 20 minutes, more preferably at least 30 minutes, still more preferably at least 40 minutes.
  • the upper limit of the centrifugation time is preferably 120 minutes or less, more preferably 90 minutes or less.
  • the centrifugation temperature is preferably 0 to 10° C., more preferably 2 to 8° C., particularly preferably 4° C.
  • the rubber particles and the cell-free protein synthesis solution are separated into the upper and lower layers, respectively, by the centrifugation.
  • the cell-free protein synthesis solution as the lower layer may then be removed to collect the rubber particles bound to the tPT family protein.
  • the collected rubber particles may be re-suspended in an appropriate buffer with a neutral pH for storage.
  • the rubber particles collected by the rubber particle collection step can be used in the same way as usual natural rubber without the need for further special treatment.
  • trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention can be recovered by subjecting the rubber particles to the solidification step described below.
  • the method for solidification in the solidification step is not particularly limited, and examples include a method of adding the rubber particles to a solvent that does not dissolve the trans-polyisoprenoid (trans rubber), such as ethanol, methanol, or acetone; and a method of adding an acid to the rubber particles.
  • a solvent that does not dissolve the trans-polyisoprenoid such as ethanol, methanol, or acetone
  • Rubber can be recovered as solids from the rubber particles by the solidification step. The obtained rubber may be dried if necessary before use.
  • trans rubber by binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro, trans rubber can be synthesized in the rubber particles, and therefore it is possible to efficiently produce trans rubber (one example of trans-polyisoprenoid) in a reaction vessel (e.g., a test tube or industrial plant).
  • a reaction vessel e.g., a test tube or industrial plant.
  • another aspect of the first invention relates to a method for synthesizing a trans-polyisoprenoid, which includes binding a protein expressed from a gene coding for a trans-prenyltransferase (tPT) family protein to rubber particles in vitro, for example in a reaction vessel (e.g., a test tube or industrial plant).
  • a reaction vessel e.g., a test tube or industrial plant.
  • tPT trans-prenyltransferase
  • trans-polyisoprenoid is a collective term for polymers containing trans-linked isoprene units (C 5 H 8 ) (in particular, the content of trans bonds is preferably at least 90%, more preferably at least 95%, still more preferably at least 97% of the total bonds).
  • Examples of the trans-polyisoprenoid include trans-sesterterpenes (C 25 ), trans-triterpenes (C 30 ), trans-tetraterpenes (C 40 ), trans rubber such as trans-1,4-polyisoprene, and other polymers.
  • isoprenoid refers to a compound containing an isoprene unit (C 5 H 8 ), and conceptually includes polyisoprenoids.
  • the method for producing a rubber product of the first invention includes: kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid of the first invention with an additive to obtain a kneaded mixture; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • the rubber product is not particularly limited as long as it is a rubber product that can be produced from rubber, preferably natural rubber, and examples include pneumatic tires, rubber rollers, rubber fenders, gloves, and medical rubber tubes.
  • the raw rubber product forming step corresponds to the step of building a green tire from the kneaded mixture
  • the vulcanization step corresponds to the step of vulcanizing the green tire.
  • the method for producing a pneumatic tire of the first invention includes: kneading a trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid with an additive to obtain a kneaded mixture; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • the trans-polyisoprenoid produced by the method for producing a trans-polyisoprenoid is kneaded with an additive to obtain a kneaded mixture.
  • the additive is not particularly limited, and additives used in production of rubber products may be used.
  • examples of the additive include rubber components other than the trans-polyisoprenoid, reinforcing fillers such as carbon black, silica, calcium carbonate, alumina, clay, and talc, silane coupling agents, zinc oxide, stearic acid, processing aids, various antioxidants, softeners such as oils, waxes, vulcanizing agents such as sulfur, and vulcanization accelerators.
  • a rubber kneading machine such as an open roll mill, a Banbury mixer, or an internal mixer may be used to perform kneading.
  • a raw rubber product (green tire in the case of tire) is formed from the kneaded mixture obtained in the kneading step.
  • the method for forming a raw rubber product is not particularly limited. Methods used to form raw rubber products may be used appropriately.
  • the kneaded mixture obtained in the kneading step may be extruded according to the shape of a tire component and then formed in a usual manner on a tire building machine and assembled with other tire components to build a green tire (unvulcanized tire).
  • the raw rubber product obtained in the raw rubber product forming step is vulcanized to obtain a rubber product.
  • the method for vulcanizing the raw rubber product is not particularly limited. Methods used to vulcanize raw rubber products may be used appropriately.
  • the green tire (unvulcanized tire) obtained in the raw rubber product forming step may be vulcanized by heating and pressing in a vulcanizer to obtain a pneumatic tire.
  • the vector of the second invention contains a nucleotide sequence in which a gene coding for a trans-prenyltransferase (tPT) family protein is functionally linked to a promoter having a promoter activity that drives laticifer-specific gene expression.
  • tPT trans-prenyltransferase
  • the gene coding for a protein involved in trans-polyisoprenoid biosynthesis in the vector can be expressed specifically in laticifers, thereby enhancing trans-isoprenoid or trans-polyisoprenoid production in the plant. This is probably because, if the expression of an exogenous gene introduced for the purpose of enhancing latex productivity is promoted in sites other than laticifers, a certain load is imposed on the metabolism or latex production of the plant, thereby causing adverse effects.
  • promoter having a promoter activity that drives laticifer-specific gene expression means that the promoter has activity to control gene expression to cause a desired gene to be expressed specifically in laticifers when the desired gene is functionally linked to the promoter and introduced into a plant.
  • the term “laticifer-specific gene expression” means that the gene is expressed substantially exclusively in laticifers with no or little expression of the gene in sites other than laticifers in plants.
  • a gene is functionally linked to a promoter means that the gene sequence is linked downstream of the promoter so that the gene is controlled by the promoter.
  • the vector of the second invention can be prepared by inserting the nucleotide sequence of a promoter having a promoter activity that drives laticifer-specific gene expression and the nucleotide sequence of a gene coding for a trans-prenyltransferase (tPT) family protein into a vector commonly known as a plant transformation vector by conventional techniques.
  • vectors that can be used to prepare the vector of the second invention include pBI vectors, binary vectors such as pGA482, pGAH, and pBIG, intermediate plasmids such as pLGV23Neo, pNCAT, and pMON200, and pH35GS containing GATEWAY cassette.
  • the vector of the second invention contains the nucleotide sequence of a promoter having a promoter activity that drives laticifer-specific gene expression and the nucleotide sequence of a gene coding for a trans-prenyltransferase (tPT) family protein, it may contain additional nucleotide sequences.
  • the vector contains vector-derived sequences in addition to these nucleotide sequences and further contains a restriction enzyme recognition sequence, a spacer sequence, a marker gene sequence, a reporter gene sequence, or other sequences.
  • the marker gene examples include drug-resistant genes such as kanamycin-resistant gene, hygromycin-resistant gene, and bleomycin-resistant gene.
  • the reporter gene is intended to be introduced to determine the expression site in a plant, and examples include luciferase gene, ⁇ -glucuronidase (GUS) gene, green fluorescent protein (GFP), and red fluorescent protein (RFP).
  • GUS ⁇ -glucuronidase
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the origin of the gene coding for a trans-prenyltransferase (tPT) family protein is not particularly limited.
  • the gene may be derived from a microorganism, an animal, or a plant, preferably a plant, more preferably any of the plants mentioned above, still more preferably a rubber-producing plant, further preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium .
  • it is especially preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , most preferably from Hevea brasiliensis.
  • trans-prenyltransferase (tPT) family protein The gene coding for a trans-prenyltransferase (tPT) family protein and the tPT family protein in the second invention are as described above concerning the first invention.
  • the vector of the second invention contains the nucleotide sequence of a promoter having a promoter activity that drives laticifer-specific gene expression and the nucleotide sequence of a gene coding for a trans-prenyltransferase (tPT) family protein, it may further contain the nucleotide sequences of genes coding for additional proteins.
  • tPT trans-prenyltransferase
  • genes coding for additional proteins include those described above concerning the first invention.
  • the promoter having a promoter activity that drives laticifer-specific gene expression is preferably at least one selected from the group consisting of a promoter of a gene coding for rubber elongation factor (REF), a promoter of a gene coding for small rubber particle protein (SRPP), a promoter of a gene coding for Hevein 2.1 (HEV2.1), and a promoter of a gene coding for MYC1 transcription factor (MYC1).
  • REF rubber elongation factor
  • SRPP small rubber particle protein
  • HEV2.1 Hevein 2.1
  • MYC1 transcription factor MYC1 transcription factor
  • rubber elongation factor refers to a rubber particle-associated protein that is bound to rubber particles in the latex of rubber-producing plants such as Hevea brasiliensis , and contributes to stabilization of the rubber particles.
  • SRPP small rubber particle protein
  • Hevein 2.1 refers to a protein that is highly expressed in the laticifer cells of rubber-producing plants such as Hevea brasiliensis . This protein is involved in coagulation of rubber particles and has antifungal activity.
  • MYC1 transcription factor refers to a transcription factor that is highly expressed in the latex of rubber-producing plants such as Hevea brasiliensis and participates in jasmonic acid signaling.
  • transcription factor means a protein having activity to increase or decrease, preferably increase, gene transcription.
  • MYC1 herein is a protein having activity (transcription factor activity) to increase or decrease, preferably increase, the transcription of a gene coding for at least one protein among the proteins involved in jasmonic acid signaling.
  • the origin of the promoter of a gene coding for REF is not particularly limited, but the promoter is preferably derived from any of the plants mentioned above, more preferably a rubber-producing plant, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium . In particular, it is further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the promoter of a gene coding for REF is preferably any one of the following DNAs [A1] to [A3]:
  • hybridize is as described above. Also, the stringent conditions are as described above.
  • promoters with nucleotide sequences having certain sequence identities to the original nucleotide sequence can also have promoter activity.
  • sequence identity to the nucleotide sequence represented by SEQ ID NO:10 is at least 60%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, further preferably at least 98%, particularly preferably at least 99%.
  • the origin of the promoter of a gene coding for SRPP is not particularly limited, but the promoter is preferably derived from any of the plants mentioned above, more preferably a rubber-producing plant, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium . In particular, it is further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the promoter of a gene coding for SRPP is preferably any one of the following DNAs [B1] to [B3]:
  • hybridize is as described above. Also, the stringent conditions are as described above.
  • promoters with nucleotide sequences having certain sequence identities to the original nucleotide sequence can also have promoter activity.
  • sequence identity to the nucleotide sequence represented by SEQ ID NO: 11 is at least 60%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, further preferably at least 98%, particularly preferably at least 99%.
  • the origin of the promoter of a gene coding for HEV2.1 is not particularly limited, but the promoter is preferably derived from any of the plants mentioned above, more preferably a rubber-producing plant, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium . In particular, it is further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the promoter of a gene coding for HEV2.1 is preferably any one of the following DNAs [C1] to [C3]:
  • hybridize is as described above. Also, the stringent conditions are as described above.
  • promoters with nucleotide sequences having certain sequence identities to the original nucleotide sequence can also have promoter activity.
  • sequence identity to the nucleotide sequence represented by SEQ ID NO:12 is at least 60%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, further preferably at least 98%, particularly preferably at least 99%.
  • the origin of the promoter of a gene coding for MYC1 is not particularly limited, but the promoter is preferably derived from any of the plants mentioned above, more preferably a rubber-producing plant, still more preferably at least one selected from the group consisting of plants of the genera Hevea, Sonchus, Taraxacum , and Parthenium . In particular, it is further preferably derived from at least one species of plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the promoter of a gene coding for MYC1 is preferably any one of the following DNAs [D1] to [D3]:
  • hybridize is as described above. Also, the stringent conditions are as described above.
  • promoters with nucleotide sequences having certain sequence identities to the original nucleotide sequence can also have promoter activity.
  • sequence identity to the nucleotide sequence represented by SEQ ID NO: 13 is at least 60%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, further preferably at least 98%, particularly preferably at least 99%.
  • the DNA which hybridizes to the above-mentioned DNA under stringent conditions or the DNA having at least 60% sequence identity to the above-mentioned DNA is a DNA having a promoter activity that drives laticifer-specific gene expression or not may be determined by conventional techniques, such as reporter assays using ⁇ -galactosidase, luciferase, green fluorescent protein (GFP), and other protein genes as reporter genes.
  • reporter assays using ⁇ -galactosidase, luciferase, green fluorescent protein (GFP), and other protein genes as reporter genes.
  • a genomic DNA is extracted from a growing plant by the cetyl trimethyl ammonium bromide (CTAB) method, then specific and random primers are designed based on the known nucleotide sequence of the promoter, and a gene including the promoter is amplified by TAIL (thermal asymmetric interlaced)-PCR using the extracted genomic DNA as a template to identify the nucleotide sequence.
  • CAB cetyl trimethyl ammonium bromide
  • the vector of the second invention (which contains a nucleotide sequence in which a gene coding for a trans-prenyltransferase (tPT) family protein is functionally linked to a promoter having a promoter activity that drives laticifer-specific gene expression) can be introduced into a plant to produce a transgenic plant transformed to express a certain protein involved in trans-polyisoprenoid biosynthesis specifically in laticifers.
  • tPT trans-prenyltransferase
  • transgenic plant in which the certain protein involved in trans-polyisoprenoid biosynthesis is expressed specifically in the laticifers, a certain function, e.g., enzyme activity, of the protein newly expressed in the plant transfected with the vector of the second invention is enhanced in the laticifers to enhance a part of the trans-polyisoprenoid biosynthesis pathway. Therefore, trans-isoprenoid or trans-polyisoprenoid production can be enhanced in the plant.
  • a certain function e.g., enzyme activity
  • transgenic plant The method for preparing the transgenic plant is explained briefly below, though such a transgenic plant can be prepared by conventional methods.
  • the plant into which the vector of the second invention is to be introduced to produce the transgenic plant is not particularly limited, but is preferably a rubber-producing plant, among others, because improved trans-polyisoprenoid productivity and increased trans-polyisoprenoid yield can be expected particularly when a tPT family protein is expressed in plants capable of biosynthesizing polyisoprenoids.
  • it is further preferably derived from at least one species of rubber-producing plant selected from the group consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum , and Taraxacum kok - saghyz , particularly preferably from Hevea brasiliensis.
  • the vector of the second invention may be introduced into a plant (including plant cells, such as callus, cultured cells, spheroplasts, or protoplasts) by any method that can introduce DNA into plant cells.
  • a plant including plant cells, such as callus, cultured cells, spheroplasts, or protoplasts
  • methods include methods using Agrobacterium (JP S59-140885 A, JP S60-70080 A, WO94/00977), electroporation (JP S60-251887 A), and methods using particle guns (gene guns) (JP 2606856 B, JP 2517813 B).
  • Agrobacterium Agrobacterium method
  • the vector of the second invention may also be introduced into, for example, an organism (e.g., a microorganism, yeast, animal cell, or insect cell) or a part thereof, an organ, a tissue, a cultured cell, a spheroplast, or a protoplast by any of the above-described DNA introduction methods to produce a trans-isoprenoid or trans-polyisoprenoid.
  • an organism e.g., a microorganism, yeast, animal cell, or insect cell
  • an organ e.g., a tissue, a cultured cell, a spheroplast, or a protoplast.
  • the transgenic plant (transgenic plant cells) can be produced by the above or other methods.
  • the transgenic plant conceptually includes not only transgenic plant cells produced by the above methods, but also all of their progeny or clones and even progeny plants obtained by passaging the foregoing.
  • progeny or clones can be produced from the transgenic plant cells by sexual or asexual reproduction, tissue culture, cell culture, cell fusion, or other techniques.
  • the transgenic plant cells, or their progeny or clones may be used to obtain reproductive materials (e.g., seeds, fruits, cuttings, stem tubers, root tubers, shoots, adventitious buds, adventitious embryos, callus, protoplasts), which can then be used to produce the transgenic plant on a large scale.
  • reproductive materials e.g., seeds, fruits, cuttings, stem tubers, root tubers, shoots, adventitious buds, adventitious embryos, callus, protoplasts
  • Whether a target protein gene is expressed in regenerated plants or not may be determined by well-known methods. For example, Western blot analysis may be used to assess the expression of a target protein.
  • Seeds can be obtained from the transgenic plant, for example, as follows: the transgenic plant is rooted in an appropriate medium, transplanted to water-containing soil in a pot, and grown under proper cultivation conditions to finally produce seeds, which are then collected. Furthermore, plants can be grown from seeds, for example, as follows: seeds obtained from the transgenic plant as described above are sown in water-containing soil and grown under proper cultivation conditions into plants.
  • the gene coding for a protein involved in trans-polyisoprenoid biosynthesis (particularly preferably the gene coding for a tPT family protein) in the vector can be expressed specifically in laticifers, thereby enhancing trans-isoprenoid or trans-polyisoprenoid production in the plant.
  • a trans-isoprenoid or trans-polyisoprenoid may be produced by culturing, for example, transgenic plant cells produced as described above, callus obtained from the transgenic plant cells, or cells redifferentiated from the callus in an appropriate medium, or by growing, for example, transgenic plants regenerated from the transgenic plant cells, or plants grown from seeds obtained from these transgenic plants under proper cultivation conditions.
  • another aspect of the second invention relates to a method for enhancing trans-isoprenoid production in a plant by introducing the vector of the second invention into the plant. Furthermore, another aspect of the second invention relates to a method for enhancing trans-polyisoprenoid production in a plant by introducing the vector of the second invention into the plant.
  • the method for producing a rubber product of the second invention includes: kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector of the second invention into a plant; forming a raw rubber product from the kneaded mixture; and vulcanizing the raw rubber product.
  • the rubber product is as described above concerning the first invention.
  • the raw rubber product forming step corresponds to the step of building a green tire from the kneaded mixture
  • the vulcanization step corresponds to the step of vulcanizing the green tire
  • the method for producing a pneumatic tire of the second invention includes: kneading a trans-polyisoprenoid produced by a transgenic plant with an additive to obtain a kneaded mixture, the transgenic plant being produced by introducing the vector of the second invention into a plant; building a green tire from the kneaded mixture; and vulcanizing the green tire.
  • a trans-polyisoprenoid produced by a transgenic plant produced by introducing the vector of the second invention into a plant is kneaded with an additive to obtain a kneaded mixture.
  • the trans-polyisoprenoid produced by a transgenic plant produced by introducing the vector of the second invention into a plant can be obtained by harvesting latex from the transgenic plant, and subjecting the latex to the solidification step described below.
  • the method for harvesting latex from the transgenic plant is not particularly limited, and ordinary harvesting methods may be used.
  • latex may be harvested by collecting the emulsion oozing out from the cuts in the trunk of the plant (tapping), or the emulsion oozing out from the cut roots or other parts of the transgenic plant, or by crushing the cut tissue followed by extraction with an organic solvent.
  • the harvested latex is subjected to a solidification step.
  • the method for solidification is not particularly limited, and examples include a method of adding the latex to a solvent that does not dissolve the trans-polyisoprenoid (trans rubber), such as ethanol, methanol, or acetone; and a method of adding an acid to the latex. Rubber can be recovered as solids from the latex by the solidification step. The obtained rubber may be dried if necessary before use.
  • the additive is not particularly limited, and additives used in production of rubber products may be used.
  • examples of the additive include rubber components other than the rubber obtained from the latex, reinforcing fillers such as carbon black, silica, calcium carbonate, alumina, clay, and talc, silane coupling agents, zinc oxide, stearic acid, processing aids, various antioxidants, softeners such as oils, waxes, vulcanizing agents such as sulfur, and vulcanization accelerators.
  • a rubber kneading machine such as an open roll mill, a Banbury mixer, or an internal mixer may be used to perform kneading.
  • the raw rubber product forming step is as described above concerning the first invention.
  • the vulcanization step is as described above concerning the first invention.
  • Total RNA was extracted from the latex of Hevea brasiliensis by the hot phenol method.
  • To 6 mL of the latex were added 6 mL of 100 mM sodium acetate buffer and 1 mL of a 10% SDS solution, and then 12 mL of water-saturated phenol pre-heated at 65° C. The mixture was incubated for five minutes at 65° C., agitated in a vortex mixer, and centrifuged at 7000 rpm for 10 minutes at room temperature. After the centrifugation, the supernatant was transferred to a new tube, 12 mL of a phenol:chloroform (1:1) solution was added, and they were agitated by shaking for two minutes.
  • the resulting mixture was centrifuged again at 7000 rpm for 10 minutes at room temperature. Then, the supernatant was transferred to a new tube, 12 mL of a chloroform:isoamyl alcohol (24:1) solution was added, and they were agitated by shaking for two minutes. After the agitation, the resulting mixture was centrifuged again at 7000 rpm for 10 minutes at room temperature. Then, the supernatant was transferred to a new tube, 1.2 mL of a 3M sodium acetate solution and 13 mL of isopropanol were added, and they were agitated in a vortex mixer. The resulting mixture was incubated for 30 minutes at ⁇ 20° C.
  • cDNA was synthesized from the collected total RNA.
  • the cDNA synthesis was carried out using a PrimeScript II 1st strand cDNA synthesis kit (Takara) in accordance with the manual.
  • the prepared 1st strand cDNA was used as a template to obtain a tPT gene.
  • PCR was performed using a KOD-plus-Neo (Toyobo Co., Ltd.) in accordance with the manual.
  • the PCR reaction involved 35 cycles with each cycle consisting of 10 seconds at 98° C., 30 seconds at 58° C., and 1 minute at 68° C.
  • the tPT gene was obtained using the following primers.
  • Primer 1 5′-ctgtattttcagggcggatatgtttcttcgaccaaggcc-3′
  • Primer 2 5′-caaactagtgcggccgcgctaatcaatccgttcgagattg-3′
  • HbSDS A tPT gene (HbSDS) was prepared as described above. The sequence of the gene was isolated to identify the full-length nucleotide sequence.
  • the nucleotide sequence of HbSDS is given by SEQ ID NO: 1.
  • the amino acid sequence of HbSDS estimated from the nucleotide sequence is also given by SEQ ID NO:2.
  • the obtained DNA fragment was subjected to dA addition and then inserted into a pGEM-T Easy vector using a pGEM-T Easy Vector System (Promega) to prepare pGEM-HbSDS.
  • Escherichia coli DH5 ⁇ was transformed with the prepared vector, the transformant was cultured on LB agar medium containing ampicillin and X-gal, and Escherichia coli cells carrying the introduced target gene were selected by blue/white screening.
  • the Escherichia coli cells transformed with the plasmid containing the target gene were cultured overnight at 37° C. on LB liquid medium. After the culture, the cells were collected, and the plasmid was collected using a FastGene Plasmid mini kit (Nippon Genetics Co., Ltd.).
  • the pGEM-HbSDS acquired in the above [Vector construction] was treated with the restriction enzyme BstZ I, and then a HbSDS fragment was collected.
  • the fragment was mixed with a pEU-E01-His-TEV-MCS-N2 cell-free expression vector that had been treated with the restriction enzymes EcoR I and Kpn I, and they were connected by in vitro homologous recombination to prepare pEU-His-N2-HbSDS.
  • Escherichia coli DH5 ⁇ was transformed with the prepared vector, the transformant was cultured on LB agar medium containing ampicillin and X-gal, and Escherichia coli cells carrying the introduced target gene were screened by colony PCR.
  • the Escherichia coli cells transformed with the plasmid containing the target gene were cultured overnight at 37° C. on LB liquid medium. After the culture, the cells were collected, and the plasmid was collected using a FastGene Plasmid mini kit (Nippon Genetics Co., Ltd.).
  • Rubber particles were prepared from Hevea latex by five stages of centrifugation.
  • To 900 mL of Hevea latex was added 100 mL of 1 M Tris buffer (pH 7.5) containing 20 mM dithiothreitol (DTT) to prepare a latex solution.
  • the latex solution was centrifuged in stages at the following different speeds: 1000 ⁇ g, 2000 ⁇ g, 8000 ⁇ g, 20000 ⁇ g, and 50000 ⁇ g. Each stage of centrifugation was carried out for 45 minutes at 4° C.
  • Cell-free protein synthesis was performed using a WEPRO7240H expression kit (CellFree Sciences Co., Ltd.). An mRNA transcription reaction was performed using the vector acquired in the above [Preparation of vector for cell-free protein synthesis] as a template in accordance with the protocol of the WEPRO7240H expression kit.
  • the resulting mRNA was purified by ethanol precipitation.
  • FIG. 3 shows an outline diagram illustrating the dialysis process.
  • the solution in the dialysis cup was transferred to a new 1.5 ⁇ L tube, and the reacted rubber particles were collected by ultracentrifugation (40000 ⁇ g, 4° C., 45 minutes) and re-suspended in an equal amount of 100 M Tris buffer (pH 7.5) containing 2 mM dithiothreitol (DTT).
  • the rubber synthesis activity of the collected reacted rubber particles was measured as follows.
  • Table 1 shows the results.
  • the “Rubber synthesis activity difference (dpm)” in Table 1 represents the difference from the radioactivity of Comparative Example 1 in which the rubber particles were bound to nothing as described later.
  • the molecular weight distribution of the very long chain polyisoprenoid (trans-1,4-polyisoprene) synthesized as described above was measured under the following conditions by radio-HPLC.
  • FIG. 4 shows the results.
  • HPLC system a product of GILSON Column: TSK guard column MP(XL) available from Tosoh Corporation, TSK gel Multipore HXL-M (two columns) Column temperature: 40° C. Solvent: THF available from Merck Flow rate: 1 mL/min UV detection: 215 nm RI detection: Ramona Star (Raytest GmbH)
  • Cell-free protein synthesis was performed using a WEPRO7240H expression kit (CellFree Sciences Co., Ltd.).
  • An mRNA transcription reaction was performed using the cell-free expression vector pEU-E01-His-TEV-MCS-N2 as a template in accordance with the protocol of the WEPRO7240H expression kit.
  • the resulting mRNA was purified by ethanol precipitation.
  • Example 2 The same procedure as in Example 1 was followed but using the prepared mRNA.
  • the reacted rubber particles were collected as in Example 1 and re-suspended in an equal amount of 100 M Tris buffer (pH 7.5) containing 2 mM dithiothreitol (DTT).
  • the rubber synthesis activity of the collected reacted rubber particles was measured as in Example 1.
  • the 1st strand cDNA prepared in [Synthesis of cDNA from total RNA] in Example 1 was used as a template to obtain a CPT gene.
  • PCR was performed using a KOD-plus-Neo (Toyobo Co., Ltd.) in accordance with the manual.
  • the PCR reaction involved 35 cycles with each cycle consisting of 10 seconds at 98° C., 30 seconds at 58° C., and 1 minute at 68° C.
  • the CPT gene was obtained using the following primers.
  • Primer 3 5′-tttggatccgatggaattatacaacggtgagagg-3′
  • Primer 4 5′-tttgcggccgcttattttaagtattccttatgtttctc-3′
  • a CPT gene (HRT1) was prepared as described above. The gene was sequenced to identify the full-length nucleotide sequence and amino acid sequence. The nucleotide sequence of HRT1 is given by SEQ ID NO:18. The amino acid sequence of HRT1 is given by SEQ ID NO:19.
  • the obtained DNA fragment was subjected to dA addition and then inserted into a pGEM-T Easy vector using a pGEM-T Easy Vector System (Promega) to prepare pGEM-HRT1.
  • the pGEM-HRT1 acquired in the above [Vector construction] was treated with the restriction enzymes Bam HI and Not I, and inserted into a pEU-E01-His-TEV-MCS-N2 cell-free expression vector that had been treated similarly with the restriction enzymes Bam HI and Not I to prepare pEU-His-N2-HRT1.
  • Cell-free protein synthesis was performed using a WEPRO7240H expression kit (CellFree Sciences Co., Ltd.). An mRNA transcription reaction was performed using the vector pEU-His-N2-HRT1 acquired in the above [Preparation of vector for cell-free protein synthesis] as a template in accordance with the protocol of the WEPRO7240H expression kit.
  • the resulting mRNA was purified by ethanol precipitation.
  • Example 2 The same procedure as in Example 1 was followed but using the prepared mRNA.
  • the reacted rubber particles were collected as in Example 1 and re-suspended in an equal amount of 100 M Tris buffer (pH 7.5) containing 2 mM dithiothreitol (DTT).
  • the rubber synthesis activity of the collected reacted rubber particles was measured as in Example 1.
  • Table 1 demonstrate that a very long chain polyisoprenoid was synthesized when rubber particles were bound to a tPT family protein (Example 1), similarly to when rubber particles were bound to HRT1, a cis-prenyltransferase family protein (Comparative Example 2). This is in contrast to when rubber particles were bound to nothing (Comparative Example 1).
  • the very long chain polyisoprenoid synthesized in Example 1 is a long chain rubber that showed the highest peak at a GPC elution time corresponding to a weight average molecular weight of about 1,000,000. It is also considered that the very long chain polyisoprenoid synthesized in Example 1 had a molecular weight distribution pattern comparable to that of the very long chain polyisoprenoid synthesized in Comparative Example 2. It should be noted that in FIG. 4 , peak heights cannot be used to compare activities because the results were not standardized among the samples.
  • FIG. 5 shows the alignment results around the conserved regions.
  • the multiple sequence alignment was carried out using software called Genetyx Ver. 11.
  • Erg20p Yeast TPT (FPPS)
  • FPPS yeast TPT
  • EuFPPS ( Eucommia ulmoides TPT) corresponds to a sequence of positions 90 to 149 or positions 225 to 283 of EuFPPS from Eucommia ulmoides represented by SEQ ID NO:4;
  • HbFPPS (HeveaTPT) corresponds to a sequence of positions 84 to 143 or positions 219 to 277 of HbFPPS from Hevea brasiliensis represented by SEQ ID NO:5;
  • Coq1p corresponds to a sequence of positions 82 to 135 or positions 249 to 307 of TPT from yeast represented by SEQ ID NO:6;
  • AtSDS1 corresponds to a sequence of positions 162 to 215 or positions 285 to 343 of AtSDS1 from Arabidopsis thaliana represented by SEQ ID NO:7;
  • HbSDS (HeveaTPT) corresponds to a sequence of positions 174 to 227 or positions 297 to 355 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2;
  • HsTPT corresponds to a sequence of positions 132 to 185 or positions 255 to 313 of HsTPT from human represented by SEQ ID NO:8;
  • MmTPT corresponds to a sequence of positions 92 to 145 or positions 215 to 273 of MmTPT from mouse represented by SEQ ID NO:9.
  • boxA corresponding to positions 183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2
  • box B corresponding to positions 310 to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 in FIG. 5 are parts of highly conserved regions of tPT family proteins derived from various organisms.
  • an amino acid sequence at positions corresponding to positions 183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 and an amino acid sequence at positions corresponding to positions 310 to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 are conserved as specific motifs (amino acid sequences (A1) or (A2) and (B)), and proteins having these motifs at the respective positions have the functions of tPT family proteins.
  • SEQ ID NO:1 Nucleotide sequence of gene coding for HbSDS from Hevea brasiliensis
  • SEQ ID NO:2 Amino acid sequence of HbSDS from Hevea brasiliensis
  • SEQ ID NO:3 Amino acid sequence of FPPS from yeast
  • SEQ ID NO:4 Amino acid sequence of EuFPPS from Eucommia ulmoides
  • SEQ ID NO:5 Amino acid sequence of HbFPPS from Hevea brasiliensis
  • SEQ ID NO:6 Amino acid sequence of TPT from yeast
  • SEQ ID NO:7 Amino acid sequence of AtSDS1 from Arabidopsis thaliana
  • SEQ ID NO:8 Amino acid sequence of HsTPT from human
  • SEQ ID NO:9 Amino acid sequence of MmTPT from mouse
  • SEQ ID NO:10 Nucleotide sequence of promoter of gene coding for rubber elongation factor from Hevea brasiliensis
  • SEQ ID NO:18 Nucleotide sequence of gene coding for HRT1 from Hevea brasiliensis
  • SEQ ID NO:19 Amino acid sequence of HRT1 from Hevea brasiliensis

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US16/466,193 2016-12-21 2017-11-21 Method for producing trans-polyisoprenoid, vector, transgenic plant, method for producing pneumatic tire and method for producing rubber product Abandoned US20190376093A1 (en)

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PCT/JP2017/041732 WO2018116726A1 (ja) 2016-12-21 2017-11-21 トランス型ポリイソプレノイドの製造方法、ベクター、形質転換植物、空気入りタイヤの製造方法及びゴム製品の製造方法

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JP2020195326A (ja) * 2019-06-03 2020-12-10 住友ゴム工業株式会社 天然ゴムの製造方法、形質転換植物、空気入りタイヤの製造方法及びゴム製品の製造方法
US20230167465A1 (en) * 2020-03-05 2023-06-01 Sumitomo Rubber Industries, Ltd. Method for producing polyisoprenoid, vector, transformed plant, method for producing pneumatic tire, and method for producing rubber product
JP2023040705A (ja) * 2021-09-10 2023-03-23 住友ゴム工業株式会社 トランス型ポリイソプレノイドの製造方法、ベクター、形質転換生物、空気入りタイヤの製造方法及びゴム製品の製造方法

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US20110201771A1 (en) * 2008-11-06 2011-08-18 University Of Akron Biosynthesis of polyisoprenoids
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JP6557990B2 (ja) * 2015-02-23 2019-08-14 住友ゴム工業株式会社 特定のプロモーター及び特定の蛋白質をコードする遺伝子を含むベクター、該ベクターが導入された形質転換植物、並びに、該ベクターを植物に導入することにより、ポリイソプレノイドの生産量を向上させる方法
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