Production method for polyisoprenoid, vector, transgenic plant, production method for pneumatic tire, and production method for rubber product

Patent metadata

Field	Value
Publication number	US-10907179-B2
Application number	US-201615579434-A
Country	US
Kind code	B2
Filing date	Jun 28, 2016
Priority date	Jun 30, 2015
Publication date	Feb 2, 2021
Grant date	Feb 2, 2021

Abstract

The present invention provides a method for producing a polyisoprenoid with which it is possible to enhance the rubber synthesis activity of rubber particles to increase natural rubber production. The present invention relates to a method for producing a polyisoprenoid in vitro, which involves the use of a gene coding for a cis-prenyltransferase (CPT) family protein and a gene coding for a Nogo-B receptor (NgBR) family protein, and further involves the use of rubber particles bound to proteins encoded by these genes; or a method for producing a polyisoprenoid, which includes introducing into a plant a vector in which a promoter having a promoter activity that drives laticifer-specific gene expression is linked to a gene coding for a CPT family protein and a gene coding for a NgBR family protein, to express proteins encoded by the genes specifically in laticifers.

First claim

The invention claimed is:

1. A method for producing a polyisoprenoid, the method comprising
the step of binding a protein expressed by a gene coding for a cis-prenyltransferase (CPT) family protein and a protein expressed by a gene coding for a Nogo-B receptor (NgBR) family protein to rubber particles in vitro.

2. The method for producing a polyisoprenoid according to claim 1 ,
wherein the cis-prenyltransferase (CPT) family protein comprises at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2 and has an enzyme activity that catalyzes a reaction of cis-chain elongation of an isoprenoid compound and
wherein the protein further comprises:
an aspartic acid residue at position 41 in the amino acid sequence of SEQ ID NO:2;
a glycine residue at position 42 in the amino acid sequence of SEQ ID NO:2;
an arginine residue at position 45 in the amino acid sequence of SEQ ID NO:2; and
an asparagine residue at position 89 in the amino acid sequence of SEQ ID NO:2.

3. The method for producing a polyisoprenoid according to claim 2 ,
wherein the cis-prenyltransferase (CPT) family protein further comprises the following amino acid sequence (A): DGNX 1 RX 2 AKK (A)
wherein X 1 and X 2 are the same as or different from each other and each represent any amino acid residue.

4. The method for producing a polyisoprenoid according to claim 2 ,
wherein the cis-prenyltransferase (CPT) family protein further comprises the following amino acid sequence (B): TX 11 X 12 AFSX 13 X 14 NX 15 X 16 RX 17 X 18 X 19 EV (B)
wherein X 11 to X 19 are the same as or different from each other and each represent any amino acid residue.

5. The method for producing a polyisoprenoid according to claim 1 ,
wherein at least one selected from the group consisting of the gene coding for a cis-prenyltransferase (CPT) family protein and the gene coding for a Nogo-B receptor (NgBR) family protein is derived from a plant.

6. The method for producing a polyisoprenoid according to claim 5 ,
wherein at least one selected from the group consisting of the gene coding for a cis-prenyltransferase (CPT) family protein and the gene coding for a Nogo-B receptor (NgBR) family protein is derived from Hevea brasiliensis.

7. The method for producing a polyisoprenoid according to claim 1 ,
wherein the binding step comprises performing protein synthesis in the presence of both rubber particles and a cell-free protein synthesis solution containing an mRNA coding for a cis-prenyltransferase (CPT) family protein and an mRNA coding for a Nogo-B receptor (NgBR) family protein to bind the CPT family protein and the NgBR family protein to the rubber particles.

8. The method for producing a polyisoprenoid according to claim 7 ,
wherein the cell-free protein synthesis solution contains a germ extract.

9. The method for producing a polyisoprenoid according to claim 8 ,
wherein the germ extract is derived from wheat.

10. The method for producing a polyisoprenoid according to claim 7 ,
wherein the rubber particles are present in the cell-free protein synthesis solution at a concentration of 5 to 50 g/L.

11. The method for producing a polyisoprenoid according to claim 1 , wherein the polyisoprenoid is natural rubber.

12. The method for producing a polyisoprenoid according to claim 1 , wherein the polyisoprenoid is very long chain polyisoprenoid.

13. The method for producing a polyisoprenoid according to claim 1 , wherein the polyisoprenoid is polyisoprenoid rubber.

14. The method for producing a polyisoprenoid according to claim 1 , the method further comprising
the step of preparing rubber particles by carrying out centrifugation at 500-1,500×g, centrifugation at 1,700-2,500×g, centrifugation at 7,000-9,000×g, centrifugation at 15,000-25,000×g, and centrifugation at 40,000-60,000×g in the stated order, and
the step of binding a protein expressed by a gene coding for a cis-prenyltransferase (CPT) family protein and a protein expressed by a gene coding for a Nogo-B receptor (NgBR) family protein to the rubber particles in vitro.

Assignees

• Sumitomo Rubber Ind
• Univ Tohoku

Inventors

• Yamaguchi Haruhiko
• INOUE Yukino
• Fushihara Kazuhisa
• Takahashi Seiji
• Yamashita Satoshi
• Nakayama Toru

Classifications

• C07K14/415 Primary

  from plants · CPC title

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C08L47/00

  Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds; Compositions of derivatives of such polymers (C08L45/00 takes precedence; of conjugated diene rubbers C08L9/00 - C08L21/00) · CPC title

• C12Y504/99

  transferring other groups (5.4.99) · CPC title

• C12N9/1085

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title