Chemically inducible expression of biosynthetic pathways
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
A01H-005/00
C12N-015/08
C12N-015/31
C12N-015/82
출원번호
UP-0376831
(2006-03-16)
등록번호
US-7732680
(2010-06-29)
발명자
/ 주소
Kourtz, Lauralynn
Peoples, Oliver P.
Snell, Kristi D.
출원인 / 주소
Metabolix, Inc.
대리인 / 주소
Pabst Patent Group LLP
인용정보
피인용 횟수 :
2인용 특허 :
48
초록▼
Methods and constructs for the introduction of multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway are provided. In one embodiment, the constructs contain two or more enzyme-encoding genes, each under the control of an inducible promoter and each with a polyadenylation signal. The
Methods and constructs for the introduction of multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway are provided. In one embodiment, the constructs contain two or more enzyme-encoding genes, each under the control of an inducible promoter and each with a polyadenylation signal. The constructs are used to produce transgenic plants, in which the expression of the enzymes are increased when a chemical inducing agent is applied, and a biosynthetic product of the series of enzymes encoded by the transgenes is produced. Constructs may be used which contain two or more enzyme-encoding genes under the control of one or more promoters activated by activator molecules or complexes expressed from a transgene or transgenes, which are themselves under the control of one or more inducible promoters and switched on following the external application of a chemical. The transgene or transgenes expressing the activator molecules or complexes may be included in the same construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. Alternatively, the transgene or transgenes expressing the activator molecules or complexes may be on a different construct from the construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. The activator molecule can be expressed using a constitutive promoter in an inactive form which is converted to the active form following application of the chemical inducing agent.
대표청구항▼
We claim: 1. A recombinant vector for the expression of a polyhydroxyalkanoate biosynthetic pathway comprising three or more elements in a plant, wherein each element comprises operatively linked in the 5′ to 3′ direction: a chemically inducible promoter that directs transcription of
We claim: 1. A recombinant vector for the expression of a polyhydroxyalkanoate biosynthetic pathway comprising three or more elements in a plant, wherein each element comprises operatively linked in the 5′ to 3′ direction: a chemically inducible promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence, wherein the nucleic acid sequences encode proteins selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, 3 hydroxy acyl ACP dehydratase, Δ 3-cis, Δ 2-trans isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes. 2. The recombinant vector of claim 1, wherein the inducible promoter in each element is selected from the group consisting of a tetracycline-inducible, pristamycin-inducible, pathogen-inducible, glucocorticoid-inducible, estrogen-inducible, copper-inducible, herbicide safener-inducible, ethanol-inducible, iso-propyl β-D-1-thiogalactopyranoside-inducible, and ecdysone-inducible promoter. 3. The recombinant vector of claim 2, wherein the promoter is an ecdysone-inducible promoter activated by a compound selected from the group consisting of ecdysone, tebufenozide, methoxyfenozide, muristerone A, ponasterone A, insect steroid hormone 20-hydroxyecdysone, and caged β-ecdysone. 4. The recombinant vector of claim 1, wherein the promoter in each element is the same promoter. 5. The recombinant vector of claim 1, wherein the promoter in each element is a different promoter. 6. The recombinant vector of claim 1 for the expression of enzymes in a polyhydroxyalkanoate biosynthetic pathway comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction; a promoter activated by an activator molecule or complex that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence, wherein the three or more elements contain nucleic acid sequences encoding beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, 3 hydroxy acyl ACP dehydratase, Δ 3-cis, Δ2-isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes. 7. The recombinant vector of claim 6, wherein the promoter is a tetracycline-responsive promoter. 8. The vector of claim 6, for expression in plants. 9. A transformed plant or plant cell comprising a recombinant vector for expression of proteins in the transformed plant or plant cell, the vector encoding a polyhydroxyalkanoate biosynthetic pathway comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction: a chemically inducible promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence, wherein the nucleic acid sequences encoding proteins selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, 3 hydroxy acyl ACP dehydratase, Δ 3-cis, Δ 2-isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, and wherein the transformed plant produces at least 10% dry weight (dwt) polyhydroxyalkanoate after inducing the inducible promoters, and wherein the plant does not exhibit a stunted chiorotic phenotype. 10. The transformed plant cell of claim 9, wherein the inducible promoter in each element is selected from the group consisting of a tetracycline-inducible, pristamycin-inducible, pathogen-inducible, glucocorticoid-inducible, estrogen-inducible, copper-inducible, herbicide safener-inducible, ethanol-inducible, iso-propyl β-D-1-thiogalactopyranoside-inducible, and ecdysone-inducible promoter. 11. The transformed plant cell of claim 10, wherein the promoter is an ecdysone-inducible promoter activated by a compound selected from the group consisting of ecdysone, tebufenozide, methoxyfenozide, muristerone A, ponasterone A, insect steroid hormone 20-hydroxyecdysone, and caged β-ecdysone. 12. The transformed plant cell of claim 9, wherein the promoter in each element is the same promoter. 13. The transformed plant cell of claim 9, wherein the promoter is an ecdysone-inducible promoter. 14. The transformed plant cell of claim 9, wherein the promoter in each element is different. 15. A transformed plant or plant cell comprising a recombinant vector for expression of proteins in the transformed plant or plant cell, the vector encoding a polyhydroxyalkanoate biosynthetic pathway comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction; a promoter activated by an activator molecule or complex that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence, wherein the nucleic acid sequences encode proteins selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, 3 hydroxy acyl ACP dehydratase, Δ 3-cis, Δ 2-isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, wherein the transformed plant produces at least 10% dwt polyhydroxyalkanoate after inducing the inducible promoters and, wherein the plant or plant cell does not exhibit a stunted chiorotic phenotype. 16. The transformed plant cell of claim 15, wherein the promoter is a tetracycline-responsive promoter. 17. A method for the production of a polyhydroxyalkanoate in a plant comprising a) introducing into the plant, a recombinant vector comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction: a chemically inducible promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence, wherein the three or more elements contain nucleic acid sequences that encode proteins selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, 3 hydroxy acyl ACP dehydratase, Δ 3-cis, Δ 2-isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, and b) activating the inducible promoters with an inducing agent, wherein the plant produces at least 10% dwt polyhydroxyalkanoate after activating the inducible promoters and, wherein the plant does not exhibit a stunted chlorotic phenotype. 18. The method of claim 17, wherein the inducible promoter in each element is selected from the group consisting of a tetracycline-inducible, pristamycin-inducible, pathogen-inducible, glucocorticoid-inducible, estrogen-inducible, copper-inducible, herbicide safener-inducible, ethanol-inducible, iso-propyl β-D-1-thiogalactopyranoside-inducible, and ecdysone-inducible promoter. 19. The method of claim 18, wherein the inducible promoter is an ecdysone-inducible promoter activated by a compound selected from the group consisting of ecdysone, tebufenozide, methoxyfenozide, muristerone A, ponasterone A, insect steroid hormone 20-hydroxyecdysone, and caged β-ecdysone. 20. The method of claim 17, wherein the promoter in each element is the same promoter. 21. The method of claim 17, wherein the promoter in each element is a different promoter. 22. The method of claim 17 wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching. 23. The plant of claim 9 selected from the group consisting of Brassica; Arabidopsis; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards, alfalfa, switchgrass, sorghum and tobacco. 24. The method of claim 17 wherein the plant is selected from the group consisting of Brassica; Arabidopsis; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards, alfalfa, switchgrass, sorghum and tobacco. 25. The vector of claim 1 encoding a β-ketothiolase (thiolase), an NADPH-acetoacetyl-CoA reductase (reductase) and a PHA synthase (synthase). 26. The plant of claim 9 encoding a β-ketothiolase (thiolase), an NADPH-acetoacetyl-CoA reductase (reductase) and a PHA synthase (synthase).
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