Chemically Inducible Expression of Biosynthetic Pathways
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12N-015/29
C12N-015/31
C12N-015/52
C12N-015/82
출원번호
US-0762941
(2010-04-19)
등록번호
US-8742203
(2014-06-03)
발명자
/ 주소
Kourtz, Lauralynn
Peoples, Oliver P.
Snell, Kristi D.
출원인 / 주소
Metabolix, Inc.
대리인 / 주소
Pabst Patent Group LLP
인용정보
피인용 횟수 :
0인용 특허 :
50
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▼
1. A recombinant plant vector for the expression of enzymes in a biosynthetic pathway comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a p
1. A recombinant plant vector for the expression of enzymes in a biosynthetic pathway comprising three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of a nucleic acid sequence; a nucleic acid sequence encoding a protein; and a 3′ polyadenylation signal sequence;wherein the promoter is selected from the group consisting of a chemically inducible promoter and a promoter activated by an activator molecule or complex, wherein at least one promoter is a promoter activated by an activator molecule or complex, and at least one promoter is a chemically inducible promoter,wherein said chemically inducible promoter is activated by external application of a chemical,wherein the biosynthetic product is a polyhydroxyalkanoate and the proteins encoded by the nucleic acid sequences are selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, dehydratase, isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, andwherein inducible expression of the recombinant plant vector in a plant produces at least 10% dwt polyhydroxyalkanoate in leaves of the plant without the plant exhibiting a stunted chlorotic phenotype. 2. A transformed plant cell comprising the recombinant vector of claim 1. 3. A transformed plant cell comprising two or more recombinant vectors, wherein at least one of the recombinant vectors comprises operatively linked in the 5′ to 3′ direction: an inducible promoter that directs transcription of one or more nucleic acid sequences encoding an activator molecule or complex; one or more nucleic acid sequences encoding an activator molecule or complex; and a 3′ polyadenylation signal sequence;andat least one of the recombinant vectors comprises 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 said inducible promoter is activated by external application of a chemical,wherein nucleic acid sequences are selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, dehydratase, isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, wherein the plant produces at least 10% dwt polyhydroxyalkanoate in leaves and wherein the plant does not exhibit a stunted chlorotic phenotype. 4. The transformed plant cell of claim 2, wherein the inducible promoter is selected from the group consisting of 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; the activator molecule or complex is a tetracycline-controlled transactivator; and the promoter activated by an activator molecule or complex is a tetracycline-responsive promoter. 5. A method for producing a polyhydroxyalkanoate in a plant comprising a) introducing into the plant two or more recombinant vectors,wherein at least one recombinant vector comprises operatively linked in the 5′ to 3′ direction: an inducible promoter that directs transcription of one or more nucleic acid sequences encoding an activator molecule or complex; one or more nucleic acid sequences encoding an activator molecule or complex; and a 3′ polyadenylation signal sequence andat least one recombinant vector comprises three or more elements, wherein each element comprises operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of two or more nucleic acid sequence, selected from the group consisting of a promoter activated by an activator molecule or complex and a chemically inducible promoter; a nucleic acid sequences encoding a protein; and a 3′ polyadenylation signal sequence; andb) activating the inducible promoters by external application of an inducing agent wherein the proteins encoded by the nucleic acid sequences are selected from the group consisting of beta-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, PHA synthase, threonine dehydratase, dehydratase, isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes, wherein the plant produces at least 10% dwt polyhydroxyalkanoate in leaves and wherein the plant does not exhibit a stunted chlorotic phenotype. 6. A method for producing a biosynthetic product in a plant comprising a) introducing into the plant the recombinant vector of claim 1, andb) activating the inducible promoters by external application of an inducing agent, wherein the plant produces at least 10% dwt polyhydroxyalkanoate in leaves and wherein the plant does not exhibit a stunted chlorotic phenotype. 7. The method of claim 5, wherein the inducible promoter is selected from the group consisting of 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; the activator molecule or complex is a tetracycline-controlled transactivator; and the promoters activated by an activator molecule or complex are selected from the group consisting of tetracycline-responsive promoters, and pristamycin-inducible promoters. 8. The method of claim 5, wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching, 9. The method of claim 6, wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching. 10. The transformed plant cell of claim 3, wherein the inducible promoter is selected from the group consisting of 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; the activator molecule or complex is a tetracycline-controlled transactivator; and the promoter activated by an activator molecule or complex is a tetracycline-responsive promoter. 11. The method of claim 6, wherein the inducible promoter is selected from the group consisting of tetracycline-inducible, pristamycin-inducible, pathogen-inducible, glucocorticoid-inducible, estrogen-inducible, copper-inducible, herbicide safener-inducible, ethanol-inducible, iso-propylp β-D-1-thiogalactopyranoside-inducible, and ecdysone-inducible promoter; the activator molecule or complex is a tetracycline-controlled transactivator; and the promoters activated by an activator molecule or complex are selected from the group consisting of tetracycline-responsive promoters, pristamycin-inducible promoters.
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