Chemically inducible expression of biosynthetic pathways

Abstract

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.

1 . A recombinant plant vector for the expression of enzymes in a biosynthetic pathway for producing an oil comprising two or more elements,
 wherein at least one of the elements 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; and   at least one of the elements comprises operatively linked in the 5′ to 3′ direction:
 a promoter activated by an activator molecule or complex that directs transcription of two or more nucleic acid sequence; 
 two or more nucleic acid sequences each encoding a protein; and 
 a 3′ polyadenylation signal sequence, 
   wherein proteins encoded by the nucleic acid sequences are selected from the group consisting of thioesterase, delta-9-desaturase, omega-3-desaturase, omega-6-desaturase, fatty acid elongase, hydroxylase, and triacylglycerol biosynthesis enzymes.   
     
     
         2 . The recombinant plant vector of  claim 1 , wherein the plant vector comprises three or more elements, and wherein at least a second 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 selected from the group consisting of thioesterase, delta-9-desaturase, omega-3-desaturase, omega-6-desaturase, fatty acid elongase, hydroxylase, and triacyl-glycerol biosynthesis enzymes; and 
 a 3′ polyadenylation signal sequence. 
 
     
     
         3 . A transformed plant cell comprising the recombinant plant vector of  claim 1 . 
     
     
         4 . 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, pathogen-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 transformed plant cell comprising the recombinant plant vector of  claim 2 . 
     
     
         6 . The transformed plant cell 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, pathogen-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. 
     
     
         7 . A method for producing an oil in a plant comprising
 a) introducing into the plant one or more recombinant plant vectors, wherein at least one recombinant vector comprises the plant vector of claim, and   b) activating the inducible promoters with an inducing agent to produce the oil.   
     
     
         8 . The method of  claim 7 , 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, pathogen-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. 
     
     
         9 . The method of  claim 7 , wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching. 
     
     
         10 . The method of  claim 7 , wherein a single recombinant plant vector is introduced into the plant. 
     
     
         11 . The method of  claim 10 , 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, pathogen-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. 
     
     
         12 . The method of  claim 10 , wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching. 
     
     
         13 . The method of  claim 12 , wherein the recombinant plant vector comprises three or more elements, and at least one additional 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. 
 
     
     
         14 . The method of  claim 13 , 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, pathogen-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. 
     
     
         15 . The method of  claim 13 , wherein the inducible promoter is activated by a chemical through a foliar spray or root drenching.