Cytochrome p450s and uses thereof

Abstract

The invention features isolated cytochrome P450 polypeptides and nucleic acid molecules, as well as expression vectors and transgenic plants containing these molecules. In addition, the invention features uses of such molecules in methods of increasing the level of resistance against a disease caused by a plant pathogen in a transgenic plant, in methods for producing altered compounds, for example, hydroxylated compounds, and in methods of producing isoprenoid compounds.

1 . A method for producing a host cell comprising an isoprenoid compound, comprising:
 a) providing a host cell that comprises heterologous nucleic acid encoding an isoprenoid synthase, and heterologous nucleic acid encoding one or more protein(s) comprising a cytochrome P450 polypeptide, wherein:   the isoprenoid synthase catalyzes production of an isoprenoid compound; and   a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of the isoprenoid compound; and   b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing an isoprenoid compound not normally produced by the host cell to produce an isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) catalyze the formation of an isoprenoid compound in the host cell, wherein the isoprenoid compound is not normally produced by the cell.   
     
     
         2 . The method of  claim 1 , further comprising isolating the host cell. 
     
     
         3 . The method of  claim 1 , wherein at least one cytochrome P450 polypeptide has dual hydroxylase activity. 
     
     
         4 . The method of  claim 1 , wherein at least one cytochrome P450 polypeptide has oxidase activity. 
     
     
         5 . The method of  claim 1 , wherein at least one cytochrome P450 polypeptide has hydroxylase activity. 
     
     
         6 . The method of  claim 1 , wherein the host cell is a yeast cell, a bacterial cell, an insect cell or a plant cell. 
     
     
         7 . The method of  claim 1 , wherein the host cell is a yeast cell. 
     
     
         8 . The method of  claim 7 , wherein the yeast is  Saccharomyces cerevisiae.    
     
     
         9 . The method of  claim 1 , wherein at least one of the cytochrome P450 polypeptides is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase. 
     
     
         10 . A host cell, comprising nucleic acid encoding an isoprenoid synthase and nucleic acid encoding cytochrome P450 polypeptides, wherein:
 the nucleic acid encoding the synthase and the nucleic acid encoding the P450 polypeptides are heterologous to the host cell;   the synthase catalyzes production of an isoprenoid compound;   the P450 polypeptides catalyze hydroxylation, oxidation, demethylation or methylation of an isoprenoid compound;   at least one of the P450 polypeptide(s) catalyzes the hydroxylation, oxidation, demethylation or methylation of an isoprenoid compound whose production is catalyzed by the synthase; and   the host cell produces a compound not normally produced by the host cell in the absence of the P450 polypeptides.   
     
     
         11 . The host cell of  claim 10 , wherein at least one of the P450 polypeptides catalyzes the dual hydroxylation of an isoprenoid whose production is catalyzed by the synthase. 
     
     
         12 . The host cell of  claim 10  that is a yeast cell, a bacterial cell, an insect cell or a plant cell. 
     
     
         13 . The host cell of  claim 10  that is a yeast cell. 
     
     
         14 . The host cell of  claim 10 , wherein at least one of the cytochrome P450 polypeptide(s) is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase. 
     
     
         15 . A method for producing an isoprenoid compound, comprising:
 a) providing a host cell that comprises heterologous nucleic acid encoding an isoprenoid synthase, and nucleic acid encoding one or more protein(s) comprising a cytochrome P450 polypeptide, wherein a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of a terpene whose production is catalyzed by the synthase; and   b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing an isoprenoid compound not normally produced by the host cell to produce an isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) catalyze the formation of an isoprenoid compound.   
     
     
         16 . The method of  claim 15 , wherein the nucleic acid encoding a cytochrome P450 is heterologous to the cell. 
     
     
         17 . The method of  claim 15 , wherein the isoprenoid compound is a diterpene or sesquiterpene. 
     
     
         18 . The method of  claim 15 , further comprising c) isolating the isoprenoid compound not normally produced by the cell. 
     
     
         19 . A method for producing a host cell comprising an isoprenoid compound, comprising:
 a) providing a host cell that comprises heterologous nucleic acid encoding an isoprenoid synthase and heterologous nucleic acid encoding one or more protein(s) comprising a cytochrome P450 polypeptide, wherein:   the isoprenoid synthase catalyzes production of an isoprenoid compound; and   a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of an isoprenoid compound;   b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing an isoprenoid compound not normally produced by the host cell to produce an isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) together catalyze the formation of an isoprenoid compound not normally produced by the cell; and   c) isolating the host cell that comprises an isoprenoid compound not normally produced by the cell.   
     
     
         20 . The method of  claim 19 , further comprising isolating an isoprenoid compound produced by the host cell. 
     
     
         21 . The method of  claim 1 , further comprising collecting fermentation medium in which the host cell is cultured; and isolating an isoprenoid compound. 
     
     
         22 . The method of  claim 15 , wherein at least one of the cytochrome P450 polypeptide(s) is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase. 
     
     
         23 . A method for producing an isoprenoid compound, comprising:
 a) providing a host cell that expresses heterologous nucleic acid encoding a first recombinant protein comprising an isoprenoid synthase, and a second recombinant protein comprising a cytochrome P450 polypeptide, wherein:   the isoprenoid synthase catalyzes production of an isoprenoid compound; and   the cytochrome P450 polypeptide catalyzes hydroxylation, oxidation, demethylation or methylation of the isoprenoid compound produced by the synthase to produce an isoprenoid compound not normally produced by the host cell nor whose production is catalyzed by the synthase;   b) culturing the host cell under conditions suitable for expressing the first recombinant protein comprising an isoprenoid synthase and the second recombinant protein comprising the cytochrome P450 polypeptide and under conditions for producing the isoprenoid compound not normally produced by the host cell, wherein the first and second recombinant protein together catalyze the formation of an isoprenoid compound; and   c) isolating an isoprenoid compound not normally produced by the host cell.   
     
     
         24 . The method of  claim 23 , wherein at least one of the cytochrome P450 polypeptide(s) is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase. 
     
     
         25 . The method of  claim 23 , wherein the host cell is a yeast cell, a bacterial cell, an insect cell or a plant cell. 
     
     
         26 . The method of  claim 23 , wherein the host cell is a yeast cell. 
     
     
         27 . A method for producing an altered isoprenoid compound, comprising:
 contacting an isoprenoid compound in vitro with one or more cytochrome P450 polypeptide(s), whereby the P450 polypeptide(s) catalyzes the dual hydroxylation, oxidation, demethylation, methylation, or any combination thereof of the isoprenoid compound to produce an altered isoprenoid compound.   
     
     
         28 . The method of  claim 27 , wherein the isoprenoid compound that is contacted with one or more cytochrome P450 polypeptides is produced by catalyzing its production from a precursor with an isoprenoid synthase to produce the isoprenoid compound. 
     
     
         29 . The method of  claim 28 , wherein the isoprenoid compound is produced in a host cell that encodes the isoprenoid synthase. 
     
     
         30 . The method of  claim 27 , wherein at least one of the cytochrome P450 polypeptide(s) is 5-epi-aristolochene hydroxylase or kaurene oxidase. 
     
     
         31 . The method of  claim 27 , wherein the isoprenoid compound is a diterpene or sesquiterpene. 
     
     
         32 . The method of  claim 27 , further comprising isolating the isoprenoid compound prior to contacting with a P450 polypeptide. 
     
     
         33 . The method of  claim 27 , wherein the isoprenoid compound is contacted with more than one cytochrome P450 polypeptide. 
     
     
         34 . A method for producing a host cell comprising an isoprenoid compound, comprising:
 a) providing a host cell that comprises heterologous nucleic acid encoding one or more protein(s) comprising a cytochrome P450 polypeptide, wherein:   a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of an isoprenoid; and   b) culturing the host cell under conditions suitable for expressing the cytochrome P450 polypeptide(s) and under conditions for producing an isoprenoid compound not normally produced by the host cell to produce an isoprenoid compound not normally produced by the host cell, wherein the cytochrome P450 polypeptide(s) catalyze the formation of an isoprenoid compound in the host cell, wherein the isoprenoid compound is not normally produced by the cell.   
     
     
         35 . The method of  claim 34 , wherein:
 the host cell comprises nucleic acid encoding more than one proteins comprising a cytochrome P450 polypeptide; and   more than one cytochrome P450 polypeptides catalyze the formation of the isoprenoid compound not normally produced by the cell.   
     
     
         36 . The method of  claim 34 , wherein at least one of the cytochrome P450 polypeptides is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase. 
     
     
         37 . The method of  claim 35 , wherein at least one of the cytochrome P450 polypeptides is selected from among 5-epi-aristolochene hydroxylase and kaurene oxidase.