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US 8,722,363 B2Granted
Cytochrome P450S and uses thereof
AssigneeUniversity of Kentucky Research Foundation
InventorsJoseph Chappell, Lyle F. Ralston
Life of this patent
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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.
Claims29
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 that is a diterpene or sesquiterpene;
- and
- a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of a diterpene or sesquiterpene;
- b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing a diterpene or a sesquiterpene isoprenoid compound not normally produced by the host cell to produce a diterpene or sesquiterpene isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) together catalyze the formation of a diterpene or sesquiterpene isoprenoid compound in the host cell, wherein the diterpene or sesquiterpene isoprenoid compound is not normally produced by the cell.
2 more elements
A host cell, comprising nucleic acid encoding a diterpene or sesquiterpene isoprenoid synthase, and nucleic acid encoding one or more cytochrome P450 polypeptide(s), wherein:
- the nucleic acid encoding the synthase and the nucleic acid encoding the P450 polypeptide(s) are heterologous to the host cell;
- the synthase catalyzes production of a diterpene or sesquiterpene compound;
- the P450 polypeptide(s) catalyze(s) hydroxylation, oxidation, demethylation or methylation of a diterpene or sesquiterpene;
- at least one of the P450 polypeptide(s) catalyzes the hydroxylation, oxidation, demethylation or methylation of a diterpene or sesquiterpene whose production is catalyzed by the synthase;
- and
- the host cell thereby can produce a diterpene or sesquiterpene compound not normally produced by the host cell nor by the synthase in the absence of the P450 polypeptide(s).
3 more elements
A method for producing 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 that is a diterpene or sesquiterpene;
- and
- a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of a diterpene or sesquiterpene;
- b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing a diterpene or a sesquiterpene isoprenoid compound not normally produced by the host cell to produce a diterpene or sesquiterpene isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) together catalyze the formation of the diterpene or sesquiterpene isoprenoid compound;
- and
- c) isolating a diterpene or sesquiterpene isoprenoid compound not normally produced by the cell.
4 more elements
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 that is a diterpene or sesquiterpene;
- and
- a cytochrome P450 polypeptide(s) catalyzes hydroxylation, oxidation, demethylation or methylation of a diterpene or sesquiterpene;
- b) culturing the host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) and under conditions for producing a diterpene or a sesquiterpene isoprenoid compound not normally produced by the host cell to produce a diterpene or sesquiterpene isoprenoid compound not normally produced by the host cell, wherein the synthase and the cytochrome P450 polypeptide(s) together catalyze the formation of a diterpene or sesquiterpene 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.
4 more elements
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 that is a diterpene or sesquiterpene;
- and
- the cytochrome P450 polypeptide catalyzes hydroxylation, oxidation, demethylation or methylation of the diterpene or sesquiterpene produced by the synthase to produce a diterpene or a sesquiterpene 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 diterpene or a sesquiterpene isoprenoid compound not normally produced by the host cell, wherein the first and second recombinant protein together catalyze the formation of a diterpene or sesquiterpene isoprenoid compound;
- and
- c) isolating a diterpene or sesquiterpene isoprenoid compound not normally produced by the host cell.
4 more elements
Dependencies are inferred from the claim text.