Cytochrome P450s and uses thereof

Patent metadata

Field	Value
Publication number	US-10184119-B2
Application number	US-201514833667-A
Country	US
Kind code	B2
Filing date	Aug 24, 2015
Priority date	Mar 9, 2001
Publication date	Jan 22, 2019
Grant date	Jan 22, 2019

Abstract

The invention relates to isolated cytochrome P450 polypeptides and nucleic acid molecules, as well as expression vectors and transgenic plants containing these molecules. In addition, the invention relates to 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.

First claim

The invention claimed is:

1. A method for producing in a recombinant host cell an isoprenoid compound not endogenously produced by a non-recombinant host cell, comprising:
a) providing a recombinant host cell that comprises heterologous nucleic acid encoding an isoprenoid synthase, and a heterologous nucleic acid encoding one or more protein(s) comprising a CYP71, CYP73, CYP82 or a CYP92 family cytochrome P450 polypeptide, wherein the nucleid acid encoding a CYP71, CYP73, CYP82 or a CYP92 family cytochrome P450 polypeptide can be amplified with degenerate primers based on any one of SEQ ID NOs: 26-29; wherein the isoprenoid synthase catalyzes production of an isoprenoid compound; wherein the isoprenoid synthase is a diterpene synthase; and wherein the cytochrome P450 polypeptide(s) catalyzes dual hydroxylation, oxidation, demethylation or methylation of the isoprenoid compound; and
b) culturing the recombinant host cell under conditions suitable for expressing the isoprenoid synthase and the cytochrome P450 polypeptide(s) under conditions for producing the isoprenoid compound; wherein the synthase and the cytochrome P450 polypeptide(s) catalyze formation of the isoprenoid compound in the host cell.

2. The method of claim 1 , wherein at least one cytochrome P450 polypeptide has oxidase activity.

3. The method of claim 1 , wherein at least one cytochrome P450 polypeptide has dual hydroxylase activity.

4. The method of claim 1 , wherein the host cell is a yeast cell, a bacterial cell, an insect cell or a plant cell.

5. The method of claim 1 , wherein the host cell is a yeast cell.

6. The method of claim 5 , wherein the yeast is Saccharomyces cerevisiae.

7. The method of claim 1 , wherein at least one of the cytochrome P450 polypeptides is 5-epi-aristolochene hydroxylase or kaurene oxidase.

8. The method of claim 1 , wherein the isoprenoid compound is a diterpene.

9. The method of claim 1 , further comprising c) isolating the isoprenoid compound.

10. A host cell, comprising nucleic acid encoding an isoprenoid synthase and nucleic acid encoding one or more protein(s) comprising a CYP71, CYP73, CYP82 or a CYP92 family cytochrome P450 polypeptides encoded by nucleic acid that can be amplified with degenerate primers based on any one of SEQ ID NOs: 26-29, 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 isoprenoid synthase is a diterpene synthase;
the P450 polypeptides catalyze hydroxylation, oxidation, demethylation or methylation of the isoprenoid compound;
at least one of the P450 polypeptide(s) catalyzes the dual hydroxylation, oxidation, demethylation or methylation of the isoprenoid compound whose production is catalyzed by the synthase; and
the isoprenoid compound produced by the host cell is not endogenously produced by a non recombinant host cell.

11. The host cell of claim 10 that is a yeast cell, a bacterial cell, an insect cell or a plant cell.

12. The host cell of claim 10 that is a yeast cell.

13. The host cell of claim 10 , wherein at least one of the cytochrome P450 polypeptide(s) is 5-epi-aristolochene hydroxylase or kaurene oxidase.

14. The method of claim 1 , wherein at least one of the cytochrome P450 polypeptide(s) is selected from among polypeptides comprising at least 80% identity to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:11.

15. The host cell of claim 10 , wherein at least one of the cytochrome P450 polypeptide(s) is selected from among polypeptides comprising at least 80% identity to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:11.

Assignees

• Univ Kentucky Res Found
• The Univ Of Kentucky Research Foundation

Inventors

• Chappell Joseph
• Ralston Lyle F

Classifications

• C12Y114/13

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13) · CPC title

• C12N9/0073 Primary

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12P7/00

  Preparation of oxygen-containing organic compounds · CPC title

• C12N15/8279

  for biotic stress resistance, pathogen resistance, disease resistance · CPC title

• C12P7/02

  containing a hydroxy group · CPC title