Cytochrome p450 and cytochrome p450 reductase polypeptides, encoding nucleic acid molecules and uses thereof

Abstract

Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells.

1 . A host cell, comprising a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
 (a) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 85% sequence identity to a P450 oxidase polypeptide set forth in SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77, or a corresponding catalytically active portion thereof;   (b) the encoded cytochrome P450 oxidase or catalytically active fragment thereof catalyzes hydroxylation or monooxygenation of santalene and/or bergamotene; and   (c) the nucleic acid molecule is heterologous to the host cell.   
     
     
         2 . The host cell of  claim 1 , wherein the encoded cytochrome P450 oxidase polypeptide exhibits at least 95% sequence identity to the cytochrome P450 oxidase polypeptide set forth in SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77. 
     
     
         3 . The host cell of  claim 1 , wherein the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof is a  Santalum album  P450 oxidase polypeptide. 
     
     
         4 . The host cell of  claim 1 , wherein the cytochrome P450 oxidase polypeptide or catalytically active fragment catalyzes formation of a santalol from a santalene, or a bergamotol from a bergamotene. 
     
     
         5 . The host cell of  claim 1 , wherein the nucleic acid molecule comprises a sequence of nucleotides selected from among:
 (a) a sequence of nucleic acids set forth in any of SEQ ID NO:2, 3, 4, 5, 67, 68, 69, 70, or 71;   (b) a sequence of nucleic acids encoding a protein having at least 85% sequence identity to a protein encoded by the sequence of nucleic acids set forth in any of SEQ ID NO:2, 3, 4, 5, 67, 68, 69, 70, or 71; and   (c) a sequence of nucleic acids comprising degenerate codons of one or more codons in the sequence of nucleic acids of (a) or (b).   
     
     
         6 . The host cell of  claim 5 , wherein the sequence of nucleic acid has at least 95% sequence identity to a sequence of nucleic acids set forth in any of SEQ ID NO:2, 3, 4, 5, 67, 68, 69, 70, or 71. 
     
     
         7 . The host cell of  claim 1 , comprising a nucleic acid molecule encoding a cytochrome P450 reductase or a catalytically active portion thereof, wherein:
 (a) the encoded cytochrome P450 reductase or catalytically active portion thereof exhibits at least 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13;   (b) the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof catalyzes transfer of two electrons from NADPH to an electron acceptor; and   (c) the nucleic acid molecule is heterologous to the host cell.   
     
     
         8 . The host cell of  claim 1 , further comprising a nucleic acid molecule encoding a santalene synthase comprising the sequence of amino acids set forth in any of SEQ ID NO:17, 52, or 53, or a sequence of amino acids that is at least 95% identical to any of SEQ ID NO:17, 52, or 53, or a catalytically active fragment thereof. 
     
     
         9 . The host cell of  claim 1 , wherein the cell is a prokaryotic cell or an eukaryotic cell. 
     
     
         10 . The host cell of  claim 1 , wherein the cell produces farnesyl diphosphate natively or is modified to produce more farnesyl diphosphate compared to an unmodified cell. 
     
     
         11 . A host cell, comprising:
 (a) a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
 the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 85% sequence identity to SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77; and 
 (ii) the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof catalyzes hydroxylation or monooxygenation of santalene and/or bergamotene; 
   (b) a nucleic acid molecule encoding a cytochrome P450 reductase or catalytically active portion thereof, wherein the encoded cytochrome P450 reductase or catalytically active portion thereof comprises the sequence of amino acids set forth in SEQ ID NO:12 or 13, or a sequence of amino acids that has at least 95% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO:12 or 13; and   (c) a nucleic acid molecule encoding a santalene synthase, wherein the encoded santalene synthase comprises the sequence of amino acids set forth in any of SEQ ID NO:17, 52, or 53 or a sequence of amino acids that is at least 95% identical to any of SEQ ID NO:17, 52, or 53 or a catalytically active fragment thereof, wherein:
 at least one of the nucleic acid molecules set forth in (a) or (b) is heterologous to the host cell. 
   
     
     
         12 . The host cell of  claim 11 , wherein the cell is a prokaryotic cell or an eukaryotic cell. 
     
     
         13 . The host cell of  claim 11 , wherein the cell produces farnesyl diphosphate natively or is modified to produce more farnesyl diphosphate compared to an unmodified cell. 
     
     
         14 . An isolated nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof, wherein:
 (a) the nucleic acid molecule is cDNA;   (b) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 85% sequence identity to SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77; and   (c) the encoded cytochrome P450 oxidase or catalytically active fragment thereof catalyzes hydroxylation or monooxygenation of santalene and/or bergamotene.   
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the encoded cytochrome P450 oxidase polypeptide exhibits at least 95% sequence identity to SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77. 
     
     
         16 . A vector, comprising the nucleic acid molecule of  claim 14 . 
     
     
         17 . A host cell, comprising a vector of  claim 16 . 
     
     
         18 . A method for producing a cytochrome P450 oxidase polypeptide or a catalytically active fragment thereof, comprising:
 (a) culturing the cells of  claim 1  under conditions suitable for expression of the cytochrome P450 oxidase polypeptide; and   (b) optionally isolating the cytochrome P450 oxidase polypeptide.   
     
     
         19 . A method for producing a santalol, bergamotol and/or mixtures thereof, comprising:
 (a) culturing a host cell of  claim 11  under conditions suitable for the formation of a santalol, bergamotol and/or mixtures thereof; wherein the host cell of  claim 11  expresses the nucleic acid molecules of part (a), (b) and (c); and   (b) optionally isolating the santalol, bergamotol and/or mixtures thereof.   
     
     
         20 . The host cell of  claim 1 , wherein the encoded cytochrome P450 oxidase polypeptide or catalytically active fragment thereof comprises a sequence of amino acids set forth in SEQ ID NO:7.