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 method for producing a santalol, a bergamotol, and/or a mixture thereof, comprising:
 (a) contacting an acyclic pyrophosphate terpene precursor with a santalene synthase or catalytically active portion thereof under conditions suitable for the formation of a santalene, a bergamotene, and/or a mixture thereof;   (b) contacting the santalene, the bergamotene, and/or the mixture thereof with a cytochrome P450 oxidase polypeptide; and   (c) optionally isolating the santalol, the bergamotol, and/or the mixture thereof; wherein steps (a) and (b) are effected simultaneously or sequentially; and   step (b) occurs in the presence of a cytochrome P450 reductase polypeptide or catalytically active portion thereof; and wherein   the cytochrome P450 reductase polypeptide or catalytically active portion thereof is encoded by a nucleic acid molecule, wherein:   the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 95% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof; and   the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor.   
     
     
         2 . The method of  claim 1 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 96% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof. 
     
     
         3 . The method of  claim 1 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 99% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof. 
     
     
         4 . The method of  claim 1 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO: 13. 
     
     
         5 . The method of  claim 1 , wherein the electron acceptor is a cytochrome P450. 
     
     
         6 . The method of  claim 1 , wherein steps (a) or (b) are effected in vitro or in vivo. 
     
     
         7 . The method of  claim 1 , wherein the acyclic pyrophosphate terpene precursor is farnesyl pyrophosphate. 
     
     
         8 . A method for producing a santalol, a bergamotol, and/or a mixture thereof, comprising:
 (a) culturing a host cell under conditions suitable for the formation of the santalol, the bergamotol, and/or the mixture thereof; and   (b) optionally isolating the santalol, the bergamotol, and/or the mixture thereof; wherein the host cell comprises a nucleic acid molecule encoding a cytochrome P450 reductase or a catalytically active portion thereof, wherein
 (i) the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 95% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof; 
 (ii) the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor; and 
 (iii) the nucleic acid molecule is heterologous to the host cell; and wherein the host cell further comprises:
 (A) a nucleic acid encoding a santalene synthase, wherein the santalene synthase catalyzes the formation of santalene and/or bergamotene; and 
 (B) a nucleic acid encoding a cytochrome P450 oxidase polypeptide. 
 
   
     
     
         9 . The method of  claim 8 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 96% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof. 
     
     
         10 . The method of  claim 8 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 99% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof. 
     
     
         11 . The method of  claim 8 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO: 13. 
     
     
         12 . The method of  claim 8 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a catalytically active fragment comprising a sequence of amino acids set forth in SEQ ID NO: 15, or a sequence that exhibits at least 95% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 15. 
     
     
         13 . The method of  claim 8 , wherein the electron acceptor is a cytochrome P450. 
     
     
         14 . The method of  claim 8 , wherein the host cell is a yeast cell that is a  Saccharomyces  genus cell or a  Pichia  genus cell. 
     
     
         15 . The method of  claim 14 , wherein the host cell is a  Saccharomyces  genus cell and the  Saccharomyces  genus cell is a  Saccharomyces cerevisiae  cell. 
     
     
         16 . The method of  claim 8 , wherein the host cell produces farnesyl diphosphate natively or is modified to produce more farnesyl diphosphate compared to an unmodified cell.