Method for producing fragrant alcohols

Abstract

This invention relates generally to methods and compositions for producing a sesquiterpene alcohol comprising contacting a sesquiterpene with a P450 polypeptide with monooxygenase activity.

What is claimed is: 
     
         1 . A method of producing an sesquiterpene alcohol comprising:
 i) contacting a terpene of Formula I:   
       
         
           
           
               
               
           
         
         with a polypeptide comprising an amino acid sequence having at least 90% sequence identify to a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 79, and SEQ ID NO: 81; and 
         ii) optionally isolating the alcohol, 
         wherein R is a saturated, mono-unsaturated or poly-unsaturated aliphatic group composed of 9 carbons and wherein R can be a branched chain or composed of one or more non-aromatic rings. 
       
     
     
         2 . The method of  claim 1 , wherein the alcohol comprises α-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol and/or mixtures thereof. 
     
     
         3 . A method of producing a sesquiterpene alcohol comprising α-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol, epi-β-santalol, lancelol and/or mixtures thereof comprising contacting α-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the sesquiterpene alcohol produced comprises at least about 36% of a cis isomer. 
     
     
         4 . The method of  claim 3 , wherein the sesquiterpene alcohol produced comprises at least 46%, 50%, 72%, 96% or 100% of a cis isomer. 
     
     
         5 . The method of  claim 3 , wherein the polypeptide comprises an amino acid sequence having at least 90%, 95%, 98%, or 100% sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73. 
     
     
         6 . The method of  claim 4 , wherein the polypeptide comprises an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73. 
     
     
         7 . An isolated polypeptide having monooxygenase activity comprising an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 79, and SEQ ID NO: 81. 
     
     
         8 . An isolated nucleic acid molecule comprising: i) the nucleic acid sequence of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 78 or SEQ ID NO: 80; or ii) a nucleic acid molecule that encodes the polypeptide of  claim 7 . 
     
     
         9 . A method for producing a polypeptide having P450 monoxygenase activity comprising transforming a host cell or non-human organism with the nucleic acid of  claim 8 ; and culturing the host cell or organism under conditions that allow for the production of the polypeptide. 
     
     
         10 . The method of  claim 3  comprising
 i) cultivating an isolated cell under conditions suitable to produce a P450 polypeptide having monooxygenase activity, wherein the cell:
 a) produces a acyclic pyrophosphate terpene precursor; 
 b) expresses a P450 reductase, 
 c) expresses a polypeptide that has α-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene synthase activity and that produces one ore more α-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene, and 
 d) expresses the polypeptide having P450 monooxygenase activity; and 
 
 ii) optionally isolating the alcohol from the cell. 
 
     
     
         11 . The method of  claim 10 , wherein the acyclic pyrophosphate terpene precursor is selected from the group consisting of geranyl-pyrophosphate (GPP), farnesyl-diphosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP). 
     
     
         12 . A vector comprising
 i) the nucleic acid molecule of  claim 8 ; or   ii) a nucleic acid encoding a polypeptide having a P450 monoxygenase activity comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 78 or SEQ ID NO: 80.   
     
     
         13 . The vector of  claim 12 , wherein the vector is a prokaryotic vector, viral vector or a eukaryotic vector. 
     
     
         14 . The vector of  claim 12 , wherein the vector is an expression vector. 
     
     
         15 . A host cell or non-human organism comprising the nucleic acid molecule of  claim 8 , or a vector comprising said nucleic acid molecule. 
     
     
         16 . The method of  claim 10 , wherein the cell is a prokaryotic cell or a eukaryotic cell. 
     
     
         17 . The method of  claim 16 , wherein the prokaryotic cell is a bacterial cell. 
     
     
         18 . The method of  claim 16 , wherein the eukaryotic cell is a yeast cell or a plant cell. 
     
     
         19 . The method of  claim 1  comprising
 i) cultivating an isolated cell under conditions suitable to produce the polypeptide having P450 monooxygenase activity, wherein the cell:
 a) produces a acyclic pyrophosphate terpene precursor; 
 b) expresses a P450 reductase, 
 c) expresses a polypeptide that has α-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene synthase activity and that produces one or more α-farnesene, β-farnesene, α-santalene, β-santalene, α-trans-bergamotene, epi-β-santalene, and/or β-bisabolene, and 
 d) expresses the polypeptide; and 
 
 ii) optionally isolating the alcohol from the cell. 
 
     
     
         20 . The method of  claim 1 , wherein step a) comprises cultivating a non-human host organism or cell capable of producing a acyclic pyrophosphate terpene precursor and transformed to express one or more of the polypeptide.