Method for producing patchoulol and 7-epi-alpha-selinene

Abstract

The present invention provides a method of producing patchoulol and 7-epi-α-selinene, said method comprising contacting at least one polypeptide with farnesyl phyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce patchoulol and 7-epi-α-selinene, compounds which can be useful in the field of perfumery. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing patchoulol and 7-epi-α-selinene is also an object of the present invention.

What is claimed is: 
     
         1 . A recombinant polypeptide having sesquiterpene synthase activity and comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the polypeptide when expressed produces a mixture of terpenes comprising one or more of patchoulol and 7-epi-α-selinene. 
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% or 98% sequence identity to SEQ ID NO: 1. 
     
     
         3 . The polypeptide of  claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         4 . An isolated recombinant nucleic acid molecule comprising
 a) a nucleotide sequence encoding the polypeptide of  claim 1 ;   b) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 or the complement thereof.   
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or the complement thereof. 
     
     
         6 . An expression vector comprising the nucleic acid molecule of  claim 4  or a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 or the complement thereof. 
     
     
         7 . The expression vector of  claim 6 , wherein the vector is in the form of a viral vector, a bacteriophage or a plasmid. 
     
     
         8 . The expression vector of  claim 6 , wherein the vector further comprises at least one regulatory sequence operably linked to the nucleic acid, and optionally at least one selection marker. 
     
     
         9 . The expression vector of  claim 8 , wherein the at least one regulatory sequence controls transcription, translation initiation or termination, and wherein the at least one regulatory sequence comprises a transcriptional promoter, operator or enhancer or an mRNA ribosomal binding site. 
     
     
         10 . A non-human host organism or a host cell transformed to harbor at least one nucleic acid molecule of  claim 4  or a vector comprising said nucleic acid, so that it heterologously expresses or over-expresses at least one polypeptide encoded by the nucleic acid molecule. 
     
     
         11 . The non-human host organism of  claim 10 , wherein the non-human host organism or host cell is a plant, a microorganism, a prokaryote, or a fungus. 
     
     
         12 . The non-human host organism or host cell of  claim 11 , wherein the non-human host organism or host cell is a bacteria or a yeast. 
     
     
         13 . The non-human host organism or host cell of  claim 12 , wherein the bacteria is  E. coli  and the yeast is  Saccharomyces cerevisiae.    
     
     
         14 . The host cell of  claim 10 , wherein the host cell is a plant cell or a fungal cell. 
     
     
         15 . A method for producing at least one polypeptide having sesquiterpene synthase activity comprising
 a) culturing a non-human host organism or a host cell transformed to express or over-express the polypeptide of  claim 1  under conditions that allow for the production of the polypeptide; and   b) optionally isolating the polypeptide from the non-human host organism or cell cultured in step a) or from culture media used in culturing the non-human host organism or a host cell.   
     
     
         16 . The method of  claim 15 , further comprising, prior to step a), transforming a non-human host organism or host cell with an expression vector comprising the nucleic acid molecule of  claim 4 , so that it harbors said nucleic acid and expresses or over-expresses a polypeptide encoded by said nucleic acid. 
     
     
         17 . A method for preparing a variant polypeptide having sesquiterpene synthase activity comprising a patchoulol and 7-epi-α-selinene synthase activity, the method comprising:
 (a) selecting a nucleic acid according to  claim 4 ; 
 (b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid; 
 (c) transforming host cells or unicellular organisms with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence; 
 (d) screening the polypeptide for at least one modified property; and, 
 (e) optionally, if the polypeptide has no desired variant patchoulol and 7-epi-α-selinene synthase activity, repeating the process steps (a) to (d) until a polypeptide with a desired variant patchoulol and 7-epi-α-selinene synthase activity is obtained; 
 (f) optionally, if a polypeptide having a desired variant patchoulol and 7-epi-α-selinene synthase activity was identified in step d), isolating the corresponding mutant nucleic acid obtained in step (c).