Production of manool

Abstract

Described herein are methods of producing (+)-manool, the methods including: contacting geranylgeranyl diphosphate with a copalyl diphosphate (CPP) synthase to form a (9S, 10S)-copalyl diphosphate and contacting the CPP with a sclareol synthase enzyme to form (+)-manool and derivatives thereof. Also described herein are nucleic acids encoding CPP synthases and sclareol synthases for use in the methods. Further described herein are expression vectors and non-human host organisms and cells including nucleic acids encoding a CPP synthase and a sclareol synthase as described herein.

1 . A method of producing (+)-manool, the method comprising:
 a) contacting geranylgeranyl diphosphate (GGPP) with a copalyl diphosphate (CPP) synthase to form a copalyl diphosphate, wherein the CPP synthase comprises
 a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or 
 b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or 
 c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and 
   b) contacting the CPP with a sclareol synthase to form the (+)-manool; and   c) optionally isolating the (+)-manool.   
     
     
         2 . The method of  claim 1 , wherein the CPP synthase comprises
 a) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or   b) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or   c) a polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.   
     
     
         3 . The method of  claim 1 , wherein step a) further comprises culturing a non-human host organism or cell capable of producing GGPP and transformed to express at least one polypeptide comprising
 a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or   b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or   c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21;   and having a CPP synthase activity, under conditions conducive to a production of CPP.   
     
     
         4 . The method of  claim 1 , wherein the method further comprises, prior to step a), transforming a non-human host organism or cell capable of producing GGPP with
 a) at least one nucleic acid encoding a polypeptide comprising
 a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or 
 b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 and SEQ ID NO: 18; or 
 c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and 
 having a CPP synthase activity, so that said organism or cell expresses said polypeptide having a CPP synthase activity; and 
   b) at least one nucleic acid encoding a polypeptide having a sclareol synthase activity, so that said organism or cell expresses said polypeptide having a sclareol synthase activity.   
     
     
         5 . The method as recited in  claim 4 , wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25. 
     
     
         6 . The method as recited in  claim 1 , further comprising processing the (+)-manool to a (+)-manool derivative using a chemical or biochemical synthesis or a combination of both. 
     
     
         7 . The method as recited in  claim 6 , wherein the derivative is an alcohol, acetal, aldehyde, acid, ether, ketone, lactone, acetate or an ester. 
     
     
         8 . The method as recited in  claim 6 , wherein the derivative is selected from the group consisting of copalol, copalal, manooloxy, Z-11, gamma-ambrol and ambrox. 
     
     
         9 . A method for transforming a host cell or non-human organism, the method comprising transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having a copalyl diphosphate synthase activity and a nucleic acid encoding a polypeptide having a sclareol synthase activity,
 wherein the polypeptide having copalyl diphosphate activity comprises
 a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or 
 b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or 
 c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 and SEQ ID NO: 21; and 
   wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25.   
     
     
         10 . The method as recited in  claim 4 , wherein the host cell or non-human organism is a plant, a prokaryote, or a fungus. 
     
     
         11 . The method as recited in  claim 4 , wherein the non-human host organism or cell is  E. coli  or  Saccharomyces cerevisiae.    
     
     
         12 . An expression vector comprising
 a) a nucleic acid encoding a polypeptide having a CPP synthase activity comprising
 a) an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or 
 b) an amino acid sequence having at least 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or 
 c) an amino acid sequence having at 98%, 99% or 100% sequence identity SEQ ID NO: 20 or SEQ ID NO: 21; or 
   b) a nucleic acid encoding a polypeptide having a CPP synthase activity comprising a nucleotide sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to a nucleic acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.   
     
     
         13 . The expression vector of  claim 12  further comprising
 a) a nucleic acid encoding a polypeptide having a sclareol synthase activity, wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25; or 
 b) a nucleic acid encoding a polypeptide having a sclareol synthase activity comprising a nucleotide sequence having at least 90%, 95%, 98%,99% or 100% sequence identity to SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 33, or SEQ ID NO: 34. 
 
     
     
         14 . A non-human host organism or cell comprising
 a) the expression vector as recited in  claim 12 ; or   b) a nucleic acid encoding a polypeptide having a copalyl diphosphate synthase activity and a nucleic acid encoding a polypeptide having a sclareol synthase activity,
 wherein the polypeptide having copalyl diphosphate activity comprises
 i. an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or 
 ii. an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or 
 iii. an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and 
 
 wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25; and 
 wherein at least one of the nucleic acids is heterologous to the non-human host organism or cell. 
   
     
     
         15 . The non-human host organism or cell of  claim 14 , wherein the non-human host organism or cell is a plant, a prokaryote, a fungus,  Escherichia coli , or  Saccharomyces cerevisiae.    
     
     
         16 . The method as recited in  claim 9 , wherein the host cell or non-human organism is a plant, a prokaryote, or a fungus. 
     
     
         17 . The method as recited in  claim 9 , wherein the non-human host organism or cell is  E. coli  or  Saccharomyces cerevisiae.