Diterpene Synthases and Method for Producing Diterpenoids

Abstract

Provided herein are diterpene synthases (diTPS) and methods for producing diterpenoids. Also provided herein are nucleic acid sequences encoding diTPS, diTPS amino acid sequences, diTPS proteins, vectors, cells, transgenic organisms, uses, compositions, methods, processes, and kits thereof.

1 . An isolated cis-abienol synthase (CAS) polypeptide selected from among:
 a) the polypeptide whose sequence is set forth in one of SEQ ID NOS:7, 50 and 55;   b) an active fragment of the polypeptide of a); and   c) a polypeptide having a sequence of amino acids that has at least 85%, 90% or 95% sequence identity with a polypeptide of a) or b), wherein:   the polypeptide or active fragment catalyzes the formation of cis-abienol from geranylgeranyl diphosphate (GGPP).   
     
     
         2 . The polypeptide of  claim 1  that has a sequence of amino acids that has at least 95% sequence identity with a polypeptide of a) or b). 
     
     
         3 . The polypeptide of  claim 1  that comprises the sequence of amino acids set forth in one of SEQ ID NOS:7, 50 and 55 or an active fragment thereof. 
     
     
         4 . The polypeptide of  claim 1  that consists of the sequence of amino acids set forth in one of SEQ ID NOS:7, 50 and 55 or an active fragment thereof. 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein the isolated polypeptide is an active fragment that is a pseudomature form. 
     
     
         6 . The isolated polypeptide of  claim 5 , wherein the polypeptide has the sequence of amino acid residues set forth in SEQ ID NO:50 or 55, or a sequence that has at least 95% sequence identity with SEQ ID NO:50 or 55. 
     
     
         7 . An isolated nucleic acid molecule, comprising a sequence of nucleotides encoding a cis-abienol synthase (CAS) polypeptide or active fragment thereof having a sequence of amino acids that has at least 85% or 90% sequence identity with a polypeptide whose sequence is set forth in one of SEQ ID NOS:7, 50 and 55 or a catalytically active fragment thereof, wherein:
 the encoded polypeptide or active fragment catalyzes the formation of cis-abienol from geranylgeranyl diphosphate (GGPP); and   the isolated nucleic acid molecule is cDNA.   
     
     
         8 . The isolated nucleic acid molecule of  claim 7 , wherein the active fragment is a pseudomature form. 
     
     
         9 . The isolated nucleic acid molecule of  claim 7 , comprising a sequence of nucleotides that has at least 85% or 90% sequence identity to the sequence of nucleotides set forth in one of SEQ ID NOS:8, 54 and 56 or the complement thereof, wherein the isolated nucleic acid molecule encodes a polypeptide that catalyzes the formation of cis-abienol from geranylgeranyl diphosphate (GGPP) or an active fragment of the polypeptide. 
     
     
         10 . A vector, comprising the nucleic acid molecule of  claim 7 . 
     
     
         11 . A host cell, comprising a nucleic acid molecule encoding a cis-abienol synthase (CAS) polypeptide or active fragment thereof having a sequence of amino acids that has at least 85% or 90% sequence identity with a polypeptide whose sequence is set forth in one of SEQ ID NOS:7, 50 and 55 or a catalytically active fragment thereof, wherein:
 the encoded polypeptide or active fragment catalyzes the formation of cis-abienol from geranylgeranyl diphosphate (GGPP); and   the encoded cis-abienol synthase is heterologous to the host cell.   
     
     
         12 . The host cell of  claim 11  that is a prokaryotic or eukaryotic host cell. 
     
     
         13 . The host cell of  claim 11 , selected from among a bacterial, fungal, plant, insect, amphibian and animal cell. 
     
     
         14 . The host cell of  claim 11  that is an  E. coli  cell or a yeast cell. 
     
     
         15 . The host cell of  claim 11  that produces GGPP. 
     
     
         16 . A method of producing cis-abienol, comprising:
 i) contacting (E,E,E)-geranylgeranyl diphosphate (GGPP) with a cis-abienol synthase (CAS) polypeptide encoded by the nucleic acid molecule of  claim 7  under conditions effective to produce cis-abienol, wherein:   contacting is effected with an isolated CAS polypeptide, or   contacting is effected in a host cell comprising the nucleic acid molecule, and the nucleic acid molecule is heterologous to the host cell; and   ii) optionally, isolating the cis-abienol produced in step i).   
     
     
         17 . The method of  claim 16 , wherein the step of contacting (E,E,E)-geranylgeranyl diphosphate (GGPP) with the cis-abienol synthase (CAS) polypeptide is effected in vitro. 
     
     
         18 . The method of  claim 16 , comprising isolating the cis-abienol. 
     
     
         19 . The method of  claim 18 , further comprising converting the cis-abienol to (−)-ambroxide. 
     
     
         20 . The method of  claim 19 , comprising isolating the ambroxide. 
     
     
         21 . The method of  claim 16 , wherein the step of contacting (E,E,E)-geranylgeranyl diphosphate (GGPP) with the cis-abienol synthase (CAS) polypeptide is effected in vivo.