Diterpenoid synthesis

Abstract

The present disclosure relates to nucleic acids that encode polypeptides with cytochrome P450 activity involved in the biosynthesis of plant derived diterpenoids or diterpenes.

1 . A transgenic cell comprising an expression vector adapted to express a nucleic acid molecule comprising at least one nucleotide sequence which is at least 70% identical to SEQ ID NO: 1 and encodes a polypeptide that has casbene oxidase activity. 
     
     
         2 . The transgenic cell of  claim 1 , wherein the vector comprises the nucleotide sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 7, or 8, wherein said nucleotide sequence encodes a casbene oxidase. 
     
     
         3 . The transgenic cell of  claim 1 , wherein said transgenic cell further comprises another expression vector comprising one or more additional nucleotide sequences encoding one or more additional polypeptides involved in the biosynthesis of diterpenoids, or diterpenoid intermediates, wherein the one or more additional nucleotide sequences comprises:
 i) a nucleic acid molecule comprising a nucleotide sequence that is at least 71% identical over the full length sequence set forth in SEQ ID NO: 10 and encodes a polypeptide with 5-keto-casbene 7,8-epoxidase activity; and/or   ii) a nucleic acid molecule comprising a nucleotide sequence that is at least 75% identical over the full length sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25 or 26 and encodes a polypeptide with cytochrome P450 activity.   
     
     
         4 . The transgenic cell of  claim 1 , wherein said transgenic cell is a plant cell. 
     
     
         5 . The transgenic cell of  claim 1 , wherein said transgenic cell is a microbial cell. 
     
     
         6 . The microbial cell of  claim 5 , wherein said microbial cell is a bacterial or fungal cell. 
     
     
         7 . The plant cell of  claim 4 , wherein said plant cell is adapted such that the nucleic acid molecule e is over-expressed when compared to a non-transgenic cell of the same species. 
     
     
         8 . A plant comprising the plant cell of  claim 4 . 
     
     
         9 . The plant of  claim 8 , wherein said plant is from the Euphorbiaceae family. 
     
     
         10 . The plant of  claim 8 , wherein said plant is of the genus  Nicotiana  spp. 
     
     
         11 . The fungal cell of  claim 6 , wherein said fungal cell is  Saccharomyces cerevisiae.    
     
     
         12 . A process for modifying of one or more diterpenoids or diterpenes, comprising:
 i) providing the transgenic plant cell according to  claim 4 ;   ii) cultivating said plant cell to produce a transgenic plant; and optionally   iii) harvesting said transgenic plant, or part thereof.   
     
     
         13 . A process for modifying one or more diterpenes or diterpenoids, comprising:
 i) providing the transgenic microbial cell according to  claim 5  that expresses at least one diterpene and/or diterpenoid metabolite;   ii) cultivating the microbial cell under conditions that modify one or more diterpenes or diterpenoids; and optionally   iii) isolating said diterpenoid from the microbial cell or cell culture.   
     
     
         14 . The transgenic cell of  claim 1 , wherein the expression vector further comprises one or more additional nucleotide sequences encoding one or more additional polypeptides involved in the biosynthesis of diterpenoids, or diterpenoid intermediates, wherein the one or more additional nucleotide sequences comprises:
 i) a nucleic acid molecule comprising a nucleotide sequence that is at least 71% identical over the full length sequence set forth in SEQ ID NO: 10 and encodes a polypeptide with 5-keto-casbene 7,8-epoxidase activity; and/or   ii) a nucleic acid molecule comprising a nucleotide sequence that is at least 75% identical over the full length sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25 or 26 and encodes a polypeptide with cytochrome P450 activity.