Construction Of Biosynthetic Pathway Of Betulinic Acid In Yeast Based On The Discovery Of The Cytochrome P450 Enzyme RoCYP-34445

It has been documented that betulinic acid(BA)is one of important anticancer constituents of many traditional Chinese medicinal herbs,including Prunella vulgaris,Pycnonotus sinensis,Nerium indicum and Rosmarinus officinalis(rosemary).And modern pharmacological studies have exhibited that BA and its derivatives semi-synthesized from BA possess bright prospects for development of both anticancer drugs and anti-HIV drugs.So far,industrial production of BA dependents on isolation from plants or semi-synthesis,which is time-costing,labor-consuming and environment-hazardous.Synthetic biology provides a new potential approaches for industrial production of BA.In this study,through deeply mined the released transcriptome of rosemary,three cytochrome P450 enzyme(CYP)candidates which could oxydize lupeol to yield BA were discovered.Phylogenetic tree analysis revealed that these candidates gene belonged to the CYP85 clan.In the experiments of functional characterization of these CYPs,it was indicated that RoCYP-34445 selectively and efficiently catalyzed conversion of lupeol to BA in yeast.The RoCYP-34445 gene was indeed a cytochrome P450 gene involved in biosynthesis of betulinic acid in rosemary by gene expression pattern analysis.In order to construct biosynthetic pathway of betulinic acid in yeast,the BA-producing yeast was established by introducing the lupeol synthase gene AtLUPs of Arabidopsis thaliana and RoCYP-34445 into yeast.The study laid the foundation for industrial production of BA by metabolic engineering approaches in the future,and provide a new strategy to fully use vast amounts of sequencing data which have been released.