Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae

Triptolide is a valuable multipotent antitumor diterpenoid in Tripterygium wilfordii, and its C-14 hydroxyl group is often selected for modification to enhance both the bioavailability and antitumor efficacy. However, the mechanism for 14-hydroxylation formation remains unknown. Here, we discover 133kb of tandem duplicated CYP82Ds encoding 11 genes on chromosome 12 and characterize CYP82D274 and CYP82D263 as 14-hydroxylases that catalyze the metabolic grid in triptolide biosynthesis. The two CYP82Ds catalyze the aromatization of miltiradiene, which has been repeatedly reported to be a spontaneous process. In vivo assays and evaluations of the kinetic parameters of CYP82Ds indicate the most significant affinity to dehydroabietic acid among multiple intermediates. The precursor 14-hydroxy-dehydroabietic acid is successfully produced by engineered Saccharomyces cerevisiae. Our study provides genetic elements for further elucidation of the downstream biosynthetic pathways and heterologous production of triptolide and of the currently intractable biosynthesis of other 14-hydroxyl labdane-type secondary metabolites. Hydroxylation at the C-14 position of triptolide is critical for its potent antitumor activity. Here, the authors report two CYP82Ds catalyze the 14-hydroxylation reaction via metabolic grid and achieve heterologous bioproduction of triptolide precursor in engineered Saccharomyces cerevisiae.

Automated summary

In this study, the authors identify two CYP82D enzymes as the 14-hydroxylases in triptolide biosynthesis and successfully produce the precursor of triptolide in engineered Saccharomyces cerevisiae. This discovery provides important genetic elements for understanding the downstream biosynthetic pathways and enabling the production of other 14-hydroxyl labdane-type secondary metabolites.