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

• Published in: Nature communications 2023-02, Vol.14 (1), p.875-875, Article 875
• Main Authors: Zhang, Yifeng, Gao, Jie, Ma, Lin, Tu, Lichan, Hu, Tianyuan, Wu, Xiaoyi, Su, Ping, Zhao, Yujun, Liu, Yuan, Li, Dan, Zhou, Jiawei, Yin, Yan, Tong, Yuru, Zhao, Huan, Lu, Yun, Wang, Jiadian, Gao, Wei, Huang, Luqi
• Format: Article
• Language: English
• Subjects: 38; 42/89; 45; 631/326/2522; 631/449/2491/2174; 631/449/2667; 631/61/318; Anticancer properties; Antitumor activity; Bioavailability; Biosynthesis; Carbon radioisotopes; Chromosome 12; Dehydroabietic acid; Diterpenes; Diterpenes - metabolism; Epoxy Compounds - metabolism; Fungi; Humanities and Social Sciences; Hydroxyl groups; Hydroxylation; Intermediates; Metabolism; Metabolites; multidisciplinary; Phenanthrenes - metabolism; Precursors; Reproduction (copying); Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Science; Science (multidisciplinary); Secondary metabolites; Triptolide; Yeast
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-36353-y

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 133 kb 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 .