Identification and catalytic mechanisms of key enzymes in the biosynthesis of asperosaponin VI from Dipsacusasperoides.

Asperosaponin Ⅵ is the major active compound in the traditional Chinese medcinal herb Dipsacus asperoides (Xu-Duan), exhibits diverse pharmacological activities, including treating osteoporosis, anti-inflammatory effects, and neuroprotective properties. However, due to limited natural sources and challenges in chemical extraction, heterologous biosynthesis offers a strategy for efficient production of asperosaponin Ⅵ and its intermediates and derivatives. Despite its potential, the biosynthetic pathway of asperosaponin Ⅵ remains poorly understood, primarily due to its complex multi-step reaction network. Here, key enzymes involved in the biosynthesis of asperosaponin Ⅵ were identified through transcriptomic, phylogenetic, and WGCNA analysis. Two β-amyrin synthases (DaOSC2 and DaOSC5), two cytochrome P450 enzymes (DaCYP714E29 and DaCYP714E30), and an arabinosyltransferase (DaUGT85J2) were characterized. Moreover, DaOSC2 and DaOSC5 represent the first reported β-amyrin synthases in the Dipsacus genus; DaCYP714E29 and DaCYP714E30 are the first cytochromes P450 enzymes identified in Dipsacaceae family to catalyze the oxidation of oleanolic acid at C-23 position; Additionally, DaUGT85J2 was identified as the first arabinosyltransferase responsible for catalyzing the 3-O-arabinosylation of hederagenin. To further elucidate the catalytic mechanisms of DaCYP714E29 and DaUGT85J2, molecular docking and mutagenesis experiments were conducted. These findings provide insights into the biosynthetic pathway of asperosaponin Ⅵ and lay the foundation for engineering efficient production pathways for this valuable compound.