Identification of Three Cytochrome P450 Enzymes Responsible for the Biosynthesis of Jujubogenin and Its De Novo Production in Yeast

Jujubosides are the principal bioactive constituents of the traditional Chinese medicine Ziziphi Spinosae Semen and are well-known for their sedative, anxiolytic, and sleep-promoting activities. These compounds are dammarane-type triterpenoid saponins characterized by a unique aglycone, jujubogenin, featuring a 5,6-spiroketal moiety that forms a rigid D/E/F tricyclic scaffold. However, the enzymatic basis underlying the formation of this distinctive spiroketal structure remains elusive. In this study, we identified and functionally characterized three cytochrome P450 enzymes from wild jujubes and Bacopa monnieri. These enzymes cooperatively catalyze an oxidative cascade in which site-selective oxidations at C-16, C-23, and C-30 drive spiroketal formation, converting dammarenediol II into jujubogenin. We further reconstituted the complete biosynthetic pathway in engineered yeast, enabling the de novo production of jujubogenin. This study establishes a foundational platform for the complete biosynthesis of jujubosides in microbial cell factories.