The Oplopanax elatus genome reveals dammaradienol synthase evolution enabling reconstruction of RK type ginsenosides biosynthesis.

RK-type ginsenosides are a subgroup of dehydrated dammarane saponins with valuable bioactivities, but they are not naturally produced in cultivated ginseng. The ginseng relative Oplopanax elatus accumulates dammaradienol, the precursor scaffold of RK-type ginsenosides, but does not produce RK-type ginsenosides. Here, we identify OeOSC14 as a specialized dammaradienol synthase that evolved through duplication and neofunctionalization of an ancestral multifunctional triterpene synthase by combining chromosome-level genome assembly, comparative genomics and biochemical analyses. Consistent with this evolutionary transition, a single N260Y substitution converts OeOSC14 from a dammaradienol-specific enzyme back into a multifunctional triterpene synthase. We further show that loss of functional C12 hydroxylase blocks the downstream oxidative step required for RK-type ginsenoside biosynthesis in O. elatus. Reconstitution of the pathway in Nicotiana benthamiana enables de novo production of ginsenosides Rk1, Rk2, and Rk3, providing evolutionary insight into triterpene diversification and establishing a plant-based route for producing these compounds.