Title : The oplopanax elatus genome reveals the evolution of a dammaradienol synthase enabling de novo biosynthesis of RK-type ginsenosides
Abstract:
RK-type ginsenosides (Rk1, Rk2 and Rk3) are rare, bioactive dammaradienol-type triterpenoid saponins, yet they are not biosynthesized in cultivated Panax ginseng. Their current supply therefore relies largely on heat-induced conversion of dammarenediol-type ginsenosides, an inefficient process that limits scalable production. The Araliaceae species Oplopanax elatus, a close relative of ginseng, accumulates the upstream triterpene dammaradienol but does not produce RK-type ginsenosides, providing a natural system to identify the missing enzymatic steps.
Using GC–MS and LC–MS metabolite profiling, we confirmed robust accumulation of dammaradienol in O. elatus leaves in the absence of RK-type ginsenosides. We then generated a chromosome-level O. elatus genome and, together with transcriptome profiling and biochemical characterization, identified an oxidosqualene cyclase responsible for dammaradienol formation (dammaradienol synthase, DDES). Comparative genomics across seven Araliaceae species indicates that DDES originated from an ancestral β-amyrin synthase via gene duplication followed by neofunctionalization and subsequent functional specialization. Notably, site-directed mutagenesis demonstrates that a single amino-acid substitution (N260Y) converts DDES into an evolutionary intermediate with broadened product specificity, yielding β-amyrin-, α-amyrin-, and dammaradienol-type activities.
Downstream pathway analysis further suggests that RK-type ginsenoside biosynthesis in O. elatus is interrupted by loss of the cytochrome P450 (CYP) ortholog required for C-12 oxidative tailoring of the dammaradienol scaffold, explaining the observed precursor overaccumulation. Guided by these insights, we reconstructed the pathway in Nicotiana benthamiana by co-expressing O. elatus DDES with ginseng CYPs and O. elatus C3/C6 glycosyltransferases, achieving de novo biosynthesis of ginsenosides Rk1, Rk2 and Rk3. Together, our results illuminate how triterpene cyclases evolve new scaffold specificities and establish a plant chassis platform for engineered production of valuable RK-type ginsenosides.
