Discovery of Class I Diterpene Cyclases Producing a Tetracyclic Cephalotene Skeleton in Plum Yews

The cephalotane diterpenoids (e.g., harringtonolide, cephanolides) from plum yews (Cephalotaxus) represent a rare class of tetracyclic C20 and C19 natural products with intriguing bioactivities. Here, we report the discovery and functional characterization of multiple diterpene synthases (diTPSs) from Cephalotaxus harringtonia and Cephalotaxus hainanensis. In addition to diTPSs responsible for ent-kaurene and biformene biosynthesis, two class I cephalotene synthases (CSs) were found to directly cyclize geranylgeranyl diphosphate (GGPP) into a unique cephalot-12-ene skeleton, which is a crucial step in the biogenesis of cephalotane diterpenoids. A plausible biosynthetic mechanism en route to tetracyclic ring formation was proposed based on an isotope labeling experiment combined with quantum chemical calculations. Site-directed mutagenesis indicated that residue F625 in CS is a key switch of diversifying the hydrocarbon backbone to bicyclic verticillene and tricyclic taxadiene. Altogether this report provides insight into the carbocation cascade reaction driven by active sites in class I diTPSs, promotes our understanding of how chemical diversity originates and evolves in gymnosperms, and lays a solid foundation for future investigation of Cephalotaxus diterpene metabolites.