The cephalotane diterpenoids (e.g., harringtonolide, cephanolides) from plum yews (Cephalotaxus) represent a rare class of tetracyclic C 20 and C 19 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.

A rapid and sensitive method using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was applied for the analysis of the metabolic profile of acarviostatin-containing aminooligosaccharides derived from Streptomyces sp. HO1518. A total of ninety-eight aminooligosaccharides, including eighty potential new compounds, were detected mainly based on the characteristic fragment ions originating from quinovosidic bond cleavages in their molecules. Following an LC-MS-guided separation technique, seven new aminooligosaccharides (10–16) along with four known related compounds (17–20) were obtained directly from the crude extract of strain HO1518. Compounds 10–13 represent the first examples of aminooligosaccharides with a rare acarviostatin II02-type structure. In addition, all isolates displayed considerable inhibitory effects on three digestive enzymes, which revealed that the number of the pseudo-trisaccharide core(s), the feasible length of the oligosaccharides, and acyl side chain exerted a crucial influence on their bioactivities. These results demonstrated that the UPLC-QTOF-MS/MS-based metabolomics approach could be applied for the rapid identification of aminooligosaccharides and other similar structures in complex samples. Furthermore, this study highlights the potential of acylated aminooligosaccharides with conspicuous α-glucosidase and lipase inhibition for the future development of multi-target anti-diabetic drugs.