Functional identification of the diterpene synthases exploring the landscape of diterpene structural diversity in...

The genus Isodon is recognized as a primary source for the production of ent-kaurane-type diterpenes and previous studies predominantly focused on ent-kaurene and miltiradiene as the most extensively investigated diterpenes in Isodon. The diversity of diterpene synthases within this genus has recently been acknowledged, while such studies have been largely restricted to single species. In this study, we systematically mined and functionally validated diterpene synthases from three Isodon species using transcriptomic and metabolomic analyses. We identified the expression profiles of genes associated with diterpene biosynthesis and integrated these data with metabolomic results to elucidate their roles within the diterpene biosynthetic pathway. By reconstructing the metabolic pathways in Escherichia coli, we functionally characterized 11 diterpene synthases and elucidated the biosynthetic pathways of several diterpene skeletons originated from nor or ent-CPP, including the previously reported skeletons like ent-kaurene and miltiradiene, as well as four skeletons (ent-13-epi-sandaracopimaradiene, ent-neoabietadiene, abieta-8(14)-en-13-ol synthase and sandaracopimaradiene) whose biogenesis was reported in Isodon genus for the first time. This study provides novel insights into the molecular basis underlying diterpene diversity in Isodon and establishes a valuable resource for the development of new bioactive molecules and potential drug lead compounds.