The direct conversion of strictosamide to pumiloside in planta expands the camptothecin biosynthetic pathway.

Cancer is a globally devastating disease that severely threatens human health and impedes social development. Camptothecin and its derivatives serve as crucial chemotherapeutic agents in clinical cancer treatment, owing to their unique inhibitory activity against DNA topoisomerase I (TOP1). Although the post-modifications of camptothecin (e.g., hydroxylation and subsequent methoxylation) have been well elucidated, the core biosynthetic pathway of camptothecin remains largely unclear. In the present study, we report an unexpected and significant finding: strictosamide can be directly converted to pumiloside both in several plant species (Nicotiana benthamiana, Salvia miltiorrhiza, and Atractylodes macrocephala) and in vitro. We term this process the "Direct Express Train" of the camptothecin biosynthetic pathway. We further demonstrate that this direct conversion proceeds more efficiently under alkaline conditions in vitro. Intriguingly, light was found to effectively facilitate this conversion. Under light exposure, exogenous flavin adenine dinucleotide (FAD) supplementation markedly promoted the reaction, with the final conversion rate reaching 38.8%. These findings not only deepen our understanding of the camptothecin biosynthetic pathway but also provide a novel strategy for the efficient biosynthesis of pumiloside (7) using plant chassis such as N. benthamiana.