Abstract

SUMMARY Camptothecin is a valuable monoterpene indole alkaloid (MIA) with antitumor activity, and strictosamide represents the first specialized intermediate in camptothecin biosynthesis. The proposed camptothecin biosynthetic pathway involves several oxidation–reduction reactions, while there have been no reports on the involvement of 2-oxoglutarate-dependent dioxygenases (2OGDs) in Ophiorrhiza pumila . In this study, the genome-wide identification of 2OGDs was performed in O. pumila , and a total of 140 2OGD genes were identified. Functional characterization revealed that five Op2OGDs catalyze the dehydrogenation of the C-3 and C-14 positions of strictosamide to produce dehydrostrictosamide. Unexpectedly, strictosamide was found to undergo a rapid flavin-mediated photocatalytic skeletal rearrangement to form pumiloside. According to the chromosomal localization of functional Op2OGD genes and camptothecin biosynthetic genes, we identified an MIA gene cluster on chromosome 5 of O. pumila , including two 2OGDs identified in this study and four previously characterized genes. These findings support that camptothecin biosynthesis in O. pumila proceeds through a biosynthetic network rather than a linear pathway. This study provides important insights into the elucidation of the camptothecin biosynthetic pathway.