Oxidation of four monoterpenoid indole alkaloid classes by three cytochrome P450 monooxygenases from Tabernaemontana litoralis

SUMMARY Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis in Tabernaemontana litoralis (milky way tree), a prolific producer of rare pseudoaspidosperma‐type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio‐ and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15‐β‐epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15‐α‐epoxytabersonine (lochnericine) found in Catharanthus roseus (Madagascar periwinkle) roots. The pseudovincadifformine 18‐hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10‐hydroxylation of the iboga MIA coronaridine also catalyzes C10‐hydroxylation of voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11‐hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification in T. litoralis , highlighting its potential for further exploration.