Biosynthesis of Monoterpene Indole Alkaloids in Tabernanthe iboga

Plants have evolved to produce specialised metabolites that enhance their ecological fitness. Tabernanthe iboga (iboga) produces monoterpene indole alkaloids (MIAs), valued for their chemical complexity and therapeutic potential. Native to West Africa, iboga holds cultural significance for its use in spiritual practices due to the psychoactive effects of ibogaine, its most well-known MIA. In addition to ibogaine, iboga produces other MIAs with intricate chemical structures, underexplored biological properties, and unknown biosynthetic pathways. This thesis elaborates on the discovery and characterization of enzymes involved in the biosynthesis of these rare MIA scaffolds in T. iboga. Through state-of-the-art plant pathway elucidation techniques combined with classical biochemical methods, this work reveals the pathway discovery of elusive MIAs and presents mechanistic proposals for their biosynthesis for the first time. Key discoveries include the elucidation of pseudo-tabersonine and pseudo-vincadifformine biosynthesis, along with a revised mechanism for the biosynthesis of coronaridine. These studies also demonstrate how biosynthetic enzymes are reused in different sequences to generate alternative scaffolds through intricate redox reactions. Additionally, the development of a streamlined screening platform using Nicotiana benthamiana leaf disks and Catharanthus roseus flowers led to the discovery of the oxidative tailoring enzymes pachysiphine synthase (TiPS) and tabersonine-16-hydroxylase (TiT16H), discoveries that illuminated plant strategies for structural diversification. Finally, the divergent biosynthesis of four MIA scaffolds from geissoschizine is explored. Oxidative rearrangements, catalyzed by P450 enzymes, drive new bond formations, including a unique C-N bond leading to mavacurane-type alkaloids in the related plant species Catharanthus roseus. The versatility of geissoschizine is investigated through functional characterization and gene silencing, revealing how neo-functionalized P450s drive scaffold divergence earlier on in MIA biosynthesis. Overall, this thesis advances our understanding of MIA biosynthesis in plants, particularly in T. iboga, and introduces new approaches to addressing challenges in plant pathway discovery, especially in non-model species.