Discovery, functional genomics, and biochemical characterization of the reserpine biosynthetic gene cluster involved in monoterpenoid indole alkaloid biosynthesis and diversification

Abstract

Monoterpenoid indole alkaloids (MIAs) are structurally complex plant metabolites with important pharmacological uses, yet their biosynthetic and evolutionary origins are incompletely understood. This thesis combines biochemical and phylogenomic approaches to dissect MIA diversification across Gentianales. We identify an ancient geissoschizine synthase (GS) biosynthetic gene cluster (BGC) and a segmentally duplicated reserpine BGC in Catharanthus roseus and Rauvolfia tetraphylla, showing the GS BGC originated near the base of Gentianales while the reserpine BGC arose later in the Rauvofioid clade. Biochemical characterization of the reserpine BGC reveals an oxidase–reductase pair (HYC3O/HYC3R) that effects a C3 stereochemical inversion to produce the 3R-MIA scaffold, and these enzymes are conserved across Gentianales. Reconstructing the evolution of these two BGCs demonstrates how genomic architecture and enzyme innovation generate chemical diversity and shows that tissue-specific expression pattern within a BGC may refine alkaloid profiles. These findings broadly advance drug discovery, synthetic biology strategies, and metabolic engineering applications for MIAs.