摘要Oxindoles are a structurally unique subclass of monoterpenoid indole alkaloids(MIAs)with significant medicinal values such as neuroprotective and anticancer properties and are predominantly found in the Rubiaceae plant family.Recent research has only just begun to uncover the biosynthetic pathways of this complex MIA subclass.Here,we report the discovery,characterization,and functional reconstitution of a four-enzyme pathway from Cephalanthus occidentalis(button bush)that enables the complete de novo biosynthesis of heteroyohimbine-type oxindole alkaloids in engineered yeast.Through transcriptomic mining and biochemical validation,we identified and characterized an ajmalicine synthase(CoAJS),a heteroyohimbine/yohimbine/corynanthe C3-oxidase(CoHYC3O),a C3-reductase(CoHYC3R),and an oxindole synthase(CoOIS)capable of converting strictosidine aglycone into 3-epi-ajmalicine and subsequently into the oxindole alkaloids mitraphylline and isomitraphylline.Saturation kinetics of CoOIS revealed comparable catalytic efficiencies for 3-epi-ajmalicine and its C20 epimer akuammigine,indicating substrate tolerance within the heteroyohimbine subclass.Yeast-based reconstruction of the pathway successfully yielded both mitraphylline epimers and the spirooxindole uncarine F.This work elu-cidates the enzymatic logic behind oxindole formation and expands the toolkit for MIA scaffold diversification and synthetic biology.