1Department of Chemistry, University of New Brunswick, Fredericton, E3B 5A3, Canada
2Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China
3ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, China
4Zhejiang Key Laboratory of Smart Biomaterials, Zhejiang University, Hangzhou, China
| Received 04 Jul 2025 |
Accepted 21 Sep 2025 |
Published 24 Oct 2025 |
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 elucidates the enzymatic logic behind oxindole formation and expands the toolkit for MIA scaffold diversification and synthetic biology.
