By identifying four key enzymes from the North American plant Cephalanthus occidentalis (button bush) and reconstituting them in yeast, the scientists achieved complete de novo biosynthesis of complex oxindole molecules that are otherwise difficult to obtain from plants or chemical synthesis.
Monoterpenoid indole alkaloids (MIAs) represent one of nature’s most diverse and pharmacologically potent chemical families, with more than 3,000 known members. Several MIAs are already used clinically, including anticancer and antiviral drugs. Within this family, oxindole alkaloids stand out for their distinctive spirocyclic structures and pronounced bioactivities in the central nervous system, as well as emerging anticancer potential. Despite their promise, oxindoles are typically found in small amounts in a limited number of plant species, mainly within the Rubiaceae family, which restricts their broader study and application. Although earlier work had identified enzymes capable of converting certain alkaloids into oxindoles, a complete, step-by-step biosynthetic pathway—from a universal plant precursor to finished oxindole products—had not been demonstrated in a heterologous system.
A study (DOI: 10.1016/j.bidere.2025.100049) published in BioDesign Research on 24 September 2025 by Yang Qu’s team, University of New Brunswick, not only clarifies how plants generate these structurally intricate alkaloids but also establishes a foundation for producing them sustainably using synthetic biology.
Using an integrated analytical, transcriptomic, and synthetic biology workflow, this study systematically elucidated oxindole alkaloid biosynthesis in Cephalanthus occidentalis and reconstructed the pathway in yeast. First, total leaf alkaloids were extracted by an acid–base protocol and profiled using LC–MS/MS, followed by preparative TLC purification and structural elucidation with 1D/2D NMR. This approach revealed five major monoterpenoid indole alkaloids, including strictosidine, ajmalicine, mitraphylline, and the previously unreported epimers 3-epi-ajmalicine and isomitraphylline, indicating active C3 and C7 epimerization in planta. To identify the underlying biosynthetic genes, leaf transcriptomes were sequenced and mined by comparative analysis with known MIA pathways, leading to the identification of an ajmalicine synthase (CoAJS), a C3-oxidase/reductase pair (CoHYC3O/CoHYC3R), and a candidate oxindole synthase cytochrome P450 (CoOIS). Functional validation was carried out by heterologous expression in an engineered yeast strain capable of producing strictosidine aglycone. Expression of CoAJS alone yielded ajmalicine and related heteroyohimbines, while subsequent introduction of CoHYC3O and CoHYC3R enabled stepwise oxidation–reduction to generate 3-epi-ajmalicine. Expression of CoOIS or the related enzyme Ms3eCIS in yeast feeding assays demonstrated their ability to convert 3R-heteroyohimbines into mitraphylline and isomitraphylline, and to further diversify akuammigine into four oxindole diastereomers, predominantly with 7R stereochemistry. Substrate screening revealed that CoOIS is highly selective for 3R-heteroyohimbines, whereas Ms3eCIS displays broader catalytic promiscuity across multiple MIA scaffolds. Enzyme kinetics showed comparable catalytic efficiencies of CoOIS toward 3-epi-ajmalicine and akuammigine, despite differences in substrate affinity and turnover. Finally, full pathway reconstruction in yeast confirmed de novo production of mitraphylline epimers and uncarine F at microgram-per-liter levels, establishing a proof-of-concept microbial platform for oxindole alkaloid biosynthesis.
By demonstrating that complex oxindole alkaloids can be produced from simple precursors in yeast, this work opens new avenues for sustainable manufacturing of high-value natural products. Microbial production could bypass the need for slow-growing or endangered medicinal plants and provide consistent access to compounds with therapeutic potential for neurological disorders, cancer, and pain management. Beyond production, the clarified enzymatic logic offers a powerful platform for generating new-to-nature oxindole analogs that may exhibit improved or entirely novel bioactivities.
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References
DOI
10.1016/j.bidere.2025.100049
Original Source URL
https://doi.org/10.1016/j.bidere.2025.100049
Funding information
This research is supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and a New Brunswick Innovation Foundation (NBIF) Research Assistantship Initiative Grant to Y.Q.
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