Brelu-Brelu, D.; Lezin, E.; Miller, E. P.; Zamar, D.-L.; Durand, M.; Pattanaik, S.; Patra, B.; Gautron, N.; Birer Williams, C.; Perrot, T.; Lanoue, A.; Oudin, A.; Le Pogam, P.; Beniddir, M. A.; Giglioli-Guivarc'h, N.; St-Pierre, B.; Massiot, G.; Papon, N.; Yuan, L.; Li, S.; Sun, C.; Besseau, S.; Courdavault, V.

For decades, medicinal plants have been an invaluable source of therapeutics for human health. Among plant specialized metabolites, monoterpene indole alkaloids (MIAs) display remarkable chemical diversity and potent bioactivities, including anticancer properties. While the biosynthetic pathways leading to major MIAs such as vincristine, vinblastine, and camptothecin have been extensively studied, the mechanisms regulating metabolic flux within these pathways remain poorly understood. Here, we uncover an unexpected regulatory layer in MIA biosynthesis involving medium-chain dehydrogenase/reductase (MDR) proteins. Using a combination of in vitro biochemical assays, pathway reconstitution in planta, protein protein interaction analyses, and metabolic engineering in yeast, we show that specific MDRs do not function as classical catalysts but instead enhance both the activity and diversify the stereochemical outputs of geissoschizine synthase (GS). These proteins physically interact with GS and strictosidine {beta}-glucosidase (SGD), forming ternary complexes that likely facilitate substrate channeling of reactive intermediates. Importantly, introduction of these MDRs into engineered yeast strains leads to a dramatic increase in the production of geissoschizine, a central MIA precursor. Collectively, our findings reveal a previously unrecognized role for MDR proteins as regulators of metabolic flux and highlight their potential as powerful tools for metabolic engineering of valuable plant natural products.