A yeast-based platform for etoposide production via yatein bioconversion.

Etoposide, a semisynthetic derivative of podophyllotoxin originally isolated from the rhizomes of mayapple (Podophyllum peltatum) and indian Podophyllum (P. hexandrum) plants, is one of the most powerful chemotherapeutic agents used to treat various types of solid tumors and blood malignancies. Despite its clinical importance, its supply is recurrently constrained due to a heavy reliance on plant extraction, where low natural precursor abundance and increasing climate-related pressures limit production scalability. Developing alternative manufacturing routes has therefore become a major objective, though reconstruction of this complex biosynthetic pathway has long posed significant challenges, even with recent advances in synthetic biology and metabolic engineering. Yeast has emerged as a robust cellular chassis for reconstituting, either partially or entirely, plant secondary metabolite pathways, and enabling cost-effective bioproduction. Here, we established an integrated biotechnological strategy for the sustainable production of advanced etoposide intermediates using engineered yeast cell factories. By combining pathway refactoring, gene copy number optimization, and tailored co-enzyme compatibility, we established an efficient heterologous pathway converting yatein into (-)-4'-desmethyl-epipodophyllotoxin (4'dEPT) in yeast. Iterative strain engineering improved metabolic flux distribution, leading to enhanced titers and accelerated production kinetics, while process engineering proved essential to maximizing overall system performance. Finally, we also demonstrated the viability of coupling bioproduction in cell factories with downstream, semisynthetic conversion by successfully isolating bioreactor-derived 4'dEPT and converting it into etoposide. In parallel, identifying resilient plant resources that can accumulate high levels of YAT provides a complementary strategy for securing the precursor supply at scale. Overall, this report validates the concept of a hybrid etoposide production platform integrating controlled plant biomass sourcing, engineered yeast cell factories, and chemical transformation steps.