A precursor-driven triterpenoid platform enables high-level biosynthesis of structurally distinct triterpenoids in Saccharomyces cerevisiae.

Sustainable microbial biomanufacturing of high-value triterpenoids in Saccharomyces cerevisiae is severely constrained by native sterol homeostasis, which rapidly channels the central intermediates 2,3-oxidosqualene (OSQ) and 2,3;22,23-dioxidosqualene (DOS) into essential ergosterol biosynthesis via lanosterol synthase (Erg7p). To break this homeostatic bottleneck without compromising cell fitness, a precursor-driven strategy utilizing post-translational ubiquitin-based N-degrons was developed to dynamically attenuate Erg7p stability. Coupled with systematic storage sink remodeling and pathway optimization, the engineered chassis achieved record-high shake-flask titers of 1.4 g/L OSQ and 3.1 g/L DOS. The versatility of this dual-precursor platform was validated through the optimized biosynthesis of pentacyclic betulinic acid (BA) and tetracyclic mogrol. For BA, balancing the gene dosage of an engineered cyclase (OeLUSM255L) and cytochrome P450 components (CYP716A155/AtCPR1) eliminated the rate-limiting betulin bottleneck, yielding 298.7 mg/L in flasks. Scale-up in a 7-L bioreactor using a two-stage fed-batch fermentation pushed total lupane-type triterpenoid titer to 8.5 g/L (including 693.5 mg/L BA). For mogrol, implementing a physical fusion architecture (AtCPR2-CYP87D18) and pathway optimization enabled a shake-flask titer of 8.4 mg/L. Scaled-up to a 50-L pilot-scale bioreactor via a simplified one-stage glucose-only fed strategy bypassed carbon-source shifting stress and compressed the fermentation time by 46-50% compared to two-stage processes, delivering a record-high titer of 124.5 mg/L with an unprecedented volumetric productivity of 1.6 mg/L/h (Qp) and a massive residual DOS pool of 21.3 g/L. This work establishes a highly generalizable metabolic rewiring paradigm, offering a scalable template for sustainable industrial biomanufacturing of complex natural products.