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

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 (OeLUS^M255L) 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 (Q_p) 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.