Reprogramming Carbon Partition for Salidroside Overproduction in <i>Saccharomyces cerevisiae</i>

Salidroside, a valuable plant-derived glycoside, holds great promise for nutraceutical and pharmaceutical applications. Although microbial biosynthesis has been established, further enhancement of its production faces a universal bottleneck in glycoside synthesis: the competition for the glycosyl donor UDP-glucose (UDPG) between essential cell wall construction and target product formation. To overcome this fundamental conflict, we constructed a high-yielding microbial cell factory through a systematic engineering strategy. We first rewired central metabolism via a thiamine diphosphate (ThDP) regeneration strategy to secure a high-level production of the precursor tyrosol. Subsequently, the introduction of a glycosyltransferase <i>Rr</i>U8GT33 from <i>Rhodiola rosea</i> enabled the conversion of tyrosol to salidroside. To address the key limitation, we enhanced UDP-glucose availability by overexpressing UDP-glucose pyrophosphorylase (UGP1) and phosphoglucose mutase (PGM1), and most critically implemented cell wall engineering to dynamically redirect carbon flux from biomass synthesis toward salidroside production by regulating β-1,3-glucan synthase (FKS1) expression. This approach effectively decouples growth pressure from the synthesis demand. Subsequent engineering steps alleviated physiological constraints, yielding a robust production host. In a bioreactor fermentation, the final strain achieved a record-breaking salidroside titer of 40.46 g/L, with a productivity of 0.24 g/(L h) and a yield of 0.27 g/g glucose. This work demonstrates the efficacy of cofactor and cell wall engineering in optimizing glycoside production and provides a scalable strategy for the microbial manufacturing of high-value natural glycosides.