Regulating protein glycosylation modification enhances the synthesis of taxadiene in Saccharomyces cerevisiae

Utilizing synthetic biology techniques to construct recombinant engineered cells for the synthesis of paclitaxel and its key precursors has emerged as an effective method to address the supply-demand imbalance and protect rare plant resources. Taxadiene, a critical precursor of paclitaxel, exhibits limited yield in eukaryotic systems, constraining its biosynthetic potential. Previous research has demonstrated that glycosylation modifications in <i>Saccharomyces cerevisiae</i> substantially impact the regulation of exogenous protein expression. In this study, we found that knocking out endogenous protein glycosylation genes in the chassis significantly improved the expression of heterologous proteins, especially the key taxadiene synthase (TS), and thereby increased the yield of taxadiene. In particular, we identified that the deletion of the glycosyltransferase gene <i>mnn11</i> increased taxadiene production levels by 65.2 %. The subsequent multi-copy integration of the key enzyme taxadiene synthase further elevated taxadiene production levels in shake flasks by more than 3-fold, reaching 113.5 mg/L. Moreover, the enhancement of geranylgeranyl diphosphate synthesis-related expression modules resulted in a 2.69-fold increase in taxadiene yield, to 420.4 mg/L. Following the optimization of fed-batch fermentation conditions, taxadiene production levels of up to 1.26 g/L were achieved, representing a 63-fold improvement over that obtained with the initial strain. Our findings provide valuable insights into enhancing heterologous taxadiene production efficiency by blocking endogenous protein glycosylation modifications in <i>S. cerevisiae</i>, establishing a critical precedent for improving compatibility between natural product synthesis and microbial cell factories.