Efficient biosynthesis of ɑ-bisabolene in S. cerevisiae by combinatorial regulation of acetyl-CoA supply, bisabolene synthase expression, and bisabolene transportation.

α-Bisabolene is an important precursor for fragrances, pharmaceuticals, and biofuels. This study presents a comprehensive metabolic engineering strategy to enhance α-bisabolene production in Saccharomyces cerevisiae. By integrating protein fusion expression, N-terminal fusion tags, dynamic regulation of competing pathways, acetyl-CoA supply enhancement, and endogenous transporter overexpression, we achieved a record α-bisabolene titer of 58.64 g/L in high-cell-density fermentation, with 95.7% extracellular proportion. Key innovations included the structural optimization (order adjustment and N-terminal modification) of the ERG20-AgBIS fusion protein to overcome downstream catalytic bottlenecks, the identification of yeast-endogenous PDR family proteins (Pdr1 and Snq2) for α-bisabolene efflux, and a systematic comparative analysis revealing the superiority of cytosolic over peroxisomal acetyl-CoA supply in S. cerevisiae. By sequentially integrating these downstream "pull" and upstream "push" strategies with diploid hybridization, this work provides a comprehensive, host-specific blueprint for optimizing high-value isoprenoids and underscores the feasibility of microbial biofuels.