Rational spatial rewiring of key enzymes enhances α-santalene production in Saccharomyces cerevisiae

Spatial compartmentalization in eukaryotic cell factories often constrains the efficiency of metabolic pathways. Here, we systematically mapped the subcellular localization of nine core enzymes in the α-santalene biosynthetic pathway of Saccharomyces cerevisiae, identifying metabolic bottlenecks associated with nuclear and endoplasmic reticulum (ER) localization. Through rational spatial engineering, including bioinformatically guided HMG1 truncation to achieve ER release and nuclear export signal (NES) tagging of key enzymes, we successfully rewired enzyme localization to enhance pathway flux. Coupled with promoter engineering to downregulate ERG9, addition-copy integration for IDI1 and ERG20 overexpression, and targeted medium optimization to improve cellular osmotolerance, we achieved substantial synergistic effects on production, leading to a 132-fold increase in α-santalene titer, reaching 568.59 mg/L in fed-batch fermentation. Our results demonstrate that combining subcellular localization engineering with classic metabolic and process optimization offers a robust and generalizable strategy for high-level terpenoid biosynthesis in S. cerevisiae. This approach not only advances the performance of S. cerevisiae cell factories but also holds promise for broader application across other yeast species and eukaryotic microbial hosts.