Alleviating the Crabtree effect for high-level production of squalene in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used biomanufacturing chassis; however, its inherent Crabtree effect severely limits energy yield and diverts carbon flux away from the desired non-ethanol products. To address this challenge, we developed a multidimensional metabolic engineering strategy to alleviate the Crabtree effect, using squalene high-level production as a model. Central metabolism was first modulated through three coordinated interventions: modulation of the SNF1/AMPK energy-sensing system, introduction of an A81D variant of the global transcriptional regulator MTH1, and enhancement of mitochondrial respiration via PET122-PET494 co-expression. These modifications reduced ethanol overflow by >13.35%, supporting an attenuated Crabtree effect. Subsequently, cellular robustness was enhanced to counter ethanol stress and redox imbalance: strengthening thiamine biosynthesis alleviated stress and favored squalene biosynthesis, while improved osmotolerance preserved redox balance, membrane integrity, and mitochondrial function during high-density fermentation. This optimized state enabled C47 to achieve a record squalene titer of 57.2 g/L in a 50-L fed-batch bioreactor, with a corresponding productivity: 0.47 g/L/h. A reduced respiratory quotient (1.3 to 1.0) during squalene production further supported the alleviation of the Crabtree effect. In summary, this study establishes a versatile framework for alleviating the Crabtree effect in S. cerevisiae, facilitating the high-level biosynthesis of squalene and other valuable non-ethanol products.