Truncation of LPD1 promoter and adaptive evolution increase cytosolic acetyl-CoA supply in yeast

Acetyl-CoA is a central metabolic intermediate that serves as a key precursor for the biosynthesis of high-value compounds such as terpenoids. However, its compartmentalization within <i>Saccharomyces cerevisiae</i> limits its availability in the cytosol, constraining production of cytosol-derived metabolites. In this study, we aimed to redirect carbon flux toward cytosolic acetyl-CoA synthesis by reducing entry into the tricarboxylic acid cycle. To achieve this, we attenuated <i>LPD1</i> expression by deleting the noncoding RNA SUT526, which is located within the <i>LPD1</i> promoter region and overlaps an upstream regulatory element. This intervention impaired cell growth and hindered the utilization of non-fermentable carbon sources such as ethanol. To address this limitation, adaptive laboratory evolution was performed in ethanol-based medium, leading to rapid recovery of growth and extended cell viability. The evolved strains exhibited enhanced acetyl-CoA synthetase activity and elevated squalene production, suggesting an increased cytosolic acetyl-CoA supply. These improvements reflect enhanced flux through acetyl-CoA-dependent biosynthetic pathways. This work presents a targeted strategy for modulating central carbon metabolism to increase cytosolic acetyl-CoA supply, providing a framework for efficient production of acetyl-CoA derived compounds in yeast.