Reprogramming Carbon Flux to Eliminate Crabtree Effect-Associated Ethanol Production for High-Yield <scp>l</scp> -Lactic Acid Biosynthesis in <i>Saccharomyces cerevisiae</i>

The Crabtree effect confers <i>Saccharomyces cerevisiae</i> a growth advantage through fast glucose utilization and ethanol-mediated inhibition of competitors, but constrains glucose conversion to nonethanol products. We reprogrammed metabolism by introducing an orthogonal cytosolic acetyl-CoA synthesis pathway and substituting alcohol dehydrogenase with organic acid dehydrogenase to maintain redox balance, thereby redirecting metabolism from ethanol to NADH-coupled organic acid biosynthesis. Using l-lactic acid as a model, the engineered strain initially showed growth defects, which were recovered by adaptive evolution. This strategy eliminated ethanol production while increasing the glucose-to-l-lactic acid yield from 0.55 to 0.90 g/g. Omics studies revealed that a start-codon mutation in the <i>PYK1</i> gene (pyruvate kinase) impaired glycolytic flux and reduced glucose consumption. This defect was partially improved through targeted reverse engineering by overexpressing genes within the glycolytic pathway that were significantly downregulated. This work provides a framework for metabolic reprogramming in yeast and guidance for NADH-coupled organic acid production.