Switching the yeast metabolism via manipulation of sugar phosphorylation

Saccharomyces cerevisiae predominantly ferments sugar to ethanol, irrespective of the presence of oxygen, which is known as the Crabtree-effect. Traditional methods rely on static controls of glycolytic flux to make S. cerevisiae Crabtree-negative, which are not favorable for future biomanufacturing applications. Considering native metabolic pathways typically harness dynamic regulatory networks, we therefore aim to develop an alternative strategy using dynamic regulation of the yeast central metabolism to generate Crabtree-negative S. cerevisiae. We report that manipulating a single step at sugar phosphorylation can alter the mode of yeast metabolism with an attenuated Crabtree-effect. By implementing catabolite-regulated sugar phosphorylation, the diauxic shift in budding yeast was effectively reduced. The Crabtree-attenuated metabolism in the engineered yeast was confirmed by multidimensional characterizations such as cell morphology, the measurements of sugar utilization rate and ethanol production, and transcriptomics. In addition, we demonstrated that the Crabtree-attenuated metabolism could substantially improve the mitochondrial synthesis of short branched-chain fatty acids from amino acid catabolism, and allow the synthesis and accumulation of retinaldehyde. Taken together, we present for the first time that manipulation of sugar phosphorylation can alter the mode of yeast metabolism, and the synthetic Crabtree-attenuated yeast factory established here might serve as a non-fermentative biomanufacturing chassis.