Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.