Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.