Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.The baker's yeast Saccharomyces cerevisiae has long served as a model for studying glucose repression, the multi-layer process by which glucose is consumed before all other carbon sources 1,2 . A wide variety of interconnected mechanisms contribute to yeast's ability to sense, respond, and optimize internal metabolism to preferentially consume glucose 3,4 . Transcriptional repressors such as Mig1, Cat8, and the Ssn6/Tup1 complex prevent transcription of glucose-repressed genes, such as those involved in gluconeogenesis and metabolism of alternative carbon sources [5][6][7][8] . The activities of these repressors are then mediated by kinases and phosphatases such as Snf1 and Glc7/Reg1, respectively 6,9,10 . Beyond these intracellular sensing mechanisms, membrane sensors such as Snf3 and Rgt2 allow yeast to sense extracellular sugar concentrations and internalize signals 11 . In sum, the S. cerevisiae glucose repression pathway is a complex network of signals and regulations comprising significant amounts of research and a continuously growing base of knowledge.Recently, a new layer of glucose repression of galactose consumption has been reported to be linked to the kinetic properties of sugar transporters 12 . Because sugars compete for cellular uptake, relative transport efficiency between two sugars will depend on extracellular sugar concentrations as well as transporter affinities (K m values) for each sugar. Consequently, it was reported that the extracellular sugar concentrations coupled with transporter substrate affinity determine the intracellular sugar concentrations 12 . As GAL ge...

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