AimsSaccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S.cerevisiae, directed evolution of the Gal2 transporter was performed. Methods and ResultsThree rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41200 single yeast cells. Gal2 variant 21 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 31 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. ConclusionsRandom mutagenesis of the S.cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. Significance and Impact of the StudyRandom mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.