AIMS: S. 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 RESULTS: Three 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 41,200 single yeast cells. Gal2 variant 2.1 harboring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3.1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose.
CONCLUSIONS: Random 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 STUDY: Random 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. This article is protected by copyright. All rights reserved.