AimsOptimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. Methods and ResultsVarious libraries were transformed to a hexose transporter deletion strain, and shuffled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusions 9,4 and 9,6), both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed the same increase in D-xylose affinity (8130mmoll(-1)) compared with Hxt2 (23721mmoll(-1)). The increased D-xylose affinity could be related to the C terminus, more specifically to a cysteine to proline mutation at position 505 in Hxt2. ConclusionsThe Hxt2(C505P) mutation increased the affinity for D-xylose for Hxt2, thus providing a way to increase D-xylose transport flux at low D-xylose concentration. Significance and Impact of the StudyThe gene shuffling protocol using the highly homologues hexose transporters family provides a powerful tool to enhance the D-xylose affinity of Hxt transporters in S. cerevisiae, thus providing a means to increase the D-xylose uptake flux at low D-xylose concentrations.