Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter

The capacity to co-transport glucose and xylose into yeast has remained a technical challenge in the field. While significant efforts have been made in transporter engineering to increase xylose transport rates, glucose-based inhibition still limit most of these transporters. To address this issue, we further engineer sugar transporter proteins to remove glucose inhibition and enable glucose/xylose co-transport. Specifically, we start with our previously derived CiGXS1 FIM mutant strain and subjugate it to several rounds of mutagenesis and selection in a hexose metabolism null strain. Through this effort, we identify several mutations including N326H, a truncation in the C-terminal tail, I171F, and M40V as additionally dominant for reducing glucose inhibition. The resulting transporter shows substantially improved xylose transport rates in the presence of high quantities of glucose including up to 70 g/L glucose. Moreover, the resulting transporter enables co-utilization of glucose and xylose with glucose rates on par with a wild-type transporter and xylose rates exceeding that of glucose. These results demonstrate that major facilitator superfamily hexose transporters can be rewired into glucose-xylose co-transporters without functional inhibition by either substrate. These results enhance the potential of using lignocellulosic biomass as a feedstock for yeast.