An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae

Background: The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like D-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes D-glucose before the D-xylose can be transported and metabolized. Low affinity D-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of D-glucose and D-xylose is desired as D-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks. Results: Evolutionary engineering of a D-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1-7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on D-xylose by the wild-type strain. Further selection for glucose-insensitive growth on D-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for D-glucose into D-xylose while maintaining high D-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1-7 and GAL2 genes. Conclusions: Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective D-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for D-xylose as compared to D-glucose while maintaining high transport rates. D-glucose and D-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.