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

Shin, Hyun Yong, Nijland, Jeroen G., de Waal, Paul P., de Jong, René M., Klaassen, Paul, Driessen, Arnold J. M.
Biotechnology for biofuels 2015 v.8 no.1 pp. 176
Saccharomyces cerevisiae, biochemical pathways, engineering, ethanol, feedstocks, fermentation, genes, glucose, hexokinase, lignocellulose, mutagenesis, mutants, pentoses, transporters, xylose, yeasts
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.