Identification and Characterization of an Efficient d‑Xylose Transporter in Saccharomyces cerevisiae

d-Xylose is the most abundant hemicellulosic monomer on earth, but wild-type Saccharomyces cerevisiae has very limited d-xylose uptake capacity. We conducted bioprospecting for new sugar transporters from the d-xylose-consuming filamentous fungus Trichoderma reesei and identified three candidates belonging to the major facilitator superfamily. When they were expressed in yeast and assayed for d-xylose uptake, one of them, Xltr1p, had d-xylose transport activity that was more efficient than that of Gal2p, an endogenous yeast transporter. Site-directed mutagenesis was used to examine the functional contributions of 13 amino acid residues for the uptake of d-xylose, and these experiments identified particular amino acids that function distinctly in d-xylose vs glucose transport (e.g., F300). Excitingly, the yeast strain expressing the N326F^Xltr1p variant was able to carry a “high efficiency” transport for d-xylose but was nearly unable to utilize glucose; in contrast, the strain with the F300A^Xltr1p variant grew on glucose but lost d-xylose transport activity.