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Abstract BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation ofd-xylose. Many yeast species within and closely related to the generaSpathasporaandScheffersomyces(both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD⁺to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressedXYLgenes from multipleScheffersomycesspecies in a strain ofSaccharomyces cerevisiae. RecombinantS. cerevisiaeexpressingXYL1fromScheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared toS. cerevisiaestrain expressingXYLgenes fromScheffersomyces stipitis. ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeastSc. xylosifermentansas a potential source for genes that can be engineered intoS. cerevisiaeto improve xylose fermentation and biofuel production.