ABSTRACTXylose is the second most abundant sugar in lignocellulose and can be used as a feedstock for next-generation biofuels by industry.Saccharomyces cerevisiae, one of the main workhorses in biotechnology, is unable to metabolize xylose natively but has been engineered to ferment xylose to ethanol with the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes fromScheffersoymces stipitis. In the scientific literature, the yield and volumetric productivity of xylose fermentation to ethanol in engineeredS. cerevisiaestill lagsS. stipitis, despite expressing of the same XR-XDH genes. These contrasting phenotypes can be due to differences inS. cerevisiae’sredox metabolism that hinders xylose fermentation, differences inS. stipitis’redox metabolism that promotes xylose fermentation, or both. To help elucidate howS. stipitisferments xylose, we used flux balance analysis to test various redox balancing mechanisms, reviewed published omics datasets, and studied the phylogeny of key genes in xylose fermentation.In vivoandin silicoxylose fermentation cannot be reconciled without NADP phosphatase (NADPase) and NADH kinase. We identified eight candidate genes for NADPase.PHO3.2was the sole candidate showing evidence of expression during xylose fermentation. Pho3.2p and Pho3p, a recent paralog, were purified and characterized for their substrate preferences. Only Pho3.2p was found to have NADPase activity. Both NADPase and NAD(P)H-dependent XR emerged from recent duplications in a common ancestor ofScheffersoymcesandSpathasporato enable efficient xylose fermentation to ethanol. This study demonstrates the advantages of using metabolic simulations, omics data, bioinformatics, and enzymology to reverse engineer metabolism.