Technical Abstract: Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI), which catalyzes conversion of xylose to xylulose. In this study, an automated two-hybrid interaction protocol was used to find yeast genes encoding proteins that bind XI to identify potential targets for improving xylose utilization by S. cerevisiae. A pDEST32 vector re-engineered for TRP selection and containing the Gal4 binding domain fused with the Piromyces sp.E2 XI open reading frame (ORF) was used as bait with a library of LEU-selectable pOAD vectors containing the Gal4 activation domain in fusion with members of the S. cerevisiae genome ORF collection. Binding of a yeast ORF protein to XI activates two chromosomally located reporter genes in a PJ69-4 yeast strain to give selective growth. Five genes, including ADH1, were identified in the two-hybrid screen, suggesting the proteins encoded by these genes bind to XI. The effect of ADH1 over-expression was examined using the pSUMOduoHisADH1 vector in an automated protocol to transform eight previously identified yeast strains that showed anaerobic growth on xylose. One transformant consumed all available glucose, xylose, and arabinose during growth on wheat straw hydrolysate.