To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. ga180 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a ga180 Delta mutant and confirmed that the ga180 Delta mutant showed a xylitol-assimilation phenotype. When the constructed ga180 Delta mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GALS. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2 Delta gal80 Delta double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the ga180 mutant with an initial cell concentration of A(660) = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30 degrees C under oxygen limitation, the ga180 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.