title: Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.
abstract: 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. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ 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 gal80 mutant with an initial cell concentration of A660 = 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°C under oxygen limitation, the gal80 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.
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Tani
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan.
authors.forename: Hisataka
authors.initials: H
authors.lastname: Taguchi
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Kazuhiro E
authors.initials: KE
authors.lastname: Fujimori
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Sahara
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Ohgiya
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kamagata
authors.affiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan. Electronic address: akamatsu@bio.sojo-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Akamatsu
mesh_terms: D000449:Aldehyde Reductase / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D005285:Fermentation / Q000187:drug effects; D017353:Gene Deletion; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009004:Monosaccharide Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D010641:Phenotype; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014993:Xylitol / Q000378:metabolism* / Q000494:pharmacology; D014994:Xylose / Q000378:metabolism / Q000494:pharmacology
chemical_list: C106901:GAL2 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D009004:Monosaccharide Transport Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C043910:galactose permease; D014994:Xylose; D000449:Aldehyde Reductase; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); C465226:TDH3 protein, S cerevisiae; D010100:Oxygen; D014993:Xylitol
keywords: Next-generation sequencing; Saccharomyces cerevisiae; Xylitol assimilation; Xyt(+) mutant; gal80 defective