Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae

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• 作者：Tatsunori Tani¹ ; Hisataka Taguchi¹ ; Kazuhiro E. Fujimori² ; Takehiko Sahara² ; Satoru Ohgiya³ ; Yoichi Kamagata³ ; Takashi Akamatsu¹ ; ^{akamatsu@bio.sojo-u.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Saccharomyces cerevisiae ; Xylitol assimilation ; Xyt+ mutant ; gal80 defective ; Next-generation sequencing
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：122
• 期：4
• 页码：446-455
• 全文大小：876 K

文摘

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant <em>Saccharomyces cerevisiaeem> 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. <em>gal80em> mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a <em>gal80Δem> mutant and confirmed that the <em>gal80Δem> mutant showed a xylitol-assimilation phenotype. When the constructed <em>gal80Δem> mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the <em>GAL80em>. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A <em>gal2Δ gal80Δem> double mutant did not show xylitol assimilation, whereas expression of <em>GAL2em> under the control of the <em>TDH3em> promoter in the <em>GAL80em> strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the <em>gal80em> 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&deg;C under oxygen limitation, the <em>gal80em> 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.