Inokuma Kentaro, Iwamoto Ryo, Bamba Takahiro, Hasunuma Tomohisa, Kondo Akihiko
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
Front Bioeng Biotechnol. 2017 Dec 20;5:81. doi: 10.3389/fbioe.2017.00081. eCollection 2017.
Xylose-assimilating yeasts with tolerance to both fermentation inhibitors (such as weak organic acids) and high temperature are required for cost-effective simultaneous saccharification and cofermentation (SSCF) of lignocellulosic materials. Here, we demonstrate the construction of a novel xylose-utilizing strain with improved fermentation ability under heat and acid co-stress using the drug resistance marker-aided genome shuffling technique. The mutagenized genome pools derived from xylose-utilizing diploid yeasts with thermotolerance or acid tolerance were shuffled by sporulation and mating. The shuffled strains were then subjected to screening under co-stress conditions of heat and acids, and the hybrid strain Hyb-8 was isolated. The hybrid strain displayed enhanced xylose fermentation ability in comparison to both parental strains under co-stress conditions of heat and acids. Hyb-8 consumed 33.1 ± 0.6 g/L xylose and produced 11.1 ± 0.4 g/L ethanol after 72 h of fermentation at 38°C with 20 mM acetic acid and 15 mM formic acid. We also performed transcriptomic analysis of the hybrid strain and its parental strains to screen for key genes for multiple stress tolerances. We found that 13 genes, including 5 associated with cellular transition metal ion homeostasis, were significantly upregulated in Hyb-8 compared to levels in both parental strains under co-stress conditions. The hybrid strain Hyb-8 has strong potential for cost-effective SSCF of lignocellulosic materials. Moreover, the transcriptome data gathered in this study will be useful for understanding the mechanisms of multiple tolerance to high temperature and acids in yeast and facilitate the development of robust yeast strains for SSCF.
木质纤维素材料的经济高效同步糖化与共发酵（SSCF）需要能够同时耐受发酵抑制剂（如弱酸）和高温的木糖同化酵母。在此，我们展示了利用耐药标记辅助基因组改组技术构建一种新型木糖利用菌株，该菌株在热和酸共同胁迫下具有提高的发酵能力。源自具有耐热性或耐酸性的木糖利用二倍体酵母的诱变基因组库通过孢子形成和交配进行改组。然后将改组后的菌株在热和酸的共同胁迫条件下进行筛选，分离出杂交菌株Hyb-8。与两个亲本菌株相比，该杂交菌株在热和酸的共同胁迫条件下显示出增强的木糖发酵能力。在38°C、含有20 mM乙酸和15 mM甲酸的条件下发酵72小时后，Hyb-8消耗了33.1±0.6 g/L木糖，并产生了11.1±0.4 g/L乙醇。我们还对杂交菌株及其亲本菌株进行了转录组分析，以筛选多重胁迫耐受性的关键基因。我们发现，与两个亲本菌株在共同胁迫条件下的水平相比，Hyb-8中有13个基因显著上调，其中5个基因与细胞过渡金属离子稳态相关。杂交菌株Hyb-8在木质纤维素材料的经济高效SSCF方面具有强大潜力。此外，本研究收集的转录组数据将有助于理解酵母对高温和酸的多重耐受性机制，并促进用于SSCF的健壮酵母菌株的开发。
