The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor. The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.

中文翻译：

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酿酒酵母的合理改造以提高对木质纤维素发酵中多种抑制剂的耐受性

将木质纤维素水解物发酵成乙醇需要强大的具有木糖能力的酿酒酵母菌株，这些菌株能够在存在微生物抑制压力的情况下运行。本研究旨在开发对预处理衍生的微生物抑制剂具有增强耐受性的工业酿酒酵母菌株，方法是鉴定新的基因组合，这些基因组合赋予对多种抑制剂的抗性（因此累积抑制剂抗性表型），同时对木糖发酵能力的影响最小。该策略由双基因盒的多个连续 delta 整合组成，该盒包含一个赋予广泛抑制剂耐受性的基因（ARI1、PAD1 或 TAL1）以及一个抑制剂特异性基因（ADH6、FDH1 或 ICT1）。在生物量生长方面将转化体的性能与亲本菌株进行比较，乙醇产量和生产力，以及合成抑制剂混合物、甘蔗渣水解物以及硬木亚硫酸盐废液的解毒能力。第一轮和第二轮 delta 整合的转化体表现出生物质和乙醇产量之间的权衡。转化体显示出相对于亲本对照增加的抑制剂抗性表型，特别是在用 2% SC 培养基中 40% 和 80% v/v 浓度的浓缩废亚硫酸盐溶液发酵中。出乎意料的是，与亲本对照相比，转化体的木糖发酵能力降低，但某些基因组合的影响较小（例如 TAL1 + FDH1）。TAL1 + ICT1 组合对生物质生长和乙醇产量都有负面影响，这可能与 ICT1 蛋白增加转化体由于细胞膜变化而对弱酸和温度的敏感性有关。所选基因的整合被证明可以增加对合成或工业水解产物中预处理抑制剂的耐受性，但它们仅限于葡萄糖的发酵。然而，一些基因组合序列对木糖转化的影响降低。