High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance. We have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4E269* to further enhance acetic acid tolerance, from a range of 0.8–1.2% acetic acid at pH 4.7, to previously unmatched levels for Saccharomyces cerevisiae. For that purpose, we crossed the WG transformant with strain 16D, a previously identified strain displaying very high acetic acid tolerance. Quantitative trait locus (QTL) mapping with pooled-segregant whole-genome sequence analysis identified four major and two minor QTLs. In addition to confirmation of snf4E269* in QTL1, we identified six other genes linked to very high acetic acid tolerance, TRT2, MET4, IRA2 and RTG1 and a combination of MSH2 and HAL9, some of which have never been connected previously to acetic acid tolerance. Several of these genes appear to be wild-type alleles that complement defective alleles present in the other parent strain. The presence of several novel causative genes highlights the distinct genetic basis and the strong genetic background dependency of very high acetic acid tolerance. Our results suggest that elimination of inferior mutant alleles might be equally important for reaching very high acetic acid tolerance as introduction of rare superior alleles. The superior alleles of MET4 and RTG1 might be useful for further improvement of acetic acid tolerance in specific industrial yeast strains.

中文翻译：

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酿酒酵母中非常高的乙酸耐受性的多基因分析揭示了复杂的遗传背景和几个新的致病等位基因。

由于木质纤维素水解产物中的高乙酸含量，高乙酸耐受性在用于第二代生物乙醇生产的工业酵母菌株中非常重要。它在第一代淀粉水解物和含有大量乙酸的酸面团中也很重要。我们之前已将 snf4E269* 鉴定为在全基因组 (WG) 转化和选择以提高乙酸耐受性后获得的菌株 MS164 中的致病等位基因。我们现在已经使用相同的 WG 转化子 MS164 进行了多基因分析，以鉴定与 snf4E269* 相互作用的新致病等位基因，以进一步增强乙酸耐受性，从 pH 4.7 的 0.8-1.2% 乙酸范围到以前无法匹配的酿酒酵母水平。为此，我们将 WG 转化体与菌株 16D 杂交，一种先前鉴定的菌株，显示出非常高的乙酸耐受性。数量性状基因座 (QTL) 作图与汇集分离的全基因组序列分析确定了四个主要和两个次要 QTL。除了在 QTL1 中确认 snf4E269* 之外，我们还鉴定了其他六个与非常高的乙酸耐受性相关的基因，TRT2、MET4、IRA2 和 RTG1 以及 MSH2 和 HAL9 的组合，其中一些以前从未与乙酸耐受性相关联. 这些基因中的一些似乎是野生型等位基因，可补充其他亲本菌株中存在的缺陷等位基因。几个新的致病基因的存在突出了非常高的乙酸耐受性的独特遗传基础和强烈的遗传背景依赖性。我们的结果表明，消除劣质突变等位基因对于达到非常高的乙酸耐受性可能与引入稀有的优质等位基因同样重要。MET4 和 RTG1 的优越等位基因可能有助于进一步提高特定工业酵母菌株的乙酸耐受性。