Guerreiro Joana F, Muir Alexander, Ramachandran Subramaniam, Thorner Jeremy, Sá-Correia Isabel
iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal.
Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, U.S.A.
Biochem J. 2016 Dec 1;473(23):4311-4325. doi: 10.1042/BCJ20160565. Epub 2016 Sep 26.
Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.
乙酸对酵母生长和代谢的抑制作用限制了工业发酵过程的生产力，特别是当木质纤维素水解产物用作工业生物技术中的原料时。食品腐败酵母对乙酸的耐受性也是酸性食品和饮料保存中的一个问题。因此，了解适应和耐受乙酸胁迫的分子机制在工业生物技术和食品工业中变得越来越重要。先前对酿酒酵母中对乙酸敏感性增加的突变体进行的遗传筛选确定了YPK1基因中的功能丧失突变，该基因编码一种由雷帕霉素靶蛋白（TOR）复合物2（TORC2）激活的蛋白激酶。我们在本研究中通过几个独立的标准表明，TORC2-Ypk1信号通路在乙酸胁迫下被激活。此外，我们证明TORC2介导的Ypk1磷酸化和激活是乙酸耐受性所必需的，并且独立于先前涉及细胞对乙酸反应的蛋白激酶Hrk1发生。此外，我们表明TORC2-Ypk1介导的l-丝氨酸：棕榈酰辅酶A酰基转移酶的激活，即催化鞘脂生物合成第一步的酶复合物激活，是乙酸耐受性所必需的。此外，使用抑制剂和突变体对鞘脂途径的分析表明，是某些复杂鞘脂的产生有助于赋予乙酸耐受性。与该结论一致，通过向生长培养基中添加外源性长链碱基前体植物鞘氨醇来促进鞘脂合成可增强乙酸耐受性。因此，对工业酵母菌株中TORC2-Ypk1-鞘脂轴的适当调节可能有助于改善含乙酸原料的发酵。
