Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

支链醇是有吸引力的先进生物燃料。然而，它们的细胞毒性阻碍了工程微生物生产高滴度的微生物。我们在酿酒酵母基因删除文库上进行了全基因组筛选，以鉴定参与异丁醇特异性耐受的细胞系统。磷酸戊糖途径基因GND1或ZWF1的缺失引起对异丁醇而不是乙醇的超敏反应。相比之下，GLN3的缺失特别增加了酵母对支链醇的耐受性。转录组学分析显示异丁醇通过GLN3和GCN4诱导氮饥饿反应，上调氨基酸的生物合成和氮清除，同时下调糖酵解，细胞壁生物发生和膜脂质生物合成。通过删除GLN3破坏这种反应足以增强耐受性，并在工程菌株中将异丁醇产量提高4.9倍。这项研究说明了耐受胁迫的适应性机制如何在化学生产的微生物发酵中导致毒性，以及遗传干预如何通过逃避此类机制来促进生产。