Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generate Saccharomyces cerevisiae mutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80, ESBP6 was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native to S. cerevisiae (Pdr12). Besides conferring tolerance to aromatic acids, ESBP6 overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.

化合物外部存在或内部产生的毒性会降低微生物细胞工厂的生长和生存力，并损害生产率。芳香族化合物通常对微生物有毒，这使其在微生物宿主中的生产具有挑战性。在这里，我们使用自适应实验室进化来产生酿酒酵母耐两种芳香酸，香豆酸和阿魏酸的突变体。在低pH（3.5）下进行进化实验，以重现工业过程的典型条件。然后，通过全基因组测序分析了耐受溶解度极限附近的芳香酸水平的突变菌株，结果显示转录激活因子（Aro80）中普遍存在点突变，该转录激活因子负责调节芳香族氨基酸作为氮源的使用。在Aro80调控的基因中，ESBP6被发现是通过将芳香酸从细胞中排出来提高对芳香酸的耐受性的原因。进一步检查Esbp6的天然功能后发现，该转运蛋白可以排泄杂酸（芳香族氨基酸分解代谢的副产物），并且该作用与酿酒酵母（Pdr12）天然的至少一种额外转运蛋白共有。除了赋予对芳族酸的耐受性外，ESBP6还显示出过表达显着改善香豆酸生产菌株中的分泌。总的来说，我们表明调节转运蛋白的活性是提高对芳香酸的耐受性的主要机制。这些发现可用于调节芳香族化合物的细胞内浓度，以优化此类产品的排泄，同时将前体分子保留在细胞内。