Adaptive laboratory evolution and reverse engineering of low pH tolerance in Saccharomyces cerevisiae

Low pH tolerance of Saccharomyces cerevisiae is a relevant trait for industrial production of carboxylic acids. However, few genetic modifications that enhance this trait have hitherto been identified. The limited success of targeted engineering approaches is largely explained by the tight maintenance of intracellular pH by complex regulatory networks. In this study, an adaptive laboratory evolution strategy was applied to decrease the minimum permissive pH of the laboratory strain S. cerevisiae CEN.PK113-7D from 2.6 to pH 2.1. independently evolved, low-pH tolerant strains showed mutations in genes involved in calcium signaling, cell-wall maintenance, membrane composition and protein turnover. Combined reverse engineering and backcrossing enabled the identification of single and combined mutations that enabled growth at extremely low pH. Specific mutations in PMR1, encoding the Golgi Ca2+/Mn2+-ATPase, already increased low-pH tolerance of S. cerevisiae. Mutations in MUK1 and MNN4 were ineffective when introduced individually, but did synergistically improve low-pH tolerance when introduced together in an unevolved background. The results of this study show the genetic complexity underlying low pH tolerance and provide further insights for the understanding and engineering of this trait.ImportanceTolerance to low pH is an important microbial feature for industrial carboxylic acids production. However, the complex, pleiotropic effects of low extracellular pH prohibits the a priori identification of genetic targets and makes the improvement of microbial tolerance to low pH via genetic engineering a daunting task. In this study, a strategy based on Adaptive Laboratory Evolution (ALE) successfully enabled the identification of such genetic targets in the industrial and model yeast Saccharomyces cerevisiae. Growth at incrementally decreasing pH over 450 generations substantially increased the low pH range of S. cerevisiae laboratory strains. Specific mutations in calcium signaling (PMR1), cell wall maintenance (MNN4) and protein degradation (MUK1) found in the evolved strains substantially improved pH tolerance when transplanted into non-evolved strains. These findings not only hold the potential to improve S. cerevisiae-based carboxylic acid production processes, but also bring new insights in the mechanisms involved in low pH tolerance.