A new laboratory evolution approach to select for constitutive acetic-acid tolerance in Saccharomyces cerevisiae and identification of causal mutations. Saccharomyces cerevisiae strain:CEN.PK113-7D

Laboratory evolution allows the selection of specific phenotypes, without the requirement for in-depth understanding of the underlying mechanisms and has been successfully used to improve the tolerance of yeast to butanol and other stresses such as freezing–thawing, temperature, ethanol, and oxidative stress. This approach has also been used to improve acetic acid tolerance in bacteria and yeast . Wright et al. (2011) used repeated batch cultivation at progressively increasing concentrations of acetic acid to select an S. cerevisiae strain with a higher acid-acid tolerance. However, after growing the evolved strain in the absence of acetic acid, the strain was no longer able to grow at high acetic acid concentrations. Although the strain was genetically stable, increased tolerance to acetic acid could only be shown after precultivation in the presence of acetic acid. These observations indicated that the increased tolerance of the evolved strain was not constitutive but required induction by acetic acid. Other studies showed also that exposure of cells to low levels of acetic acid increases acetic-acid tolerance and that thus adapted cells showed a less dramatic decrease in intracellular pH than non-adapted cells upon exposure to acetic acid . Adaptation to acetic acid was also shown to protect yeast cells from acetic acid-mediated programmed cell death. To benefit from inducible acetic-acid tolerance in industrial processes, an adaptation step would have to be implemented prior to the fermentation, making the whole process more complex. Yeast strains with a constitutive, high-level tolerance, which do not require such an adaptation, would therefore clearly be preferable for industrial application.The aim of this study is to investigate how laboratory evolution and mutagenesis can be used to obtain Saccharomyces cerevisiae strains with constitutive acetic-acid tolerance. To maximize the selective pressure on constitutively tolerant strains and simultaneously minimize the competitiveness of cells with inducible acetic-acid tolerance, laboratory evolution was performed by alternatingly growing S. cerevisiae in medium with and without acetic acid. In parallel, mutagenesis and screening was applied to obtain constitutively tolerant mutants without laboratory evolution. Subsequently, a combination of whole-genome sequencing, crossing and segregation studies was used to investigate the mutations underlying the constitutive acetic-acid tolerant phenotype. The impact of these mutations on acetic-acid tolerance was confirmed by reverse engineering the evolved phenotype into the non-evolved reference strain.