Fig. 3

Description of laboratory evolution for constitutive acetic acid tolerance in microaerobic serum flasks and characterization of resulting mutants. a Laboratory evolution for constitutive acetic acid tolerance (evolution experiment MUT1). After UV mutagenesis S. cerevisiae CEN.PK113-7D cells were grown in a series of sequential microaerobic batch cultures, alternatingly in the presence (grey squares) and absence (black circles) of acetic acid. Over a series of 50 transfers, the acetic acid concentrations in the stressed culture cycles were progressively increased from 9 to 18 g/L (grey line). b Fraction of cells able to grow on SMG agar plates with 9 g/L acetic acid (pH 4.5) after three consecutive microaerobic batch cultures on SMG (pH 6.0) without acetic acid. The first culture was inoculated from the final serum bottle in the presence of 18 g/L acetic acid from the evolution lines of MUT1, MUT2, MUT3, HAT1 and HAT2. To mimic the growth conditions of the mutants, prior to the three cultures without acetic acid also CEN.PK113-7D was induced in SMG with 9 g/L acetic acid and subsequently grown in SMG with 18 g/L acetic acid, corresponding to the final concentration of acetic acid during the mutants evolution