*3.2.1 Selective pressures*

During an evolutionary experiment, the mutations and consequently the diversity generated are completely random and cannot be predicted or controlled [65]. Therefore, it is crucial to identify the selective pressures that will best conduct the selection of positive mutations toward the desired phenotype. Once they are clearly identified, a proper initial experimental characterization should be performed to identify the intensity that these selective pressures should have. If too low there will be no selection, and if too high, yeast will struggle to continue in culture. Additionally, if a further industrial application is predicted, evolutionary engineering should be performed in conditions as close as possible to the actual conditions in which the yeast will later perform. By doing so, yeasts will not only face the selective pressures chosen to drive evolution but also all the other constraints naturally present [66]. For instance, in wine fermentation conditions, yeasts need to cope with stress factors as diverse as low pH, nutrient deprivation, ethanol, osmotic, and oxidative stress which are commonly present [67]. Therefore, it is preferable to use natural or synthetic must as a media that closely mimics realistic conditions while allowing the control and modification of specific parameters [68].
