**3.3 From the bench to the cellar**

Once a positive evolution is detected in a wine evolutionary approach, a thorough work of validation needs to be done before an evolved wine yeast can actually be used in a cellar. The first step, often at laboratory scale, is to submit evolved yeasts to the evolutionary conditions in direct comparison with the parental strain, separately or in competition, to evaluate the relative improvement of the phenotype [73]. If acceptable, this comparison should also be validated in different realistic conditions where the yeast might perform. Typically, natural or synthetic musts are used in order to better reproduce enological fermentation conditions [68]. Aside from the characterization itself, this first screening allows for the search of possible trade-offs. A trade-off occurs when a particular phenotypic trait gets improved at the expense of one or more other phenotypic traits that get worsen. This is well illustrated in a study by Wenger et al. [74] who successfully evolved *S. cerevisiae* for a higher fitness in anaerobic glucose-limited media. Despite this, when in aerobic, carbon-rich environments, the evolved clones performed less well than their ancestor due to a trade-off. Similarly, yeast cells evolved for efficient galactose consumption which presented trade-offs when grown on glucose as a carbon source [75]. In winemaking context it is fundamental for yeast traits such as aroma production or fermentation efficiency to be kept at high standards and free of trade-offs. To note that in an evolutionary approach, the higher the number of generations occurred, the higher the chances of unrelated mutation accumulation. This reinforces the fact that the approach should be stopped as soon as a positive evolution is detected to avoid the accumulation of potential trade-offs.

Another fundamental test is to propagate and dry the yeast under industrial conditions, often the method used to produce commercialized wine yeast strains. Propagation and drying represent as the major sources of stress for yeast including oxidative, osmotic, and desiccation stresses which the evolved strains need to endure at least as well as the parental strains [21, 76–78]. The final stage of validation is the scale-up to pilot and industrial fermentation volumes, often performed by cellars with tanks of several hectoliters. If the evolved wine yeast strain performance is satisfactory both for the evolved phenotype and the remaining important traits, the evolutionary engineering process is then a success from an industrial point of view, and the yeast can be commercialized. From an academic point a view, new knowledge can also be generated by studying the new genetic profile in correlation to the evolved phenotype and how the evolved strains differ from the parental one. Approaches to conduct this characterization include genome microarray hybridization and direct DNA sequencing [75, 79, 80].

#### **3.4 Successful evolutionary engineering in winemaking**

To illustrate the potential of evolutionary engineering approaches in winemaking, few examples can be used where technical or field problematics were successfully solved by using this approach with validated evolved strains.

While using a long-term batch culturing on gluconate (a carbon source poorly assimilated by *S. cerevisiae*), Cadière et al. [19] evolved a commercial wine yeast strain obtaining interesting results. Evolved clones presented a carbon flux through the pentose phosphate pathway which increased by 6% when compared to the parental strains. This also resulted in a higher fermentation rate, lower levels of acetate production, and increased production of aroma compounds. As the process was carried out at a laboratory scale but in realistic (enological) conditions, the same phenotypic improvements were verified when the evolved strain was used in pilot-scale trials [81]. It was identified that the evolved strain produced higher levels of phenyl ethanol, isobutanol, isoamyl alcohol, ethyl acetate, isoamyl acetate, and ethyl esters [82].

Other authors were able to obtain a stable wine yeast strain with slightly enhanced glycerol production. By employing sulfite as a selective agent in an alkaline pH, Kutyna et al. [83] obtained evolved clones with an increase of 41% in glycerol production, which can have a benefic impact in wine organoleptic properties. To reduce the final ethanol content in wine, Tilloy et al. [18] submitted a wine strain to hyperosmotic stress for 200 generations, which yielded evolved clones that grew better under osmotic stress and glucose starvation and produced markedly more glycerol but also succinate and 2,3-butanediol. The approach was then complemented with an intra-strain breeding strategy that further increased the glycerol yield and reduced ethanol production in wine by up to 1.3% (v/v).

More recently, López-Malo et al. [80] performed an evolutionary process aiming for a higher performance for low-temperature fermentations (12°C). It was discovered that inositol and mannoprotein limitations were responsible for an evolution toward shorter fermentation times and higher final populations. After genome sequencing, it was discovered that an SNP in the gene *GAA1*, fundamental in inositol and mannoprotein synthesis, was at the basis of the improvement.
