# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 2 Genetic Improvement of _S. cerevisiae_
### Adaptive Laboratory Evolution  
Adaptive laboratory evolution (also known as experimental evolution) consists of recreating in the laboratory, in an accelerated way, the natural selection process, promoting the selection of the mutations that are most useful to our purpose. This technique shares several features with random mutagenesis [27], including some limitations, such as that it cannot be easily targeted to specific genes, that dominant mutations are favoured, and the difficulties of connecting readily selectable phenotypes to technological traits. However, it also offers advantages, as the strains obtained in this way do not fall under GMO regulations. In this case, mutations appear spontaneously in the population during each round of duplication (although it can eventually be combined with random mutagenesis). Yeasts are grown in a selective environment in which the desired metabolic changes would favour their growth. Once such a mutation appears, and after several generations under the same selection conditions, many individuals in the population will harbour the desired mutation. In addition, new variants can arise from the original one, and in this way the favourable mutations for the intended technological objective can be accumulated in the evolving lineage. The random nature of spontaneous mutations, and the fact that many of them can be added over time during experimental evolution, gives some advantages to this approach over genetic engineering. As indicated above, the challenge of this technology is to anticipate the metabolic pathways to be altered in order to reach the expected technological output, and the growth conditions that will give advantage to the yeast cells improved in that way. In the context of alcohol level reduction, adaptive laboratory evolution has been used to channel carbon flux towards the pentose phosphate pathway (PPP; Figure 1). The selective pressure was established using gluconate as the only carbon source [28]. Gluconate is a substrate poorly assimilated by _S. cerevisiae_ and metabolized by the PPP. The rationale for targeting PPP was that, compared to glycolysis, an additional CO\({}_{2}\) molecule is released for each glucose molecule entering the pathway (meaning that this carbon will not contribute to ethanol production). The selected strain showed a 1.5-fold increase in flux through the PPP compared to the ancestral strain, corresponding to a reduction of 2 g/L in ethanol levels. A fivefold increase in flux through the PPP would have been necessary to reduce the ethanol level of wine by approximately 1% (vol/vol). However, selected evolved strains did not show a reduced ethanol yield, but an increased production of esters [29], which can be useful, but not for the original purpose. Other authors have used experimental evolution to push carbon flux towards glycerol production. The process was based on a long-standing strategy for the industrial production of glycerol based on sulphite addition [30]. The reaction between sulphite and acetaldehyde decreases the availability of acetaldehyde for ethanol production and NADH oxidation, and cells must compensate the redox balance by increasing glycerol production. [PERSON] et al. [31] used this principle to drive the evolution of a laboratory strain, with sodium sulphite as a selective agent, at an alkaline pH. Strains evolved in this way did show a relative increase in glycerol yields, but there was a very limited impact on ethanol yield. In addition, genetic analysis showed that the sulphite tolerance and glycerol yield were not linked in the evolved strains. Glycerol is the major osmolyte in yeasts [32]. [PERSON] et al. [33] targeted glycerol production to reduce the ethanol yield from a wine yeast strain. In this case, the selective pressure was osmotic stress and the HOG intracellular signal transduction pathway. Strains evolved in a hypersaline medium were selected and further improved by sporulation and back-crossing.