# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 2 Genetic Improvement of _S. cerevisiae_  
### Genetic Engineering  
Since the 1990s, it has been a constant in wine biotechnology that product improvement and problem solving tend to be addressed, in the first instance, by the genetic engineering of wine yeasts. However, the wine industry is not the ideal breeding ground for genetically modified organisms due to restrictions to GMOs on food production in most countries, and their negative perception by wine consumers [5]. The only two recombinant wine yeasts that have been commercialised so far do not seem to have become bestsellers in the markets they were introduced in [5; 6]. Therefore, none of the recombinant strains described in this section were intended for direct commercialization. Nevertheless, their study provided useful information for the improvement of yeast strains by alternative approaches. Indeed, in several instances, the reduction in alcohol levels appeared as a side-effect of genetic manipulations which were not intended for that purpose. As mentioned above, when it comes to reducing the alcohol level, a key issue to guide genetic engineering is the choice of carbon sinks. Figure 1 shows a summary of the main enzymatic reactions involved in alcoholic fermentation, including those referred to on the following lines. Alcohol dehydrogenase (ADH) catalyses the final step in the production of ethanol during alcoholic fermentation. It is therefore a rational deletion target for limiting ethanol production. However, cell metabolism is complex and depends on a set of biochemical reactions that must be balanced in several ways, especially regarding the redox balance. For this reason, simple logics often lead to unwanted results. For example, alcohol dehydrogenase deletion leads to a reduction in ethanol production and an increase in that of glycerol, but these recombinant strains are unable to grow under anaerobic conditions [7]. This genetic modification also results in higher levels of acetic acid and acetaldehyde, far beyond levels suitable for commercial wines. In addition to the modulation of the expression of endogenous genes by shutting down or unregulating their expression, genetic engineering allows for the heterologous expression of genes from other biological species. In 1994, [PERSON] and [PERSON] [8] expressed lactate dehydrogenase from _Lactobacillus casei_ in _S. cerevisiae_ to divert some of the pyruvate formed in glycolysis to lactate formation. The engineered yeast strain simultaneously performed alcoholic and lactic fermentations. The redox balance was not affected, as the formation of lactic acid is compensated by the disappearance of ethanol in equimolecular amounts, for the same consumption of NADH. Thus, a reduction of 1% ethanol leads to an increase in 15 g/L lactic acid. The increased lactate content of these wines contributed to a higher total acidity. This feature could be useful to compensate for low acidity, which is a problem affecting some grape varieties and growing regions, and is also related to global warming. However, this also sets a limit to this approach, as excessive acidity would make the wines unpalatable. Glycerol contributes positively to the viscosity, body, and sweetness of wine. It has been a preferred carbon sink for the genetic engineering of wine yeasts. The overexpression of _GPDI_, coding for glycerol 3-\(P\) dehydrogenase, results in a significant increase in glycerol (plus 28 g/L) and a reduction in ethanol (minus 15 g/L), and also in the appearance of excess acetate and acetoin to restore the intracellular redox balance during fermentation [9]. Researchers identified Ald6 as the aldehyde dehydrogenase isoenzyme responsible for most of the acetate production of _S. cerevisiae_[10]. However, the knockout of _ALD6_ resulted in an additional increase in acetoin production which, at high concentrations, negatively affects wine aroma, providing buttery notes [11].