# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 2 Genetic Improvement of _S. cerevisiae_
### Genetic Engineering
Following this line of research, the problem of acetoin overproduction was tackled by overexpressing _BDH1_, which codes for butanediol dehydrogenase [12]. This enzyme catalyses the conversion of acetoin to 2,3-butanediol, a compound with no apparent sensory impact. Using this recombinant strain, wines show a significant reduction in ethanol, and above 26 g/L of glycerol content. The number of different genetic modifications required to achieve this objective is in itself an indication of the difficulty of using this as a general approach to developing wine yeast strains with a low ethanol yield. The different adjustments that were required to develop the above-described recombinant yeasts respond to metabolic constraints concerning the redox balance and the availability of an adequate pool of cofactors. Therefore, some authors have tried to act directly on the reactions of the redox system. By cloning an NADH oxidase for the regeneration of the reducing power, and with controlled oxygenation, not only is a decrease in ethanol obtained but also a significant increase in the levels of acetaldehyde, acetic acid, and acetoin [13]. There are two enzymatic steps from pyruvate to ethanol during alcoholic fermentation (Figure 1). The final one catalysed by alcohol dehydrogenase has been discussed above. The first, from pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase (PDC), was also taken as an improvement target by some researchers. In the absence of PDC activity, the Crabtree effect is abolished, but this activity is required for acetyl-CoA synthesis in the cytoplasm and NAD\({}^{+}\) regeneration. It is therefore essential for _S. cerevisiae_ growing on glucose [14]. The deletion of _PDCI_ resulted in a fourfold reduction of the PDC activity, with an increase in pyruvate levels, but no reduction of ethanol levels [10]. The deletion of _PDC2_, coding for a transcription factor involved in PDC gene expression, results in 19%PDC activity, with a clear reduction in the ethanol content, combined with an increase in glycerol and pyruvate release [15]. This effect is enhanced by increased glycerol-3-phosphate dehydrogenase activity, through the overexpression of _GPD1_, without the over-production of acetic acid and acetaldehyde associated with _GPD1_ over-expression in the original strain. [PERSON] et al. [16] replaced one of the native _PDC2_ alleles in different diploid wine yeast strains by a truncated _PDC2_ allele. These engineered strains showed a 2% ABV reduction without any increase in acetate production. An alternative way to produce cytosolic acetyl CoA in _pdc_ strains was developed by substituting Acs1 and Acs2 (acetyl CoA synthetase) with a heterologous pyruvate carboxylase and a transacetylase [17]. This strain produces glycerol, succinate, pyruvate and acetate from glucose, but not ethanol. The additional deletion of gpp1 and gpp2 (glycerol 3p phosphatase) avoids the production of these metabolites, including glycerol, and increases the growth rate. Crabtree-negative strains of _S. cerevisiae_ were also constructed by substituting all of the active hexose transport genes with a single chimeric one [18]. This strain does not produce ethanol as long as oxygen is available for respiration. However, its growth kinetics are extremely slow. Further possibilities of using the respiratory metabolism of yeast to reduce the ethanol yield during wine fermentation will be examined below in a specific section. Triose-phosphate isomerase, encoded by _TPII_, is the enzyme catalysing the interconversion between the two products resulting from the breakdown of fructose 1,6-bisphosphate: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (Figure 1). The topology of the pathway suggests that _TPII_ deletion would result in equimolar levels of glycerol and ethanol precursors. However, in practice, such deletion strains are unable to grow on glucose due to a redox imbalance [19]. In order to improve industrial glycerol yields, additional genetic modifications have been introduced in strains deleted for _TPII_[20].