# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 2 Genetic Improvement of _S. cerevisiae_
### Genetic Engineering
These authors deleted _ADHI_ (coding for alcohol dehydrogenase) and _TPII_ in a yeast strain while overexpressing _GPD1_ (coding for glycerol-3-phosphate dehydrogenase), _FPS1_ (coding for a glycerol transporter) and _ALD3_ (coding for an aldehyde dehydrogenase). These strains produce very low levels of ethanol (about 25% of the parent strain), and their glycerol levels are multiplied by 30. However, their growth rates were strongly reduced. Another alternative carbon sink is yeast biomass. Despite the portion of carbon going to biomass being quantitatively small during wine fermentation, some authors have targeted reserve sugars to divert the carbon flux from ethanol. A moderate overexpression of _TPS1_, the gene coding for trehalose-6-phosphate synthase, involved in trehalose biosynthesis, resulted in a clear reduction in the ethanol yield, without an apparent impact on the redox balance, according to the profile of the byproducts analysed [21]. Global transcription machinery engineering (gTME) technology has recently been employed to construct a low-alcohol yeast strain. One strain with a reduced ethanol yield was selected from a library of mutants in _SPT15_ (coding for a transcription factor) generated by random directed mutagenesis [22]. The pleiotropic effect of this mutation downregulates hexose transport and ethanol production, and upregulates glycerol production. Acetic acid is not increased, but the mutant shows slower growth kinetics than the parent strain. The treatment of must with glucose oxidase is an alternative method to decrease the alcohol content of wine that will be discussed in the corresponding section. Based on this method, [PERSON] et al. [23] cloned a glucose oxidase gene from _Aspergillus niger_ in a laboratory strain of _S. cerevisiae_. Wines produced with the recombinant strain contained 2% less ethanol. The recombinant strain showed antimicrobial activity against acetic and lactic acid bacteria, probably due to the H\({}_{2}\)O\({}_{2}\) released by the oxidation reaction. The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9) genetic modification system has already been implemented in _S. cerevisiae_, including wine yeasts [24; 25]. The basic CRISPR/Cas9 system consists of a guide RNA molecule (gRNA) and an endonuclease (Cas9). The endonuclease mediates a double strand break guided by the hybridization of gRNA with genomic DNA. This triggers the DNA repair mechanisms of the host cell. The system can be designed to target the nuclease to the gene of interest, and to perform more or less large insertions and deletions. An advantage of this system is that, unlike other genetic engineering techniques, it can easily be tuned to leave no trace of the ancillary genetic material. In addition, it is less dependent on traditional transformation markers, and makes easier the manipulation of diploid loci. Nonetheless, microorganisms obtained with this system still fall under European Union GMO regulations. The genes _GPD1_ and _ATF1_ have been cloned in a haploid wine strain using this technique [26], with no differences in ethanol production between the strains, contrary to the results obtained in previous works [9; 15]. Other works applying this technique for the reduction of wine alcohol content will certainly appear soon.