# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 4 CRISPR System Applied to Wine Yeasts
Low urea-metabolizing ability may result from the low activity of urea amidolyase, inhibition of amidolyase activity by the presence of high levels of ammonia, deficiencies of cofactors required by amidolyase, or low activity due to hyperactive arginase [60]. Genetic as well as environmental factors influence the amount of urea released by the cells. There are, already, some commercial yeast strains, Lallemand 71B(r), Red Star SC1120(r), and Premier Cuvee (PdM)(r), described as producing low levels of urea [61]. [PERSON] et al. [47] applied the CRISPR/Cas9 system to two commercial strains of _S. cerevisiae_, EC1118 and AWRI796, eliminating the \(CAN1\) arginine permease pathway and the production of arginine permeases Can1p (Figure 4A) to generate strains with reduced urea production, achieving a reduction of 18.5% for EC1118 and 35.5% for AWRI796. Both _S. cerevisiae_ starter strains and \(CAN1\) mutants were able to complete the fermentation process, and recombinant strains containing the can1 mutation did not produce urea, indicating that the genetic modification reduced the arginine metabolism. More recently, [PERSON] et al. [49] evaluated the coactive effect of the multiregulation of yeast urea metabolism, hydrolysis, and transport on EC accumulation during Chinese rice wine fermentation. They used the modified N85[_DUR1,2_] yeast strain, which was already studied, and were able to decrease urea content in Chinese rice wine [62]. The mentioned scientists [49] applied a modified CRISPR/Cas9 vector containing the _DUR3_ overexpression cassette and sgRNA to the N85[_DUR1,2_] yeast strain. After the fermentation of Chinese rice wine samples by the engineered strain N85_DUR1,2/DUR3_, analysis of the fermentation broth showed that urea content was 43.7% and 92.0% lower than that of the parental strain N85_DUR1,2_ and original strain N85, respectively. The constructed strain N85_DUR1,2/DUR3_ was able to decrease 16.8% of the EC concentrations in Chinese rice wine when compared to the parental strain. Figure 4: (**A**) Argin permeases in yeast cells [55] and _Saccharomyces cerevisiae_ S288c Pathway: urea cycle with predicted enzymes and subtracts. Retrieved from [[https://biology.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PWY-4984](https://biology.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PWY-4984)]([https://biology.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PWY-4984](https://biology.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PWY-4984)). Accessed 22 November 2020. (**B**) Precursors and mechanism of ethyl carbamate (EC) formation. The disposal of EC precursors depends on the microorganism present during fermentation. _S. cerevisiae_ degrades arginine to urea and Lactobacillus converts arginine into citruline. Both compounds react with ethanol producing EC [59]. Glycerol is an important by-product of alcoholic fermentation. In _S. cerevisiae_, this polyol controls intracellular redox balance and reduces osmotic stress [63] besides being responsible for the velvet sensation in the mouth persistence and mellowness [64]. Under fermentation conditions, glycerol synthesis makes it possible to reoxidize surplus NADH produced by anabolism and during the synthesis of organic acids [65] (Figure 5A). Glycerol is formed in two enzymatic steps: (i) the reduction of dihydroxyacetone phosphate to generate glycerol-3-phosphate by the action of NAD-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p), regulated by two homologous genes _GPD1_ and _GPD2_[66]; and dephosphorylation by glycerol-3-phosphatase, regulated by _GPP1_ and _GPP2_ genes [67]. These isoenzymes have different biological roles: the Gpd1p-Gpp2p mixture is mainly involved in glycerol production during osmotic stress, whereas Gpd2p-Gpp1p is involved in adjusting the NADH-NAD redox balance under anaerobic conditions [68].