# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 4 CRISPR System Applied to Wine Yeasts
Glycerol 3-phosphate dehydrogenase activity controls the amount of glycerol produced [69] and the expression of _GPD1_ is upregulated after the exposure of cells to hyperosmotic stress [67] and controlled by the HOG (high osmolarity glycerol) MAP kinase pathway [69] (Figure 5B). The _S. cerevisiae ATF1_ gene translates for the alcohol acetyltransferase kinase I, which catalyzes the formation of acetate esters from acetyl coenzyme A (Figure 6) [70; 71]. Some of these acetate esters can present a glue-like aroma (ethyl acetate), but most of them present a fruity-like aroma (isoamyl acetate) or even a flower-like aroma (phenylethyl acetate) and are important flavor components of fermented beverages, such as beer and wine. If, during  
Figure 5: (**A**) Biochemical pathways leading to the production of glycerol (taking place in the cell cytosol) results in a depletion of the NADH cofactor. (**B**) Aldehyde dehydrogenases (ALDH) (converting acetaldehyde to acetic acid) are implicated in replenishing the NADH levels as higher acetic acid levels are observed with _GPD1_ overexpression. ferentation, the balance of acetate esters is disturbed, it can lead to severe off-flavors such as the formation of acetic acid with the concomitant vinegar scent [72]. Much attention has, therefore, been drawn to the different factors that control the formation of volatile acetate esters [73]. To increase ester and glycerol production in the AWRI1631 wine yeast strain, [PERSON] et al. [26] used the CRISPR/Cas9 editing technique to combine different yeast phenotypes. The yeasts were firstly transformed (obtention of plasmid-cured cells with incorporated _ATFI_ and _GPDI_ genes), and after, transformed cells were grown in YPD liquid media and isolated. The newly _S. cerevisiae_ strains were designated AWRI1631[_ATFI_] and AWRI1631[_GPDI_]. An AWRI1631[_GPDI_]MAT\(\alpha\) strain was then incubated in 1 mL of liquid YPD with AWRI1631[_ATFI_]MAT\(\alpha\) overnight. Single colonies and diploid strains identified by mating-type PCR screening were obtained. Diploids were plated on 1% potassium acetate agar, spores from _asci_ were isolated, and resulting spores were PCR-screened if both promoter swap features were presently generating the strain AWRI1631[_ATFI_GPDI_]. This newly made strain was able to overexpress both _GPD1_ and _ATFI_. After an analysis of the wine was obtained, increased glycerol and acetate ester levels were found, and significantly less acetic acid was formed during fermentation when comparing the results obtained in wine made with the yeast strain that only overexpressed _GPD1_[26]. Another article that covers glycerol production by wine yeasts was published by [PERSON] et al. [74]. The article title is \"The Use of CRISPR-Cas9 Genome Editing to Determine the Importance of Glycerol Uptake in Wine Yeast During Ice-wine Fermentation.\" The high concentration of sugars in ice wine must cause remarkable stress for the fermenting yeast, causing cells to lose water and shrink. Glycerol helps the yeast to resist this kind of stress. Therefore, yeast cells can increase the internal glycerol concentration by actively  
Figure 6: For _ATFI_ overexpression, acetyl-CoA is condensed with alcohols to form acetate esters. As more acetate esters are produced, much less acetyl-CoA is available to convert to acetic acid. transporting glycerol into the cell from the environment or by synthesizing glycerol. The H\({}^{+}\)/glycerol symporter, Stl1p, is glucose repressed and inactivated, despite osmotic stress induction [75]. To investigate the role of Stl1p in ice wine fermentation, [PERSON] et al. [74] developed a rapid, single-plasmid, CRISPR-Cas9-based genome-editing method to construct the _S. cerevisiae_ K1-V1116 lacking _STL1_. In an assay with ice wine fermentation, the \(\Delta\)_STL1_ strain presented elevated glycerol and acetic acid production compared to the parent strain, validating that glycerol uptake by H\({}^{+}\)/glycerol symporter has an important role during ice wine fermentation in K1-V1116.