## 2 Microbiota of Vineyard and Fermenting Must
### Saccharomyces cerevisiae  
_S. cerevisiae_ is the main yeast involved in wine fermentation and the most commonly employed microorganism for bread and beer production. Moreover, as a result of strong domestication, winemakers can select the best-performing _S. cerevisiae_ strains to improve wine production in terms of flavor and fragrance from an enormous diversity of strains [36]. As previously reported, _S. cerevisiae_ may be primarily found in soil and grape berries and at the initial stage of fermentation; however, it is not completely ruled out if it may be found on winery machinery, such as stemmer-crushers, pumps, pipes, or fermentation vessels, where it could contribute to enhancing biodiversity ecology [37]. From an enological point of view, _S. cerevisiae_ is important for several traits, among which are tolerance to high ethanol concentrations and the production of killer factors. As previously mentioned, thanks to the Crabtree effect, even with aerobic conditions, _S. cerevisiae_ does not employ its respiratory system to metabolize saccharides and increase biomass growth, but instead, through pyruvate, it releases ethanol and other two-carbon molecules. As a result of this, _S. cerevisiae_ creates and accumulates ethanol, which is deadly to most other microbial species capable of competing with it for sugar molecules and hence eliminates competition. After that, it continues to consume the produced ethanol, thus improving its own growth [38]. Focusing on must fermentation, higher alcohols (fusel alcohols), from a quantitative point of view, are the most important compounds produced by _S. cerevisiae_. Their biosynthesis is performed via amino acid catabolism, through the Ehrlich pathway [38]. Initially, transaminases, encoded by _aro8_, _aro9_, _bat1_, and _bat2_ genes, deaminate amino acids to the corresponding \(\alpha\)-ketoads. Secondly, \(\alpha\)-ketoads are converted into their corresponding aldehydes by enzymes encoded by one of the five decarboxylase genes (_Pdc1p_, _Pdc5p_, _Pdc6p_, _Aro10p_, and _Thi3p_) present in _S. cerevisiae_. Finally, the alcohol dehydrogenases catalyze the reduction of aldehydes into corresponding higher alcohols [39]. Recently, the selection of indigenous strains of _S. cerevisiae_ has been widely applied, especially to improve the aroma profile and enhance the typicity of wines [40; 41]. Inoculation of _S. cerevisiae_ starters is a common practice, with dozens of different strains available on the market, selected for good fermentation kinetics (tolerance to osmotic and ethanol stresses and good nutritional efficiency in using available nitrogen sources). In addition, different starter cultures respond variably to the applied fermentation techniques and contribute differently to the development of sensory characteristics; as a result, yeast selection has become a key tool for developing and differentiating wines [42]. According to the International Organization of Vine and Wine (OIV), industrial starters can be marketed as: Active Dry Yeast (ADY), Active Frozen Yeast (AFY), Compressed Yeast (COY), Cream Yeast (CRY), Encapsulated (beads) or Immobilised Yeasts (ENY), and _\"levain de tirage\"_ for sparkling wines [43]. The application of biotechnological tools, such as genetic engineering, in _S. cerevisiae_ opens new possibilities for the development of novel, enhanced, or changed wine traits, qualities, tastes, aromas, or manufacturing methods [44]. In addition, more sophisticated genetic engineering techniques have gained increasing attention over the past ten years, mostly CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which uses the \"adaptive immunity\" mechanism naturally found in bacteria and archaea to construct a precise tool for genome editing [45; 46]. In wine yeasts, this technique has been successfully applied to create engineered strains with lowered urea production [47], higher osmotic tolerance [48], or to improve wine aromatic complexity [46]. Moreover, a strain of _S.