# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 1 Introduction  
The _Brettanomyces_ genus was first described by [PERSON] and [PERSON] in 1921, and its primary taxonomic framework was established in 1964 by [PERSON] [1]. It is the anamorphic form of the _Dekkera_ genus belonging to the Pichiaceae family [2,3]. The phylogenetic classification of _Brettanomyces_ and _Dekkera_ was first performed in 1987 by [PERSON] et al., and then in 1993 by [PERSON] et al. [2]. Subsequently, in 2011, [PERSON] et al. reclassified the genus to include five species: _D. bruxellensis_, _D. anomalous_, _B. custersianus_, _B. nanus_, and _B. naardenensis_[4]. Recently, a new species named _B. acidotrans_ was isolated from olive oil and has been characterized as a strongly acetic-acid-tolerant yeast [5]. In disagreement with the Melbourne Convention (International Code of Nomenclature for algae, fungi, and plants), which states that a single valid name must be assigned under the new code for a fungal species [6], both _\"Brettanomyces\"_ and _\"Dekkera\"_ have been used in the scientific literature. However, the designation _\"Brettanomyces\"_ is more commonly applied in the food and biotechnology industries, wherein the species _B. bruxellensis_ is immediately associated with wine by all the stakeholders of the sector [7,8]. Thus, we preferentially use _Brettanomyces_ in this review, but also use the original names given in the literature cited. _B. bruxellensis_ is the best-known species within the genus; it is a facultative anaerobic yeast that has been isolated from different sources including fruit peels, beer, wine, cheese, kombucha, kefir, tea, olives, sodas, and wooden barrels [9]. This species has a significant role in the production of Belgium-style beer, especially in the aromatic profiles of Lambic and Gueuze beers, via the release during its proliferation of different aromatic compounds such as fused alcohols and esters (responsible for the floral, fruits, and spicy characteristics) [10, 11, 12, 13, 14, 15, 16]. In contrast, _B. bruxellensis_ is considered to be a spoilage yeast in the wine sector, causing negative sensory properties for productsaged in wood [17]. Contamination by this yeast causes the \"Brett\" character, consisting of unpleasant aromas that are perceived when specific thresholds are exceeded [18]. These compounds include secondary metabolites such as nitrogenous compounds (e.g., 2-acetyl-3,4,5,6-tetrahydropropylidine, 2-acetyl-1,2,5,6-tetrahydropyridine, and 2-ethyl-3,4, 5,6-tetrahydropyridine) and/or volatile phenols (4-vinylphenol, 4-vinylguiacol, 4-ethyl phenol, and 4-ethylguiacol), the latter being associated with \"horse sweat\", \"leather\", \"medicinal\", \"barmyard\", and other similar descriptors [18; 19; 20]. Karyotype analyses, intron splice site amplification (ISS-PCR), and microsatellite genetic profiling performed on strains derived from different microbial collections worldwide have revealed that _B. bruxellensis_ strains exhibit a high intraspecific variability, which is also supported by the chromosome number (from four to nine) and the ploidy state [21; 22; 23]. Karyotype, ploidy, and the source of isolation can be useful in classifying the different strains (e.g., dissimilar _B. bruxellensis_ strains have been obtained from tequila/ethanol, wine, and kombucha environments). Similarly to other yeasts, changes in ploidy are believed to be a driver of adaptation [24]. _B. bruxellensis_ isolated from wine shows specific adaptations, such as tolerance to SO2, which are linked to their diploid or triploid state [23; 25]. This aspect deserves a thorough investigation; indeed, while gene duplication in _Saccharomyces cerevisiae_ has been suggested to bring evolutionary and adaptive benefits to the species, recent studies have revealed that non-_Saccharomyces_ yeast species (namely _Kluyveromyces marxianus_) face environmental stresses via only the up-/downregulation of multiple pathways (anti-osmotic, antioxidative, etc.) rather than a ploidy change [24; 26]. _B.