# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 2 Development of Molecular Tools for the Genetic Modification of _B. bruxellensis_  
To date, eight different _B. bruxellensis_ strain genomes have been sequenced and deposited in the NCBI (National Center for Biotechnology Information) database (some information can be seen in Table 2). First, the DNA of the wine-isolated _B. bruxellensis_ CBS2499 strain was partially sequenced by [PERSON] et al. in 2007 [38]. Later, [PERSON] et al. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Features** & & **References** \\ \hline Growth rate (\(\mu\), h\({}^{-1}\)) & 0.037–0.114 & [29] \\ Ethanol yield (g/g glucose) & 0.44–0.46 & [29] \\ Glycerol yield (g/g glucose) & 0.0–0.026 & [28] \\ Crabtree positive & yes & [9; 20; 28] \\ Custers effect & yes & [9; 22; 28; 29] \\ Presence of respiratory complex 1 & yes & [9; 20; 28] \\ Nitrate assimilation & strain specific & [9; 22; 28] \\ Ethanol tolerance & 14\% & [28] \\ Whole–genome sequencing & yes & [8; 36; 37] \\ _AOX1_ & yes & [9; 20; 28] \\ Phenol metabolism & strain specific & [9; 28] \\ Enzymatic activities (_VPR; CD;_ \(\beta\)–glucosidase) & strain specific & [19; 28] \\ SO2 tolerance & strain specific & [37] \\ \hline \hline \end{tabular} _AOX1: Alcohol oxidase I; VPR: Vinyl Phenol Reductase; CD: cinnamate decarboxylase._  
\end{table} 
Table 1: Physiological and metabolic features of _B. bruxellensis_ species. (2012) completed its sequencing and discovered that it has the typical characteristics of the hemiascomyc (gene number, intron size and number, intergenic length, and gene content) [36]. The genome analysis identified 5600 predicted genes, of which several were duplicated, suggesting a diploid form. In the same year, [PERSON] et al. (2012) fully investigated the genome of another wine-spoilage strain, the widespread triploid AWRI1499 [37], in which a high density of single-nucleotide polymorphisms (SNPs) and an enrichment in genes for membrane proteins and oxidoreductase enzymes were revealed. Thereafter, the genomes of other _B. bruxellensis_ strains were sequenced: (i) CBS2796 (ATCC 52904) strain, isolated from sparkling wine, with a genome length of 11.77 Mb [38]; (ii) UMY321 strain (corresponding to L17) [22] recognized to be diploid, with 82,632 SNPs revealing a high level of heterozygosity [39; 40]; (iii) LAMAP2480 strain, with the largest genome found so far (26.99 Mb), has been the subject of study focusing on its spoilage role and biotechnological potential. The genome analysis showed the presence of several genes related to stress tolerance, nutrient uptake, ethanol production, and lignocellulosic assimilation [41]; and (iv) UCD 2041 strain isolated from fruit wine, the DNA sequence of which was compared with strains belonging to different _Brettanomyces_ species, such as _B.anus_ CBS1945, _B. anomala_ CBS8139, _B. naardenensis_ CBS6042, and _B. custersianus_ CBS4805, revealing a large genetic distance within the genus. Moreover, relevant marker genes of domestic adaptation and fermentation were identified in UCD 2041, CBS1945, and CBS8139. Peculiar horizontal gene transfer events, which are probably responsible for the ability of these strains to utilize sucrose, were also detected [42]. Furthermore, CBS11270 is an example of another sequenced strain isolated from industrial ethanol production, with a genome size of 15.39 Mb across four chromosomes and a high level of SNPs (40.6% of which are in coding regions). Genomic analysis showed that this strain is diploid, with several genes replicated in chromosomes 1 and 4, highlighting interchromosomal gene duplications and loss of heterozygosity in some of them [43; 44]. Lastly, the CRL-50 strain, which is one of the two oldest known _Brettanomyces_ isolates, was collected from a Carlsberg beer sample in Denmark between the years 1904 and 1908 [44] and sequenced by [PERSON] et al. [45]. A comparative genome analysis was carried out in order to compare this strain with other _B. bruxellensis_ strains isolated from beer, wine, kombucha, sodas, olives, and bioethanol production plants.