# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 2 Development of Molecular Tools for the Genetic Modification of _B. bruxellensis_
### Transformation Protocols  
Over the past 30 years, molecular tools and transformation protocols have been designed and exploited mainly for _S. cerevisiae_ species. Nevertheless, these tools and protocols can be taken as models and applied to the manipulation of non-_Saccharomyces_ yeasts (e.g., _Yarrowia lipolytica_, _Pichia pastoris_ syn. _Komagataella phaffii_, _Hansenula polymorpha_ syn. _Ogataea angusta_, _Kluyveromyces lactis_, _Candida albicans_, and _C. glabrata_) in order to study or modify metabolic traits that are useful in industrial processes [55]. In Table 5, a comparison of the different methods available for the transformation of _B. bruxellensis_ strains is presented, considering that the well-known high genetic variability that exists in the species may create limitations. Various approaches have been proposed by a few research groups: the LiAc/PEG transformation protocol [49], electroporation treatment [56; 49], the spheoroplast transformation procedure, and the spheoroplast intraspecific fusion methodology [49; 57]. Lithium acetate treatment causes a transitional opening in the cell membranes of yeasts to permit the acquisition of genetic material from the outside; this method is highly efficient in _B. bruxellensis_ when using up to 100 ug of DNA [49]. The electroporation method is quick and simple, whereby the exogenous DNA enters through pores in the cell wall and membrane created by a high-voltage pulse. Its efficiency significantly depends on the yeast species, cell growth phase, and final density of the culture, as well as the electroporation pulse parameters [49]. This protocol was optimized in 2015 in a study by [PERSON] et al. [56], in which the electroporation pulse parameters (1.8 kV and 5 ms) and the optimal osmotic stabilizer (1M sorbitol) were established. Thereafter, this technique was utilized to transform several strains with cassettes containing genes encoding for fluorescent proteins to discriminate subpopulations by flow cytometry and fluorescent microscopy [56]. While spheoroplast transformation was successfully applied in _S. cerevisiae_ using 1 M sorbitol as the osmotic stabilizer, it resulted in a procedure unsuitable for _B. bruxellensis_, contrary to what was observed for the electroporation approach. Indeed, this chemical has a negative effect on this yeast species, inducing a state in which cells remain viable but become nonculturable, similar to what is observed after cell exposure to sulfite [49; 58]. Another transformation technique that includes spheorplast formation is recursive intraspecific fusion based on genome shuffling. Genome shuffling is a feasible method for the rapid manipulation of complex phenotypes using whole cells and organisms. This procedure can be useful for generating stable changes in the genomes of _Brettanomyces_ strains, and its application as an effective whole-cell engineering strategy could be advantageous for the rapid improvement of industrially important microbial phenotypes [58]. This overview shows that the most efficient strategy for _B. bruxellensis_ transformation is the electroporation protocol proposed by [PERSON] et al. in 2015 [56] and  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Vectors** & **Drug Resistance** & **Promotor** & **Terminator** & **Insertion** & **References** \\ \hline pMK–T\(-\)_TDH1_pr–_kanMAX_ & Kanamycin & Bb_TDH1_ & Ag_TEF2_ & Bb_TDH1_, Ag_TEF2_ & [53; 54] \\ pMK–T\(-\)_TDH1_pr–_natMAX_ & Kanamycin & Bb_TDH1_ & Ag_TEF2_ & Bb_TDH1_, Ag_TEF2_ & [53; 54] \\  & Nourseothricin &