# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 2 Development of Molecular Tools for the Genetic Modification of _B. bruxellensis_
### Construction of Molecular Cassettes for B. bruxellensis Manipulation  
The current molecular tools for the genetic manipulation of _B. bruxellensis_ face many issues, hindering the immediate implementation of this species in biotechnological applications. As mutagenesis through homologous integration is difficult to perform in many non-_Saccharomyces_ yeasts [48], in 2013, [PERSON] et al. first attempted and implemented the insertion of a specific genetic element into _B. bruxellensis_ cells via nonhomologous recombination [49]. The authors transformed the CBS2499 strain with a heterologous DNA fragment of 1.9 kb, containing the _kanMX4_ sequence encoding for G418 resistance (G418\({}^{\text{R}}\)) and flanked with the regions 5\({}^{\prime}\)Sc and 3\({}^{\prime}\)Sc (220 bp of the 5\({}^{\prime}\) end and 165 bp of the 3\({}^{\prime}\) end of the _YMR224C_ ORF from _S. cerevisiae_[49]. A low transformation efficiency was detected, confirming the results obtained in _S. cerevisiae_ following the nonhomologous integration of heterologous transforming DNA [50]. This could be attributed to a tolerance to DNA mismatches, a tendency to repair the DNA damage throughout the nonhomologous end-joining (NHEJ) pathway or the illegitimate recombination apparatus, or a lower efficiency of the short sequence recombination (SSR) system [51]. This result supports the phylogenetic proximity of _B. bruxellensis_ to _P. pastoris_, which exhibits low transformation frequencies when homologous integration with ends-out vectors is carried out [51]. As a consequence, when proceeding with the genetic manipulation of a non-_Saccharomyces_ yeast, and with the construction of associated molecular tools, the natural inclination towards nonhomologous recombination has to be considered. The first circular vector proposed for _B. bruxellensis_ transformation was presented by [PERSON] et al. in 2014. It contained CIGO 1, 2, and 3 sequences to promote the autonomous replication of the plasmid [19]. Later, [PERSON] et al. (2016) inserted _CEN1_ and _CEN2_ sequences of _B. bruxellensis_ in several vectors [51]. In this case, the transformation efficiency was lower than that obtained using the linearized plasmid P892. Although this outcome confirmed that integrating vectors have a higher stability than replicating vectors in non-_Saccharomyces_ yeasts, the experiment suggested that the centromeric plasmids did not contain strong autonomously replicating sequences [51; 52]. In her doctoral thesis, [PERSON] (2017) proposed other cassettes for _B. bruxellensis_, i.e., pMK-T-_TDH1_pr-_kan_MX_ and pMK-T-_TDH1_pr-_natMX_ cassettes, including the _B. bruxellensis TDH1_ promoter (Bb_TDH1_) and the Ag_TEF2_ terminator (derived from _Ashbya gossypii_) [53]. When these elements were integrated into the genomes of AWRI1499, AWRI1608, and AWRI1626 strains, they brought about antibiotic resistance without affecting the growth rates of the recombinants in comparison to the wild types [53]. The search for useful autonomously replicating vectors recently led to the construction of a series of pMA plasmids containing the aforementioned cassettes for G418, Nat, or Hyg as selectable markers [53; 54]. The pMA-_TDH1_pr-_natMX_ vector was then modified to produce the fluorescent proteins GFP (green fluorescent protein) and TagBFP (blue fluorescent protein); both were flanked by the strong _S. cerevisiae_ promoter ScFBA1p and its terminator ScPGK1. The obtained plasmids were named pMA-_TDH1_pr-_natMX_::GFP and pMA-_TDH1_pr-_natMX_::BFP. Additionally, other plasmids containing the heterologous Ag_TEF2_ promoter instead of the homologous Bb_TDH1_ were constructed. However, the transformation experiments carried out with all the constructs revealed low efficiency [54]. This outcome is probably associated with the heterologous elements inserted into the vectors used, in which the Bb_TDH1_ promoter ensured an efficiency more than seven times higher than that of Ag_TEF2_. In fact, the tailored cassette for _B.