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In biotechnology, budding yeast Brettanomyces (Dekkera) bruxellensis plays several important roles. On the one hand, it spoils wine by synthesizing “barnyard” and “band-aid” aromas, but it also adds coveted Brett character to some specialty beers and red wines. Furthermore, it can drive bioethanol fermentations and shows potential for second-generation bioethanol production. However, this potential awaits largely untapped, in part because we are still unable to tailor its physiology to the needs of the bioprocess. To tackle this challenge, our group developed the first protocol for effi- cient genetic transformation of B. bruxellensis and we are continuing our work by building much needed genetic toolbox. Here we will discuss challenges and pitfalls encountered during the development of the transformation system, starting from the optimization of transformation method to the complex fate of the transforming DNA which integrates randomly in the genome despite extensive homologies at its ends. We will detail our attempts to evade this obstacle by introducing genes which drive efficient gene targeting in S. cerevisiae. Moreover, we will outline the path for implementing state-of-the-art techniques of genetic manipulation, such as RNA interference and CRISPR/Cas9, as well as roadblocks that could arise on the way

Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, a good ethanol producer found in wine and a strain resistant to inhibitors found in lignocellulosic hydrolysates. Compared to parental strains, hybrids grew faster in the absence, as well as in the presence of acetic and levulinic acid and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates. It was shown that the overexpression of YAP1 gene, involved in a stress response, further increased the survival. Furthermore, hybrids displayed different fermentative potential in a CO2 production test suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with use of genetic engineering techniques is a promising approach for an improvement or development of new biotechnologically relevant strains of S. cerevisiae.

Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous (hybrid) diploid S. cerevisiae strains by mating two haploid isolates having those desirable traits. In comparison to parental haploids, hybrid diploids were more resistant to several inhibitors frequently found in different lignocellulosic hydrolysates, such as acetic and levulinic acid and 2-furaldehyde. Interestingly, hybrids showed different fermentation ability in a CO2 production test. Our results suggest that construction of intraspecies hybrids coupled with the use of different genetic engineering techniques is a promising approach for the improvement or development of new biotechnologically relevant strains of S. cerevisiae. Additionally, we also show that in two biotechnologically interesting natural isolates percentage of successful gene targeting (gene targeting fidelity) is much lower than in widely used laboratory strains. Furthermore, the most frequent off-targeted event was duplication of a targeted chromosome yielding aneuploids having two copies of targeted chromosome, one containing transformed and the other containing untransformed allele. These results warn us that it could be more difficult to make precise genetic modifications of natural S. cerevisiae strains than expected.

Bioethanol production from raw lignocelullosic material requires a strain of a microorganism which tolerates high ethanol concentrations and presence of growth inhibitors. Therefore we constructed hybrid diploid S.cerevisiae strains by mating two haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration and the other has been shown to be resistant to several growth inhibitors. These haploids were mated and resulting homozygous and heterozygous (hybrid) diploids were tested for the ability to produce ethanol and to grow in the presence of several growth inhibitors frequently found in different lignocelullosic hydrolysates (acetic and levulinic acid, 2-furaldehyde). The results showed that some hybrid diploids were more resistant to tested inhibitors than parental haploid strains and homozygous diploids. It is also interesting that constructed strains showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed hybrids. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains.