engleski

Development of molecular-genetic tools for yeast Dekkera/Brettanomyces bruxellensis

Yeast Dekkera/Brettanomyces bruxellensis is long known as a spoilage microorganism in wine where it produces volatile phenols giving the wine a distinct offflavor described as horse sweat, mice or burnt plastic. This yeast produces and tolerates high ethanol concentrations which enables it to spoil the wine at late stages of its production, making the economic losses even greater. But besides being able to produce high ethanol concentrations (around 14%), it was shown that B. bruxellensis can ferment cellobiose and be adapted to growth inhibitors typically present in lignocellulose hydrolasate. Therefore, it is probably one of the most promising non- Saccharomyces yeast species for second- generation bioethanol production. In hand with growing scientific interest in physiological features and industrial potential of D. bruxellensis, there is also a growing need for development of molecular-genetic tools. The aim of our research is to develop a set of genetic markers, vectors, yeast strains and methods which will ultimately enable routine genetic modifications and protein expressions in D. bruxellensis. We showed that D. bruxellensis transformants can be obtained by transformation with linear DNA fragment containing KanMX4 selection marker, but initially transformation efficiencies achieved by standard LiAc/PEG or electroporation procedures were very low and yielded only several transformants per sample. By optimizing a series of parameters (growth phase at which the cells are harvested for transformation, pretreatment of cells, the final cell density in the transformation sample, pulse strength and DNA mass) we developed the electroporation procedure for D. bruxellensis. The optimized procedure consistently yields 300-500 transformants per sample and results in transformation efficiencies of 2.8x10E3 up to maximum 2.7x10E4 transformants per microgram of DNA. Establishing a reliable and efficient transformation procedure presents the first critical step toward developing full spectra of genome-editing tools for D. bruxellensis. Our current research is focused on assessing possibilities of homologous recombination in D. bruxellensis, as well as isolation of auxotrophic strains and development of other dominant selection markers. Taken together, these results will facilitate both fundamental and applied research of this interesting yeast species.

Yeast, transformation

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