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Genetic transformation and characterization of yeast Dekkera/Brettanomyces bruxellensis

Yeast Dekkera/Brettanomyces bruxellensis is a notorious wine spoilage yeast that can cause great economic losses, but it is also a potential industrial organism for the production of bioethanol and acetic acid. However, for the construction of strains that could potentially be used in biotechnology as well as for the detailed genetic characterization of this species it is necessary to establish the protocol for genetic transformation, i.e. for the introduction of the foreign DNA fragment into the cell. We have developed two such protocols by varying parameters of transformation methods commonly used for the transformation of Saccharomyces and non- Saccharomyces yeasts. Since the transforming DNA fragment carried kanMX4 sequence, transformants were selected on the geneticin-containing medium. Integration of the transforming DNA fragment into the genome was confirmed by Southern blot and the stability of the transformants was 93-100%. We have also isolated ura3 mutant enabling selection of transformants on the medium without uracil instead of using antibiotic-based selection. Surprisingly, restriction analysis of the mutated ura3 gene suggested presence of the two ura3 alleles. Therefore, we performed bioinformatics analysis on the genome of the wild type D. bruxellensis, RFLP (restriction fragment length polymorphism) of the URA3 loci, as well as complementation of the ura3 E. coli strain with the wild type URA3 alleles. Results obtained confirmed the existence of two functional URA3 alleles in the wild type genome supporting the hypothesis that D. bruxellensis strain CBS2499 is rather partial diploid than haploid. Moreover, our transformation results and isolation of the ura3 mutant will encourage and enable construction of biotechnologically applicable strains.

Dekkera/Brettanomyces bruxellensis; transformation; auxotrophy

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