engleski

Genetic transformation of yeast Dekkera/Brettanomyces bruxellensis

Yeast Dekkera/Brettanomyces bruxellensis is a notorious wine spoilage yeast, but it is also a potential industrial organism for production of bioethanol and acetic acid. However, for detailed genetic characterization and construction of new strains with potential application in biotechnology, it is necessary to develop protocol for genetic transformation, i.e. for the introduction of for- 115 Book of Abstracts: “Vera Johanides” – Biotechnology in Croatia by 2020 eign DNA into the cell. We have developed procedure for transformation of yeast D. bruxellensis by varying parameters of the three frequently used methods for transformation of Saccharomyces and non- Saccharomyces yeasts: transformation by alkali cations, electroporation, and spheroplast transformation. Obtained transformation efficiency was up to 20 transformants/μg linear DNA. As a selective marker sequence kanMX4 was used, allowing for selection of transformants on geneticin-containing medium. Molecular analysis confirmed presence of the transforming DNA fragment in the genome. Stability of transformants varied between 93.6% and 100%. To enable selection of transformants without the use of antibiotics, we have isolated strain with inactive URA3 gene therefore allowing for selection using medium lacking uracil. These results represent the first and necessary step in the development of methodology for precise genetic modification of D. bruxellensis which could enable its use in biotechnological production.

Dekkera/Brettanomyces bruxellensis; genetic transformation; sorbitol; electroporation; LiAc/PEG transformation method; ura- auxotroph

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