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Genetic side-effects during gene replacement in yeast Saccharomyces cerevisiae

Gene replacement is one of basic techniques used for the construction of producer strains in biotechnology. This method is based on the introduction of linear, non-replicative transforming DNA fragments to replace the specific genomic region by homologous recombination, using cell’s endogenous proteins evolutionary conserved across all eukaryotic organisms. We have investigated gene replacement in yeast Saccharomyces cerevisiae, a model organism used for research of almost all processes in eukaryotic cells and one of the most important industrial microorganisms. Despite the general belief 105 Book of Abstracts: “Vera Johanides” – Biotechnology in Croatia by 2020 that gene replacement in yeast occurs in more than 99 % of cases, we have observed that up to 10 % of transformants are due to aberrant genetic events that include duplication of targeted chromosome, additions of the transforming DNA fragment next to the targeted sequence and random, illegitimate integrations of the transforming DNA fragment. Moreover, it was observed that both the percentage and the spectrum of aberrant genetic events depend on the type of modification being introduced to the genome. Results of this research give guidelines that facilitate the increase in successful gene replacement and at the same time warn that newly constructed strains should be properly checked using appropriate molecular genetic techniques.

gene targeting; chromosome duplication; Saccharomyces cerevisiae

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