| Popis: |
Gene targeting is a genetic technique that allows the modification of an endogenous gene by homologous recombination. It is achieved by the introduction of linear, non-replicative DNA fragments that should replace the homologous (targeted) sequence in the genome. It is generally assumed that gene targeting in the conventional yeast Saccharomyces cerevisiae almost exclusively occurs by homologous recombination and that it is usually higher than 90 %. Non-conventional yeast species are isolated from different niches and have interesting properties such as utilization of different carbon sources, fermentation potential, tolerance to different growth inhibitors and they are emerging as potential producers in different biotechnological processes. However, the efficiency of gene targeting in most of those species is still unknown. Using linear transformation cassette, a DNA that contains antibiotic resistance gene in the middle flanked by regions homologous to the target sequence in the genome, we have determined the efficiency of gene targeting in Brettanomyces/Dekkera bruxellensis, Kluyveromyces marxianus and Scheffersomyces stipitis. Depending on the yeast species gene targeting efficiency, obtained using transformation cassettes with flanking homologies longer than 1 kb, varied from less than 0, 1 % to 75 %. Our results suggest that in some species (e. g. K. marxianus) use of flanking homologies, longer than 1 kb, allows the introduction of targeted modifications in the genome, while in some other yeast species, advanced gene-editing techniques e.g. CRISPR/Cas system might be mandatory to allow precise modifications of the genome. |
