The development of molecular tools and methods for genetic modification of organisms plays a very important role in the production of new metabolites and development of new host organims and their transfer into biotechnological processes. Bacteria Escherichia coli and yeast Saccharomyces cerevisiae have so far featured as the most successful industrial hosts, but more attention is nowadays being paid to the use of non-conventional yeasts. We focused on the non-conventional yeast species Hanseniaspora uvarum, which is most often found on grape berries, where it also plays a very important role in the alcoholic fermentation and can consequently affect the quality of wine. Through our research, we asembled two plasmids in yeast S. cerevisiae with 700-1000 bp long homologous region of ribosomal RNA or transposon Ty3, which allowed integration of the gene cassettte into the H. uvarum genome. The assembled plasmid also carried sequences for a selection marker, green fluorescent protein and elements that allow plasmid replication in both S. cerevisae and E. coli. Plasmid was then inserted into H. uvarum by combined transformation with electroporation and lithium acetate, where the gene cassette was integrated into the genome by homologous recombination. Because of low transformation efficiency we tried to improve the transformation method for H. uvarum, but unfortunately we did not succeed,. However, we achieved insertion of the gene for green fluorescent protein into H. uvarum by homologous recombination and we also confirmed its successful expression. In the future, this approach could be utilized for strain labeling, cellular localization, as well as growth and viability monitoring.
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