In this B.Sc. thesis, we investigated the establishment of a transformation protocol for non-conventional and genetically poorly characterized yeast species Hanseniaspora uvarum and Hanseniaspora opuntiae. Both species are of interest due to their potential application in mixed fermentations with Saccharomyces species, where they can particularly contribute to the aromatic complexity of wine. To validate the transformation procedure, we used an established protocol for Saccharomyces cerevisiae transformation using the plasmid p414_HisCas9, whereas an adapted electroporation transformation procedure was developed for the Hanseniaspora strains. For the transformation, we used the plasmid pJJH3200_His_Cas9, which features a species-adapted origin of replication. S. cerevisiae transformants grew successfully, as did H. uvarum transformants after repeating the experiment with a higher initial optical density of cells, whereas no transformants were obtained for H. opuntiae. Determination of the total protein concentration of H. uvarum transformants mostly yielded negative or low values; therefore, the presence and expression of the Cas9 protein could not be reliably confirmed. The results indicate that the transformation of representatives of the genus Hanseniaspora is methodologically challenging and that further optimization of transformation, PCR validation of transformants, and improvements in protein detection approaches will be required to establish a reliable system.
|
