GENETIC-TRANSFORMATION OF AUXOTROPHIC MUTANTS OF THE FILAMENTOUS YEAST TRICHOSPORON-CUTANEUM USING HOMOLOGOUS AND HETEROLOGOUS MARKER GENES

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully in spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per-mu-g of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementing cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroplasts with an efficiency of 50-200 colonies per-mu-g of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.