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Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes
Author(s) -
Ochsner Urs A.,
Glumoff Virpi,
Kälin Markus,
Fiechter Armin,
Reiser Jakob
Publication year - 1991
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320070511
Subject(s) - cosmid , biology , transformation (genetics) , mutant , auxotrophy , selectable marker , gene , microbiology and biotechnology , genetics , heterologous , plasmid , genomic library , peptide sequence
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 ( arg B gene) as selection marker with an efficiency of 5–100 transformants per μg of DNA. In these transformants the heterologous arg B 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 μg 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.