Genetics of the yeast, Phaffia rhodozyma

The aim of this thesis is studying the genetics of Phaffia and to develop a genetic transformation system for this yeast. The genetic properties of Phaffia were studied on the gene and genome level.As a first step the molecular structure of the Phaffia actin gene was analyzed. Actin genes are highly conserved throughout nature, and as such they have been used for the classification of significantly diverging eukaryotic groups, like (in-)vertebrates, plants and fungi.We anticipated that the analysis of the primary structure of Phaffia actin gene and comparison with the actin genes from fungi, including 2 ascomycetous filamentous fungi, 2 basidiomycetous yeasts and 5 ascomycetous yeasts would provide further phylogenetic information on this yeast.It was found that the Phaffia actin gene encoded a protein consisting of 375 amino acids. In addition 4 (non-coding) intervening sequences were present. Comparison of both the coding DNA sequence and its predicted protein product with their fungal counterparts, revealed that least homology was found with the ascomycetous yeasts, like Saccharomyces cerevisiae and Kluyveromyces lactis . It was also shown, that based on these comparisons Phaffia is closer related to the filamentous ascomycetous fungi Thermomyces lanuginosus and Aspergillus nidulans , whereas most homology was found with the basidiomycetous yeast Filobasidiella neoformans (perfect stage of Cryptococcus neoformans ).In addition to the phylogenetic analysis of the actin exons, the architecture of the introns (splice site consensus sequences, size, position in the gene) was compared. it was shown that the Phaffia introns most resembled that of Filobasidiella neoformans where as least resemblance occurred with the ascomycetous yeasts. This result was in agreement with the actin exon homology studies. Furthermore, the presence of multiple introns in the Phaffia actin gene resembled the situation in the actin genes from F. neoformans and the filamentous fungi, whereas the ascomycetous yeasts only carry one intron in their actin genes.Similar results were obtained by (phylo-)genetic analysis of the five introns containing Phaffia glyceraldehyde-3-phosphate dehydrogenase gene.The genomic organization of the multiple rDNA genes in Phaffia was elucidated. It was found that Phaffia carries the rDNA genes in three clusters, of 12, 14 and 35 copies, on three different chromosomes. In the ascomycetous yeasts and fungi the rDNA is mainly present on one chromosome.The significant differences on the gene and genome level with the ascomycetous yeasts affected the strategy for the development of a transformation system for Phaffia . Whereas several marker gene sequences or sequences for plasmid replication and maintenance can be readily interchanged between most ascomycetous species as a result of high homologies, it was shown that this was not the case for Phaffia . Therefore an almost entirely homologous transformation sytem was developed using plasmids carrying the dominant G418 resistance gene (Km R), driven by either the Phaffia actin or the gpd promoter and a Phaffia ribosomal DNA (rDNA) fragment for homologous integration.It was found that the rDNA clusters could serve as a target for high copy number integration. This integrative transformation system was used to determine the ploidy of Phaffia , strain CBS 6938, by monitoiring chromosomal shifts as a result of multiple integrations. It was found that this strain was haploid.Plasmids carrying the gpd promoter driven Km Rgene transformed Phaffia with significant higher efficiencies than constructs with the actin promoter. Furthermore, the plas-mid copy number and transformation efficiencies of the first were found to be influenced by the presence of the gpd terminator downstream the Km Rgene.It was shown that plasmid amplification occurred independent from selection pressure to an extend that appeared to be negatively related to the effectiveness of expression of the Km Rgene. This observation indicated that the rising metabolic burden, as a consequence of amplification, imposes limits to the number of plasmid copies.The effectiveness, stability, and plasmid amplifying properties of the Phaffia transformation system offer possibilities for the use of recombinant DNA technology in developing industrially attractive Phaffia strains.