Integrative transformation of the dimorphic yeast arxula adeninivorans LS3 based on hygromycin B resistance

A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA. For this purpose a cassette was constructed which contains the E. coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A. adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene. This cassette was fused into the 25S rDNA of A. adeninivorans. Linearization of this vector was required for high transformation frequencies. The vector was integrated in multiple copies into the 25S rDNA by homologous recombination. Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions. Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.