Cloning and sequence analysis of the Candida tropicalis URA3 gene encoding orotidine-5'-phosphate decarboxylase.

An integrative transformation system was established for a phenol-utilizing strain of Candida tropicalis M4. The system is based on an auxotrophic mutant host of C. tropicalis U-6 that is defective in orotidine-5'-phosphate decarboxylase (ODCase). As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene, which codes for ODCase. The gene was cloned by complementation of the ura3 mutation of Sachharomyces cerevisiae SHY-3 and the pyrF mutation of Escherichia coli. The C. tropicalis U-6 was transformed by plasmid containing the C. tropicalis URA3 gene at a frequency of 1 to 10 transformants per microgram of plasmid DNA. When the URA3 gene was expressed in E. coli minicells, a 30-kDa protein was identified. Nucleotide sequence analysis revealed the presence of an open reading frame, encoding a protein of 268 amino acids with a calculated molecular mass of 29.7 kDa. The nucleotide sequence of URA3 gene and its deduced amino acid sequence showed significant homology to those of the ODCase of other fungal species.