Cloning and Sequence Analysis of the Candida tropicalis URA3 Gene Encoding Orotidine-5`-Phosphate Decarboxylase

Abstract.

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 thatis defective in orotidine-5`-phosphate decarboxylase (ODCase). As aselectable marker, we isolated and characterized the C. tropicalisURA3 gene, which codes for ODCase. The gene was cloned bycomplementation of the ura3 mutation of Saccharomycescerevisiae SHY-3 and the pyrF mutation of Escherichiacoli. The C. tropicalis U-6 was transformed by plasmidcontaining the C. tropicalis URA3 gene at a frequency of 1 to10 transformants per microgram of plasmid DNA. When the URA3gene was expressed in E. coli minicells, a 30-kDa protein wasidentified. Nucleotide sequence analysis revealed the presence of anopen reading frame, encoding a protein of 268 amino acids with acalculated molecular mass of 29.7 kDa. The nucleotide sequence ofURA3 gene and its deduced amino acid sequence showedsignificant homology to those of the ODCase of other fungal species.