Molecular analysis of the Aureobasidium pullulans ura3 gene encoding orotidine-5'-phosphate decarboxylase and isolation of mutants defective in this gene.

Orotidine-5'-phosphate decarboxylase (OMP decarboxylase) catalyses the final step in the pyrimidine biosynthesis, the conversion of orotidine-5'-phosphate (OMP) to uridine-5'-phosphate. The ura3 gene of Aureobasidium pullulans. encoding OMP decarboxylase, was isolated from an Aureobasidium genomic library constructed in the plasmid pBlueskriptSK-. The ura3 gene of A. pullulans has an open reading frame of 271 amino acid residues. Analysis of the sequence revealed the presence of two introns. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Aspergillus niger, Neurospora crassa, Phycomyces blakesleeanus and Homo sapiens. The ura3 gene is the third Aureobasidium gene that has been cloned and analysed. We have also isolated ura3 mutants by selection of ethyl methanesulphonate mutagenised cells on 5-fluoroorotic acid. Transformation of these A. pullulans mutant strains to prototrophy showed the functionality of the cloned gene.