CLONING AND EXPRESSION OF THE ARO3 GENE ENCODING DAHP SYNTHASE FROM CANDIDA-ALBICANS

In Saccharomyces cerevisiae, the primary step in the aromatic (ARO) amino acid (aa) biosynthetic pathway is catalyzed by two isozymes of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS). The activity of one DAHPS isozyme (encoded by the ARO3 gene) is feedback inhibited by phenylalanine, whereas the other (encoded by the ARO4 gene) is inhibited by tyrosine. The expression of these genes is also regulated at the transcriptional level by the general control activator GCN4. We took advantage of the high degree of aa sequence homology between DAHPSs from several species to isolate ARO3 homologues from the pathogenic yeast Candida albicans. An ARO3/ARO4-specific sequence was generated from C. albicans genomic DNA by polymerase chain reaction amplification and used as a probe to screen a C. albicans cDNA library. A 1.3-kb cDNA clone was isolated and sequenced. The cDNA contains a long open reading frame predicting a 368-aa protein with significant homology to known DAHPSs, including both the S. cerevisiae ARO3 and ARO4 products (68.5% and 58.5% identity, respectively). Northern analysis of yeast and mycelial poly(A)(+)RNA. revealed equivalent expression of a 1.3-kb transcript in both cell types. A genomic clone was isolated by crosshybridization, and analysis of the 5' untranslated region revealed the presence of a putative GCN4-binding site. This clone complemented an aro3 mutation in S. cerevisiae; functional complementation was inhibited by the presence of excess phenylalanine (but not tyrosine) in the growth medium, confirming that the cloned gene is the C. albicans homologue of ARO3.