ANALYSIS OF THE REGULATION OF THE ASPERGILLUS-NIDULANS ACUD GENE, ENCODING ISOCITRATE LYASE, BY CONSTRUCTION OF A HYBRID PROMOTER

In order to confirm functionally that a 208 bp fragment of the 5'-flanking sequence of the acuD gene of Aspergillus nidulans is the region responsible for acetate inducibility and catabolite repression, a hybrid promoter was constructed by insertion of this fragment into the promoter of the (highly expressed) oliC gene of A. nidulans. Analysis of expression of the lacZ reporter gene fused to the oliC/acuD promoter showed induction by acetate at much higher levels than wild-type acuD expression. Acetate inducibility of the hybrid promoter was dependent on the facB gene, demonstrating that a facB-dependent upstream activating sequence (UAS) for acetate must be located in the 208 bp acuD fragment. In parallel, partial relief of the transcriptional repression of acetate inducibility by sucrose and glucose was observed in a creA(-) background, showing that the 208 bp acuD fragment also responds to the a creA gene. In addition, the results show that combination of a regulatory element from a low-expression promoter (acuD) with a high-expression constitutive promoter (oliC) leads to amplification of the level of regulated expression.