Abstract

The glucoamylase gene of Aspergillus niger, glaA , is expressed at high levels in the presence of starch or maltose and repressed at the level of transcription by glucose. The promoter region of glaA was subjected to deletion and site-specific mutation analysis. Using autonomously replicating vectors with the A. niger lacA as the reporter gene, regulatory elements that control glaA expression in A. niger were mapped. Expression analysis showed that two elements that closely match the AnCF-binding consensus sequence were important for glaA expression. Site-directed mutagenesis of four elements that matched the CreA binding consensus showed that only two of these sites were important for regulation and that both sites were essential for glaA expression in maltose and glucose. Interestingly, one of the essential CreA sites overlapped the upstream portion of the single AmyR consensus element while the other site was immediately downstream from this site. The results of this study, therefore, suggested that both AnCF and AmyR were required for transcription of glaA and that regulation involved competition for overlapping binding sites between the transcriptional repressor CreA, and starch responsive transcription activator AmyR. The highly expressed pyruvate kinase gene of Aspergillus niger, pkiA , encodes an enzyme in glycolysis. This study found that pyruvate kinase expression was approximately 8-fold higher under glycolytic growth conditions, as compared to gluconeogenic growth conditions. Deletion analysis of the pkiA promoter region found three regions important for expression. One of the regions contains a 17 base pair sequence that is conserved in A. niger and A. nidulans . Another region contains a sequence closely matching the AnCF-binding consensus