CLONING AND CHARACTERIZATION OF THE PEPE GENE OF ASPERGILLUS-NIGER ENCODING A NEW ASPARTIC PROTEASE AND REGULATION OF PEPE AND PEPC

have cloned the pepE gene of Aspergillus niger, encoding an aspartic protease (PEPE): by screening a h genomic DNA library with a heterologous probe, the Neurospora crassa gene coding for a vacuolar proteinase. Sequencing of pepE genomic and cDNA clones revealed that the gene contains three introns, which are 91, 56 and 58-bp long. The deduced protein consists of 398 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature part of PEPE has extensive homology with other aspartic proteinases such as pepsins, cathepsins and, in particular, with proteinase A of Saccharomyces cerevisiae and pepsin 1 of Candida albicans. Northern blot analyses revealed that cells contain an abundant pepE transcript whose amount does not change upon carbon or nitrogen limitation, the presence of proteins in the medium or changes in the pH of the medium. We also show that pepC, the A. niger homologue of yeast protease B, is also expressed constitutively under these conditions.