DISRUPTION OF 3 ACID PROTEASES IN ASPERGILLUS-NIGER - EFFECTS ON PROTEASE SPECTRUM, INTRACELLULAR PROTEOLYSIS, AND DEGRADATION OF TARGET PROTEINS

Three acid protease genes encoding two extracellular proteases (PEPA a nd PEPB) and one intracellular protease (PEPE) were disrupted in Asper gillus niger. Northern-blot analysis showed the absence of wild-type p rotease mRNAs in the disruptants while western-blot analysis proved th e absence of the encoded proteases. Characterization of the residual p roteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16 % and 94 % for the Delta pepA and the Delta pepB disruptants, respectively. In the Delta pepE disruptan t. the total intracellular proteolytic activity was reduced to 32 %. A part from the reduced intracellular pepstatin-inhibitable aspartyl pro tease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the Delta pepE strain. This may in dicate the presence of a cascade activation mechanism for several vacu olar proteases, triggered by the PEPE protein, similar to the situatio n in Saccharomyces cerevisiae. Disruption of a single protease gene ha d no effects on the transcription of other non-disrupted protease gene s in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was obser ved. By recombination, we also constructed Delta pepA Delta pepB, Delt a pepB Delta pepE and Delta pepA Delta pepE double disruptants as well as a Delta pepA Delta pepB Delta pepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity wa s the highest in the triple disruptant and the Delta pepA Delta pepB r ecombinant.