Disruption of three acid proteases in Aspergillus niger: effects on protease spectrum, intracellular proteolysis, and degradation of target proteins

• Published in: European journal of biochemistry Vol. 247; no. 2; pp. 605 - 613
• Main Authors: Hombergh, J.P.T.W. van den, Sollewijn Gelpke, M.D, Vondervoort, P.J.I. van de, Buxton, F.P, Visser, J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.07.1997
• Subjects: Aspartic Acid Endopeptidases - biosynthesis; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - metabolism; Aspergillus; Aspergillus niger; Aspergillus niger - enzymology; Aspergillus niger - genetics; Blotting, Southern; cascade activation; disruption; Endopeptidases - metabolism; Fungal Proteins; Genetic Linkage; Kinetics; Moleculaire genetica van industriële micro-organismen; Molecular Genetics of Industrial Micro-organisms; Molecular Sequence Data; Mutagenesis; plant biochemistry; plant breeding; plant genetics; plant physiology; Plasmids; protease; proteolytic degradation; Restriction Mapping; VLAG
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1997.00605.x

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic 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, repectively. In the delta pepE disruptant, the total intracellular proteolytic activity was reduced to 32%. Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the delta pepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed delta pepA delta pepB, delta 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 was the highest in the triple disruptant and the delta pepA delta pepB recombinant.