Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases.
In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated. The major protease, which is responsible for 80-85% of the total activity, is aspergillopepsin A, a protein of ca. 43 kDa, the activity of which is inhibited by pepstatin. In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin. Several protease-deficient mutants were obtained by in vivo UV mutagenesis. In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy. In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted. In three UV-induced mutants, aspergillopepsin A is also missing. One of these mutants, AB1.18, is mutated in the pepA gene, which is located on chromosome I. One of the other mutants, AB1.13, which has only 1-2% of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B. The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene. Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII. Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.
Mattern
I E
IE
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
van Noort
J M
JM
van den Berg
P
P
Archer
D B
DB
Roberts
I N
IN
van den Hondel
C A
CA
D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis / Q000235:genetics
D005656:Fungal Proteins; D016282:Aspartic Acid Endopeptidases; C029094:aspergillopepsin I