NEW PROTEASE MUTANTS IN ASPERGILLUS-NIGER RESULT IN STRONGLY REDUCED IN-VITRO DEGRADATION OF TARGET PROTEINS GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF 7 COMPLEMENTATION GROUPS

Several mutants of Aspergillus niger, deficient in extracellular prote ase expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutag enesis of conidiospores and selected by halo-screening on a new dual-s ubstrate plate assay, belong to at least seven different complementati on groups. These seven prt loci were assigned to linkage groups using master strains with marked chromosomes. One prt locus (prtC) could be assigned to linkage group I, three (prtB, prtE and prtG) to linkage gr oup III, one (prtF) to linkage group V and the two remaining loci (prt A and prtD) to linkage group VIII. Extracellular proteolytic activitie s varied from 2 to 3% up to 80% of the protease activity of the parent al strain. Assigning the different prt mutants to structural or regula tory genes is difficult since only one structural gene, pepA, has been mapped unambiguously on linkage group I but is not identical to prtC. All prt mutants except for prtC are likely to be regulatory mutants o r else belong to a proteolytic cascade because residual activities sho wed that more proteolytic activities were affected simultaneously. Dou ble mutants were constructed both by recombination and by a second rou nd of mutagenesis. In both cases mutants with further reduced extracel lular proteolytic activities were isolated. A sensitive in vitro degra dation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used t o show extremely reduced proteolytic PELB degradation in the culture m edia of some of these mutants.