van den Hombergh J P, van de Vondervoort P J, van der Heijden N C, Visser J
Molecular Genetics of Industrial Microorganisms Agricultural University, HA Wageningen, The Netherlands.
Curr Genet. 1995 Sep;28(4):299-308. doi: 10.1007/BF00326427.
Several mutants of Aspergillus niger, deficient in extracellular protease expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutagenesis of conidiospores and selected by haloscreening on a new dual-substrate plate assay, belong to at least seven different complementation 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 group III, one (prtF) to linkage group V and the two remaining loci (prtA and prtD) to linkage group VIII. Extracellular proteolytic activities varied from 2 to 3% up to 80% of the protease activity of the parental strain. Assigning the different prt mutants to structural or regulatory 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 or else belong to a proteolytic cascade because residual activities showed that more proteolytic activities were affected simultaneously. Double mutants were constructed both by recombination and by a second round of mutagenesis. In both cases mutants with further reduced extracellular proteolytic activities were isolated. A sensitive in vitro degradation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used to show extremely reduced proteolytic PELB degradation in the culture media of some of these mutants.
已分离出几种黑曲霉细胞外蛋白酶表达缺陷的突变体，并对其进行了遗传学和生物化学特征分析。这些突变菌株是通过对分生孢子进行体内紫外线诱变后获得的，并通过在一种新的双底物平板测定法上进行光晕筛选来选择，它们至少属于七个不同的互补群。使用带有标记染色体的主菌株将这七个prt基因座定位到连锁群。一个prt基因座（prtC）可定位到连锁群I，三个（prtB、prtE和prtG）定位到连锁群III，一个（prtF）定位到连锁群V，其余两个基因座（prtA和prtD）定位到连锁群VIII。细胞外蛋白水解活性从亲本菌株蛋白酶活性的2%至3%到80%不等。由于只有一个结构基因pepA已明确定位在连锁群I上，但与prtC不同，因此很难将不同的prt突变体归为结构基因或调控基因。除prtC外，所有prt突变体可能都是调控突变体，或者属于蛋白水解级联反应，因为残留活性表明有更多的蛋白水解活性同时受到影响。通过重组和第二轮诱变构建了双突变体。在这两种情况下，都分离出了细胞外蛋白水解活性进一步降低的突变体。开发了一种基于同源果胶裂解酶B（PELB）蛋白的灵敏体外降解测定法，用于分析黑曲霉中的蛋白水解降解，并用于显示其中一些突变体培养基中PELB的蛋白水解降解极度降低。
