DOI 10.1007/s00253-005-0013-y APPLIED MICROBIAL AND CELL PHYSIOLOGY

Abstract Although filamentous fungi have a unique prop-erty of secreting a large amount of homologous extracel-lular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mu-tant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased gluco-amylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein