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A new method for screening and isolation of hypersecretion mutants in Aspergillus niger

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Author: Weenink, X.O. · Punt, P.J. · Hondel, C.A.M.J.J. van den · Ram, A.F.J.
Type:article
Date:2006
Institution: TNO Kwaliteit van Leven
Source:Applied Microbiology and Biotechnology, 6, 69, 711-717
Identifier: 239125
doi: doi:10.1007/s00253-005-0013-y
Keywords: Biology · Biotechnology · Enzymes · Fungi · Organic acids · Screening · Starch · Aspergillus niger · Extracellular proteins · Glucoamylase proteins · Mutagenesis · glucan 1,4 alpha glucosidase · laccase · detection method · fungus · microbiology · article · Aspergillus niger · catalysis · catalyst · correlation analysis · enzyme activity · fungus growth · fungus isolation · fungus mutant · isolation procedure · nonhuman · phenotype · Pleurotus ostreatus · protein synthesis · screening · Aspergillus niger · Biomass · Glucan 1,4-alpha-Glucosidase · Laccase · Mutation · Pleurotus · Protein Transport · Recombinant Proteins · Starch · Aspergillus niger · Fungi · Pleurotus ostreatus

Abstract

Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular 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 (GlaA 512-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 mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase 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. © Springer-Verlag 2005.