Title
A new method for screening and isolation of hypersecretion mutants in Aspergillus niger
Author
Weenink, X.O.
Punt, P.J.
van den Hondel, C.A.M.J.J.
Ram, A.F.J.
TNO Kwaliteit van Leven
Publication year
2006
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.
Subject
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
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http://resolver.tudelft.nl/uuid:215fc8ae-12bf-49db-9e68-be10ffd6ae1b
DOI
https://doi.org/10.1007/s00253-005-0013-y
TNO identifier
239125
ISSN
0175-7598
Source
Applied Microbiology and Biotechnology, 69 (6), 711-717
Document type
article