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

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 (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 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.

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Acknowledgements

The authors thank Dr. R.J. Gouka from Unilever, The Netherlands, for providing pUR7894 containing the cDNA sequence of the pox2 gene and Dr. D. Archer who kindly provided the A. niger acid amylase gene. We thank Dr. B.C. Lokman and Dr. V. Joosten for valuable discussions and R. Timmermans for his help with the isolation and characterization of the mutants. This work was supported by a grant from STW (Dutch Foundation for Technical Research).

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Correspondence to Arthur F. J. Ram.

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Weenink, X.O., Punt, P.J., van den Hondel, C.A.M.J.J. et al. A new method for screening and isolation of hypersecretion mutants in Aspergillus niger . Appl Microbiol Biotechnol 69, 711–717 (2006). https://doi.org/10.1007/s00253-005-0013-y

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Keywords

  • Heterologous Protein
  • Laccase Activity
  • Laccase Production
  • Starch Medium
  • Glucoamylase Gene