• filamentous fungi;
  • fermentation;
  • protease inhibition;
  • GFP;
  • process parameters;
  • fractional factorial design


Filamentous fungi such as Aspergillus niger are attractive hosts for recombinant DNA technology because of their ability to secrete bioactive proteins with post-translational processing such as glycosylation. Foreign genes can be incorporated into the chromosomes of the filamentous fungi, providing superior long-term genetic stability. However, heterologous protein production is often severely hampered by fungal proteases. In this work, a recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP]#11 which carries a glucoamylase (GLA)-green fluorescent protein (GFP) fusion gene was selected as a model system to study the effects of bioprocess parameters—agitation intensity, initial glucose concentration, initial yeast extract concentration, and dissolved oxygen tension (DO)—on extracellular protease inhibition and heterologous protein production. Based on previous experimental experience and results, a 24–1 fractional factorial design was applied to the experiments. Each parameter was tested at two levels. It was found that agitation affected the GFP production most significantly. Higher agitation rate resulted in higher GFP production. Protease activity was most influenced by initial glucose concentration and DO. Fungal morphology was also affected by these parameters. The effects of these parameters on pellet size and pellet porosity are discussed. Copyright © 2003 Society of Chemical Industry