Aspergillus as a Cell Factory for Protein Production: Controlling Protease Activity in Fungal Production (2007)

CONTENTS 26.1 Introduction ............................................................................................................................. 441 26.2 Strain Development ................................................................................................................ 44226.2.1 Classical Methods to Screen for Protease Mutants ................................................. 442 26.2.2 Molecular Genetic Methods to Construct Protease Mutants ................................... 44326.2.2.1 Protease Genes ........................................................................................... 443 26.2.2.2 Protease Regulators ................................................................................... 44426.2.3 A Novel and Effi cient Method for Isolation of Protease-Defi cient Fungi ............... 446 26.3 Fermentation Conditions ........................................................................................................ 44626.3.1 pH Regulation ........................................................................................................... 447 26.3.2 Carbon Catabolite Control ....................................................................................... 448 26.3.3 Nitrogen Metabolite Control .................................................................................... 448 26.3.4 Sulfur and Phosphorus Metabolite Repression ........................................................ 449 26.3.5 Induction of Protease by Protein .............................................................................. 449 26.3.6 Bioprocess Engineering ........................................................................................... 450 26.3.7 System Biology Approach ........................................................................................ 45026.4 Conclusion ............................................................................................................................... 451 Acknowledgments .............................................................................................................................. 451 References .......................................................................................................................................... 45226.1 Introduction Since ancient times microorganisms have been used in a variety of traditional food processes (e.g., the production of alcoholic beverages, cheese, and bread). Fungi are applied in cheese-making and in traditionally oriental food such as soy sauce, tempeh, and sake. However, the presence and role of these microorganisms was for most processes only identifi ed in recent times. Fungi, like Aspergillus oryzae in the production of sake, were discovered to play a key role in the production of this product by the excretion of enzymes. In 1894, the fi rst microbial enzyme that was commercially produced appeared on the market, called “takadiastase”; it was in fact fungal amylase produced by Aspergillus oryzae [1]. Nowadays, a large number of fungal enzymes are commercially available and their application extends well beyond their traditional use in food processes. Glucoamylase, α-amylases, cellulase, lipase, and protease are only a few examples of enzymes produced by fi lamentous fungi that are commercially available. Aspergillus species, and particularly A. niger and A. oryzae, play a dominant role in the production of many of these enzymes [for a list of commercial enzymes see the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) at www.amfep.org].