The development of Aspergillus niger var. awamori as a host for the expression and secretion of heterologous gene products.

Filamentous fungi possess unique features which make them attractive as hosts for the production of heterologous gene products. For example, certain fungal species are capable of secreting large quantities of protein in submerged culture. Selected strains of Aspergillus niger can produce greater than 20 g of glucoamylase I ~ I in industrial fermentations [ 11. Some filamentous fungi, including A. niger, have a long history of safe use for the production of enzymes, food products. antibiotics. and biochemicals. Additionally, there is an extensive knowledge base covering low-cost fermentation and large scale recovery of proteins from filamentous fungi. During the past five years, several laboratories have explored the use of filamentous fungi for the expression and secretion of heterologous proteins (for recent reviews, see [2-71). Our laboratory has developed techniques to use the filamentous Deuteromycete fungus A. niger var. awamori as a host organism for the expression and secretion of bovine chymosin and Rhizomucor miehei aspartyl protease (RmAP), two milk-clotting enzymes used commercially in cheese manufacturing. Chymosin is synthesized in the fourth stomach of suckling calves as a zymogen precursor (preproenzyme) with a 16 amino acid signal peptide and a 42 amino acid propeptide which is autocatalytically removed at low pH [8]. Chymosin is the preferred enzyme for cheesemaking, primarily because it preferentially cleaves a specific Phe-Met bond in tc-casein, resulting in a high degree of milk clotting with a very low amount of non-specific