Filamentous Fungi as Expression Systems for Heterologous Proteins

Abstract

A wide variety of heterologous genes have been introduced into filamentous fungi with the purpose of producing the encoded protein, usually in a secreted form. There are several reasons for electing to use filamentous fungi as expression hosts and principal amongst them are that many species naturally secrete native proteins (mainly hydrolytic enzymes that facilitate their saprophytic lifestyle) with apparent efficiency, there is extensive commercial experience of large-scale fungal cultivation and extremely high yields of some native proteins have been achieved. There are also some commercial success stories with a few heterologous proteins providing hope that other proteins will be produced with the same facility and to equally high yields. In the main, such hopes have not been realised and it is the purpose of this chapter to discuss some of the pitfalls of fungal expression and present a summary of recent advances in the science underlying the production of heterologous proteins from filamentous fungi that is hoped will lead to rational improvement of fungi as expression hosts. Our focus on recent advances is because this topic has been reviewed previously (Archer and Peberdy 1997; Gouka et al. 1997a; Radzio and Kück 1997; Maras et al. 1999; Archer 2000; Conesa et al. 2001a; Peberdy et al. 2001; Punt et al. 2002) and we will cite these reviews where they summarise key issues and provide useful reference to the original literature. We will not specifically discuss yeast expression systems although several yeast species (e.g. Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris) have been successfully used as expression hosts at the laboratory and commercial scales. Even S. cerevisiae which is not normally noted for its capacity to secrete proteins, has been developed into a production system for commercially acceptable yields of heterologous proteins such as insulin and human serum albumin. Thus, it is feasible to modify an organism through a combination of mutagenesis and targeted gene manipulation in order to achieve a goal. S. cerevisiae has an advantage over all other fungi in being by far the best studied and furnished with the best-developed genetics underpinned with the complete genome sequence and increasing information on genome structure and function. We can, therefore, learn from the yeast systems and apply this knowledge to filamentous fungi where appropriate provided we recognise, as will be seen below that there are clear differences. The challenge is to explore and develop the hyphal fungi to become better producers of heterologous proteins. To achieve this aim we have improved tools (e.g. transformation, gene deletion), improved knowledge on basic mechanisms of gene expression and protein secretion and genome sequence data for several species. The emphasis throughout the chapter is on the molecular basis of protein production and its improvement by gene manipulation.