Removing boundaries for therapeutic protein production in the filamentous fungus Trichoderma reesei: Reduction of the host protease load

The filamentous fungus Trichoderma reesei is an important protein production hosts in the enzyme industry. It produces native cellulase and hemicellulase enzymes with extremely high efficiency; the highest enzyme yields exceed 100 g/l. The success in production of mammalian proteins has thus far been much more limited. Antibody Fab fragments and bovine chymosin have been produced at over 100 mg/l levels in earlier work. We report results from a new effort to develop a T. reesei platform for therapeutic protein production. This effort has been much more successful, leading to production levels of several g/l for several therapeutic molecules. The production development of two proteins, full-length IgG antibodies and interferon α2b will be reported. After constructing the first expression strains it became clear that the host proteases formed a major boundary for their production. The secreted T. reesei proteases were characterised with respect to their biochemical properties, their activity against the target proteins and their abundance at mRNA and protein levels. The data obtained was used to make a series of T. reesei strains with multiple protease deletions. The general protease activity in the best deletion strains was ca. 30-fold reduced as compared to the original strain. In the best constructed strains both IgG antibodies and interferon ?2b could be produced at above 7 g/l. Interestingly, the protease deletion strains had a faster growth rate than the original strains. This study shows the excellent potential of T. reesei as a host for therapeutic protein production.