Valkonen, M.; Girych, M.; Havukainen, S.; Sveholm, E.; Luoto, J. C.; Schmidt, G.; Adlung, N.; Aalto, A.; Ollila, O. H. S.; Mattila, H.; Paasela, T.

Microbes are powerful cell factories for making molecules that are difficult or impossible to produce by other means. Filamentous fungi such as Trichoderma reesei are superior hosts for recombinant protein production, yet secreted proteases often degrade target proteins, reducing yields and limiting process robustness. Preventing proteolysis without relying on expensive commercial inhibitors or compromising strain fitness has been a longstanding challenge in fungal biotechnology, particularly for scaling up production to industrially relevant levels. Here, we report the identification of a native inhibitory protein from T. reesei, TrI9, and show that directing its secretion into the culture medium markedly reduces extracellular protease activity and enables production of the highly protease-sensitive spider silk-like protein CBM-AQ12-CBM, which could be used in high-performance biomaterials. Computational and in vitro analyses provide mechanistic insight, showing that TrI9 functions as a multi-target inhibitor of subtilisin-like proteases (SLPs), including SLP2, a protease that cannot be eliminated by gene deletion due to its crucial role in normal growth and development. This TrI9-based strategy for protease mitigation presents a novel approach for strain improvement, protecting a wide range of protease-labile products beyond silk proteins during large-scale fermentation. Protecting sensitive proteins without added inhibitors offers a cost-effective alternative for scaling up protein production across multiple applications, from protein-based materials manufacturing to pharmaceuticals, as well as enzyme and food applications.