Protease suppression by native I9 inhibitor improves recombinant protein production in Trichoderma reesei

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. SignificanceThe discovery and characterization of an independently encoded I9 inhibitor (TrI9) in the filamentous fungus Trichoderma reesei reveals a new layer of biological regulation of subtilisin-like protease activity in fungi. We show that overexpressing and secreting TrI9 into the culture supernatant can suppress extracellular proteolysis, including activity from the essential SLP2 protease thereby overcoming a major barrier to production of protease-sensitive recombinant proteins without compromising strain fitness. For industrial biomanufacturing, this translates into higher effective titers, more consistent product integrity and reduced reliance on costly commercial protease inhibitors, hence, improving process robustness and economics. As a broadly applicable strain-and-process strategy, TrI9-enabled protease control strengthens T. reesei as a scalable platform for precision fermentation of diverse, otherwise hard-to-produce proteins.