Proteolytic and non-proteolytic mechanisms of keratin degradation in <i>Onygena corvina</i> revealed by a proteogenomic approach

Keratin-rich by-products from the poultry, textile, and leather industries pose a significant challenge for sustainable waste management due to their highly recalcitrant nature. While microbial degradation of keratin has been studied and may offer a viable solution, the complex enzyme machineries that are potentially needed to degrade these recalcitrant by-products remain partly unknown. In this study, we employed a high-resolution proteogenomic approach to characterize the keratinolytic machinery of <i>Onygena corvina</i>, a non-pathogenic saprophytic fungus. Using a membrane agar plate method with insoluble substrates, we obtained secretomes enriched in secreted and substrate-bound proteins during growth on α- and β-keratin-rich substrates, specifically wool and feather meal. Our findings reveal that <i>O. corvina</i> has a richer proteolytic machinery than previously reported, including enzymes that are used across keratin types, as well as enzymes that are specifically targeted to either α- or β-keratin. In addition to proteases, the secretomes contain numerous other proteins, including cell wall-modifying enzymes, oxidoreductases, esterases, phosphatases, and sialidases that are involved in the deconstruction of keratin. We propose that these additional enzymes destabilize keratin through a combination of mechanical keratinolysis, removal of post-translational modifications, reduction of disulfide bonds, and cleavage of isopeptide bonds, thereby enhancing proteolytic accessibility. Interestingly, keratin degradation by <i>O. corvina</i> was most efficient when using mixed substrates containing both feather and wool meal. These novel insights into the keratinolytic system of <i>O. corvina</i> underscore the importance of considering synergistic enzyme interactions when developing biotechnological approaches for the valorization of keratin-rich byproducts.IMPORTANCEKeratin-rich by-products from agro-industrial processes are generated in large volumes and present a significant environmental burden due to their recalcitrant nature. Microbial degradation offers a promising solution, but the mechanisms involved in keratin decomposition remain elusive. In this study, we show that the saprophytic fungus <i>O. corvina</i> secretes a diverse and specialized enzymatic arsenal when grown on keratin-rich substrates, such as feather (β-keratin) and wool (α-keratin) meal. Its secretome includes both shared and keratin-type specific proteases, along with accessory proteins, such as oxidoreductases, esterases, phosphatases, and sialidases, that aid in substrate destabilization. Our findings uncover the complex enzymatic system driving keratinolysis in this fungus and provide a foundation for developing sustainable, enzyme-based strategies to valorize keratin-rich waste.