Functional characterization of fungal laccases via siRNA silencing enables targeted biodegradation.

Edible fungi serve as efficient bioconverters, transforming lignocellulosic wastes into valuable fungal proteins by extracellular enzyme systems (e.g., laccases). However, the functional characterization of these lignocellulolytic enzymes is still a major challenge, limiting the strategic application of fungal systems in targeted biodegradation. Here, we developed a versatile, non-transformative siRNA delivery platform for rapid gene function analysis in the edible fungus Pleurotus ostreatus. Using spray-induced (SIGS) in mycelia, injection-induced (IIGS) in fruiting bodies, and co-culture-induced (CIGS) silencing during fermentation, we successfully achieved sustained suppression of two extracellular laccases PoLac2 and PoLac10. Transient gene silencing (12-24 h) was achieved with single treatments, whereas CIGS applications prolonged the suppression of laccase activity, sustaining it for 96 h or more. Our results revealed distinct substrate-specific roles: PoLac2 was critical for lignin degradation and bisphenol A (BPA) removal (85.8% reduction in degradation upon silencing), whereas PoLac10 primarily mediated cellulose and wheat straw decomposition. This method proves to be an efficient, high-throughput tool for functional genomics in fungi. It offers a practical route to identifying enzymes crucial for managing agricultural wastes and degrading phenolic pollutants, supporting more sustainable environmental practices.