Weighted sum matrix approach and proteomics insights identify a broad spectrum hyper-cellulolytic and thermostable...

Efficient and stable enzyme systems are crucial for lignocellulosic biomass conversion, yet they remain a significant limitation in second-generation biofuel production. Here, we applied a weighted sum matrix (WSM)-based multi-criteria screening strategy to identify superior cellulolytic fungus from a diverse collection of natural isolates. This approach revealed Talaromyces marneffei as a previously unreported hyper-cellulolytic organism. The T. marneffei secretome exhibited nearly two-fold higher biomass saccharification efficiency than a commercial enzyme preparation under industrially relevant conditions. Label-free quantitative nano-LC-MS/MS analysis identified 549 secreted proteins, including 201 carbohydrate-active enzymes (CAZymes) spanning multiple glycoside hydrolase and auxiliary activity families. Quantitative profiling revealed a high abundance of key cellulolytic enzymes, particularly GH7 cellobiohydrolase I (CBH1), along with diverse accessory enzymes enabling synergistic biomass deconstruction. The enzyme concoction displayed broad operational robustness, with optimal activity at acidic pH (4-4.5), elevated temperature optima (60-70 °C), and exceptional storage stability, retaining over 96% activity after 60 days at ambient temperature. Molecular modeling and dynamics simulations of CBH1 revealed an open, flexible active-site architecture with enhanced ligand interactions, providing mechanistic insight into its thermostability. Collectively, this study highlights T. marneffei as a promising source of industrially relevant lignocellulolytic enzymes and demonstrates the value of proteomics-driven secretome analysis for enzyme discovery.