Parallel proteomic and phosphoproteomic analyses reveal cellobiose-dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

Abstract

High cost of lignocellulases restricts the commercialization of biofuel and bio-product production from lignocellulosic biomass. Constitutively expressed lignocellulases are considered to degrade cellulose to release small amount of soluble cellodextrins such as cellobiose for further large-scale production of lignocellulases; however, the underlying mechanism remains to be elucidated. Here, a triple β-glucosidase mutant of the model fungus Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to perform parallel analyses of proteome and phosphoproteome changes in response to cellobiose and Avicel cellulose. The results revealed shared proteome and phosphoproteome responses to cellobiose and Avicel, corroborating the idea that cellobiose mediates the regulation of lignocellulase expression and secretion. The results further suggest that this regulation is achieved at multiple levels, including epigenetic, transcription, post-transcription, translation, and post-translation. Proteome profiling revealed that the proteins upregulated by cellobiose and Avicel were over-represented in cellulose degradation and degradation product transport pathways. Phosphoproteome profiling revealed that the proteins differentially phosphorylated by cellobiose and Avicel were over-represented by the pathways such as transcriptional control, protein processing and export, cell wall biogenesis, and cellular signaling. Deletion mutation analysis further suggests that the ER chaperon protein Hsp70-6, the translocation complex subunit Sec66/Sec71, and the signal peptidase subunit Spc2 are involved in lignocellulase secretion, particularly translocation across the endoplasmic reticulum. Altogether, the results offer a new insight into how cellobiose mediates the regulation of lignocellulase expression and secretion, providing a potential strategy for the strain engineering to improve lignocellulase production.