Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

Abstract

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R² = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

Keywords: Neurospora; cellobiose; cellulase; cellulose; glycosylphosphatidylinositols; protein folding; protein translocation; protein transport; proteomics; ribosome synthesis.

FIG 1

Venn diagram analysis comparing proteins affected by alternate carbon sources. (A) Comparison of proteins that increased in abundance upon exposure to cellobiose (CB) versus no carbon (NC), upon exposure to Avicel (AV) versus NC, and under carbon starvation (NC versus Suc). (B to E) Comparison of proteins that increased in abundance by exposure to cellobiose or Avicel with the targets of the CLR-1 direct regulon (B), the CLR-2 direct regulon (C), the XLR-1 direct regulon (D), or the Ire-1/HAC-1 regulon (E). Quantification of proteins was performed with a P value of <0.05.

FIG 2

Functional enrichment analysis of the gene/protein sets with correlated or uncorrelated abundance of proteins and mRNAs. (A) Correlation between changes in mRNA levels and protein abundances upon exposure to Avicel (AV) versus no carbon (NC). (B) Functional enrichment analysis of the gene/protein sets whose protein levels correlated with mRNA levels (correlated) and those regulated differentially only at the protein level but not at the mRNA level (uncorrelated).

FIG 3

Phenotypic analysis of the deletion mutants of the selected proteins that increased in abundance in the Δ3βG strain upon exposure to cellobiose (CB versus NC). (A) Growth rates of wild type (WT) and mutant strains. A suspension of 10³ conidia from the strains indicated was inoculated into race tubes containing VMM and incubated for 3 days. Mycelium length was measured every 24 h. (B and C) Total secreted protein levels (B) and endoglucanase activities (C) in the supernatant of 96-h cultures in VMM with 2% Avicel after a shift from a 16-h VMM culture. Shown are the mean values of at least three replicates. Error bars show the standard deviations between these replicates. The significance of differences between the mutants and the WT was based on t test analysis. Asterisks indicate significant differences (*, P < 0.001). Azo-CMC, azo-carboxymethyl cellulose.

FIG 4

Schematic model illustrating cellobiose-elicited action of ER stress and lignocellulase synthesis. Cellobiose, the major cellulolytic product by endo-glucanases (EG) and exo-glucanases (CBH), is transported by the cellodextrin transporters/transceptors CDT-1 and CDT-2. Cellobionic acid, derived from catalytic reactions of CDH and LPMO, is transported into the cell by CBT-1. Signals, derived from cellobiose, a modified version of cellobiose, and/or cellobiose interaction with CDT-1 and/or CDT-2 (40) or other intracellular proteins results in the activation of downstream events, including transcriptional activation and gene expression, modulation of intracellular metabolism, and translation and secretion of lignocellulases. In particular, cellobiose signaling induces the activation of the Ire-1/HAC-1-mediated UPR pathway (29), particularly proteins involved in translocation, folding, and protein glycosylation. Upright arrows indicate increases in protein abundance in response to cellobiose.