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.

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

“…A shared phosphorylation response to cellobiose and Avicel was identified as reflected by that 728 phosphoproteins exhibited differential phosphorylation in response to both cellobiose and Avicel (Figure 1b). A shared proteome response to cellobiose and Avicel was also shown by our quantitative proteome analysis (Liu et al, 2020). Gene Ontology (GO) enrichment analysis revealed that the differential phosphoproteome in response to cellobiose and Avicel was over‐represented by the pathways related to cellular signaling, transcriptional control, and protein translocation and excretion (Figure 2; Table S3), while the proteome with increased abundance was over‐represented by cellulose catabolism, carbohydrate metabolism, and sugar transport (Liu et al, 2020).…”

“…Correlation analysis suggests that there is only a moderate correlation between changes in protein and mRNA levels in N . crassa in response to cellulose (Figure 3; Liu et al, 2020). Comparison of proteome and transcriptome changes revealed that the proteins with increase in both protein abundance and mRNA levels of their encoding genes were primarily associated with cellulose degradation and degradation product transport (Figure 2; Table S5; Liu et al, 2020), corroborating the finding that cellulose degradation and utilization are controlled primarily at transcriptional level (Huberman et al, 2016).…”

“…crassa in response to cellulose (Figure 3; Liu et al, 2020). Comparison of proteome and transcriptome changes revealed that the proteins with increase in both protein abundance and mRNA levels of their encoding genes were primarily associated with cellulose degradation and degradation product transport (Figure 2; Table S5; Liu et al, 2020), corroborating the finding that cellulose degradation and utilization are controlled primarily at transcriptional level (Huberman et al, 2016). It was also noted that some proteins showed increased abundance in early response to cellobiose and Avicel but no change of the mRNA levels of their encoding genes (Figure 3; Table S5; Liu et al, 2020).…”

“…crassa was inoculated onto slants of Vogel's minimal medium (VMM; Vogel, 1956) with 1.5% agar and 2% sucrose and grown for 3 days at 30℃ in the dark followed by 7 days at 25℃ in light. Conidia were inoculated into 100 ml of liquid VMM with 2% (w/v) sucrose at 10 6 conidia/ml and grown at 25℃ in constant light and shaking for 16 h. Mycelia were harvested by filtrating and washed with VMM without a carbon source, followed by growth in fresh VMM with either 0.5% sucrose, 0.2% cellobiose, 0.5% Avicel ® PH‐101 (Sigma Aldrich), or with no carbon source added, as described previously (Liu et al, 2020).…”

“…The liquid chromatography‐mass spectrometry (LC‐MS) and data analysis were performed as described in our previous study (Liu et al, 2020). An appropriate volume of 5% acetonitrile (v/v) solution with 0.1% (v/v) trifluoroacetic acid was added to the dried peptide samples.…”