Quantitative proteomic approach for cellulose degradation by Neurospora crassa

Conversion of plant biomass to soluble sugars is the primary bottleneck associated with production of economically viable cellulosic fuels and chemicals. To better understand the biochemical route that filamentous fungi use to degrade plant biomass, we have taken a quantitative proteomics approach to characterizing the secretome of Neurospora crassa during growth on microcrystalline cellulose. Thirteen proteins were quantified in the N. crassa secretome using a combination of Absolute Quantification (AQUA) and Absolute SILAC to verify protein concentrations. Four of these enzymes including 2 cellobiohydrolases (CBH-1 and GH6-2), an endoglucanase (GH5-1), and a β-glucosidase (GH3-4) were then chosen to reconstitute a defined cellulase mixture in vitro. These enzymes were assayed alone and in mixtures and the activity of the reconstituted set was then compared to the crude mixture of N. crassa secretome proteins. Results show that while these 4 proteins represent 63-65% of the total secretome by weight, they account for just 43% of the total activity on microcrystalline cellulose after 24 h of hydrolysis. This result and quantitative proteomic data on other less abundant proteins secreted by Neurospora suggest that proteins other than canonical fungal cellulases may play an important role in cellulose degradation by fungi.