Enzymatic Degradation of Cellulose by the Filamentous Fungus Neurospora crassa
Lignocellulosic biomass is an abundant renewable resource that can be used as a feedstock for production of second-generation biofuels. Currently, the bottleneck to generation of such fuels lies in the expensive and technically challenging process for converting biomass to fermentable sugars. Filamentous fungi are efficient at depolymerizing plant biomass. These fungi are also used for the production of industrial enzymes due to their ability to secrete large quantities of enzymes. The filamentous fungus Neurospora crassa is a genetically tractable model organism and a proficient degrader of plant biomass. In this dissertation, the enzymatic depolymerization of cellulose by Neurospora crassa was investigated using a combination of quantitative proteomics, genetics, and biochemistry.
Chapter 1 is an introduction to lignocellulosic biomass and the organisms and enzymes that degrade it. In Chapter 2, a quantitative proteomic approach was taken to characterize the secretome of Neurospora crassa during growth on microcrystalline cellulose. Depolymerization of cellulose occurs by endoglucanases that hydrolyze internal glycosidic bonds and cellobiohydrolases that hydrolyze cellobiose from the reducing or non-reducing chain ends of cellulose. In addition to these canonical cellulases, a number of other proteins were quantified including a beta-glucosidase, a cellobiose dehydrogenase (CDH), and glycosyl hydrolase family 61 (GH61) enzymes. The beta-glucosidase and 3 cellulases were purified and biochemically characterized. While these 4 enzymes represent more than 85% of the cellulase in the secretome, they were significantly impaired in their rate of cellulose degradation relative to the complete set of enzymes in the secretome. This result suggested that proteins other than canonical cellulases may be important in cellulose depolymerization by fungi.
The deletion of a gene encoding cellobiose dehydrogenase in N. crassa is described in Chapter 3. Cellobiose dehydrogenase (CDH) catalyzes the oxidation of cellobiose to cellobionolactone, but the biological function of this protein was previously unknown. Deletion of cdh-1 reduced cellulase activity 37-49% and addition of purified CDHs to the cdh-1 deletion strain resulted in a 1.6 to 2.0-fold stimulation in cellulase activity. The stimulatory effect of CDH required the presence of molecular oxygen and other secreted metalloproteins. The discovery that cellobiose dehydrogenase plays an integral role in degradation of cellulose marked a significant shift from previous models centered on the degradation of cellulose by mixtures of glycosyl hydrolases.
To determine the molecular mechanisms by which CDH can enhance cellulase activity, a fractionation strategy was employed as described in Chapter 4 to identify synergistic metalloproteins in the N. crassa secretome. CDH was shown to enhance cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs), previously called GH61 enzymes. These enzymes catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage and facilitate elimination of the adjacent carbohydrate moiety. A further discussion of the mechanism of copper-dependent PMOs is discussed in Chapter 5. Here, the action of different PMOs was shown to be regiospecific resulting in oxidized products modified at C1 on the reducing end or C4 on the non-reducing end. CDHs and proteins related to the PMOs are found in cellulolytic species throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose depolymerization and could significantly reduce the cost of biofuel production.
In chapter 6, the development of N. crassa as a host for recombinant expression of secreted enzymes is described. Expression of the endocellulase GH5-1 is used to complement the phenotype of a gh5-1 deletion strain. Experiments with GH5-1 fused to GFP (green fluorescent protein) were used to visualize the binding of this endocellulase to plant biomass. Additional applications for N. crassa as an expression host for fundamental studies of biomass depolymerizing enzymes are described.