Driving biomass breakdown through engineered cellulosomes

Abstract

Extraction of sugar is the rate-limiting step in converting unpretreated biomass into value-added products through microbial fermentation. Both anaerobic fungi and anaerobic bacteria have evolved to produce large multi-cellulase complexes referred to as cellulosomes, which are powerful machines for biomass deconstruction. Characterization of bacterial cellulosomes has inspired synthetic "designer" cellulosomes, consisting of parts discovered from the native system that have proven useful for cellulose depolymerization. By contrast, the multi-cellulase complexes produced by anaerobic fungi are much more poorly understood, and to date their composition, architecture, and enzyme tethering mechanism remain unknown and heavily debated. Here, we compare current knowledge pertaining to the cellulosomes produced by both bacteria and fungi, including their application to synthetic enzyme-tethered systems for tunneled biocatalysis. We highlight gaps in knowledge and opportunities for discovery, especially pertaining to the potential of fungal cellulosome-inspired systems.

Keywords: CBM, Carbohydrate Binding Module; ELISA, Enzyme-Linked Immunosorbent Assay; GH, Glycoside Hydrolase; GST, Glutathione S-Transferase; SLH, Surface Layer Homology; anaerobic fungi; biofuels; cellulase; cellulosome; lignocellulose.

Figure 1.

Enzymes required for hydrolysis scale with the complexity of the biomass substrate. A wide variety of enzymes are required to depolymerize the components of crude, unpretreated biomass. For complete conversion of cellulose into glucose, a cocktail of β-glucosidases, endoglucanases, and exoglucanases are required. Hydrolysis of hemicellulose requires enzymes with additional functionality, including xylanases and mannanases. To access these sugar polymers from crude biomass, it is often necessary to solubilize lignin, which is crosslinked within cellulosic and hemicellulosic fibers. For this process, accessory enzymes such as polysaccharide deacetylases, peroxidases, and esterases are required. Bacterial cellulosomes typically contain enzymes required only for cellulose degradation while fungal enzyme complexes contain a richer diversity of enzymes to enable degradation of crude plant material.

Figure 2.

Synergistic Action of Cellulases within a Cellulosome. Cellulases assemble in close proximity on a noncatalytic protein called a scaffoldin. The endoglucanse reduces the degree of crystallinity of the cellulose substrate and liberates 2 cellulose chain ends. The exoglucanase processes along a free chain, freeing cellobiose with each cleavage. This cellobiose is then transferred to a nearby β-Glucosidase, which hydrolyzes it into 2 glucose monomers.