S4: Free and complexed enzymes exhibit synergistic mechanisms in cellulose deconstruction: Implications for enzyme cocktail design for biofuels production

Nature has evolved multiple enzymatic strategies for the degradation of plant cell wall polysaccharides, which are central to carbon flux in the biosphere and an integral part of renewable biofuels production. Many biomass-degrading organisms secrete synergistic cocktails of individual enzymes with one or a few catalytic domains per enzyme, whereas some bacteria synthesize large multi-enzyme complexes, termed cellulosomes, which contain multiple catalytic units per complex. Both enzyme systems employ similar catalytic chemistries, but the physical mechanisms by which these enzyme systems degrade polysaccharides are still unclear. Here we examine a prominent example of each type, namely a free-enzyme cocktail expressed by the fungus Hypocrea jecorina and a cellulosome secreted from the bacterium Clostridium thermocellum. We observe striking differences in activity wherein at the same loading, free enzymes are more active on pretreated biomass whereas cellulosomes digest purified cellulose much faster. When combined, these systems display synergistic enzyme activity.  To gain further insights, we imaged free enzyme- and cellulosome-digested cellulose and biomass by transmission electron microscopy, which revealed evidence for different mechanisms of cellulose deconstruction by free enzymes and cellulosomes. Specifically, the free enzymes employ an ablative, fibril-sharpening mechanism, whereas cellulosomes physically separate individual cellulose microfibrils from larger particles for enhanced access to cellulose surfaces. Interestingly, when the two enzyme systems are combined we observed changes to the substrate that suggests mechanisms of synergistic deconstruction. Insight into the different mechanisms underlying these two polysaccharide deconstruction paradigms will enable new strategies for enzyme engineering to overcome biomass recalcitrance.