Abstract
BackgroundThe development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium Clostridium thermocellum is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass.ResultsIn this study, 60 cellulosomal components were recombinantly produced in E. coli and systematically permuted in synthetic complexes to study the function–activity relationship of all available enzymes on Kraft pulp from pine wood as the substrate. Starting from a basic exo/endoglucanase complex, we were able to identify additional functional classes such as mannanase and xylanase for optimal activity on the substrate. Based on these results, we predicted a synthetic cellulosome complex consisting of seven single components (including the scaffoldin protein and a β-glucosidase) and characterized it biochemically. We obtained a highly thermostable complex with optimal activity around 60–65 °C and an optimal pH in agreement with the optimum of the native cellulosome (pH 5.8). Remarkably, a fully synthetic complex containing 47 single cellulosomal components showed comparable activity with a commercially available fungal enzyme cocktail on the softwood pulp substrate.ConclusionsOur results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of C. thermocellum can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.
Highlights
The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy
We show the rapid adaptation of a fully synthetic cellulosome complex on an industrial substrate based on delignified softwood from Kraft pulp process and present a screening strategy to identify enzymatic functions necessary within the cellulosome complex to enhance substrate degradation
Clostridium thermocellum was able to grow on Kraft softwood fibers over several days under anaerobic conditions
Summary
The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. When the components are separately produced recombinantly, one of the major advantages over fungal enzyme cocktails is the possibility to quickly adapt the composition of synthetic cellulase complexes by selectively adding new enzymatic functions or to change the stoichiometry of components added. Another advantage of the bacterial components from thermophiles is their higher temperature optimum compared to the fungal enzymes, a key feature to increase solubility of substrate and by-products, to increase diffusion rates, and to decrease viscosity. Despite many decades of research in this field, the commercial use of these native enzyme complexes is mainly hampered by the low production yield from anaerobic bacteria [9]
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