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Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization
- Leis, Benedikt, Held, Claudia, Andreeßen, Björn, Liebl, Wolfgang, Graubner, Sigrid, Schulte, Louis-Philipp, Schwarz, Wolfgang H., Zverlov, Vladimir V.
- Biotechnology for biofuels 2018 v.11 no.1 pp. 220
- Clostridium thermocellum, Escherichia coli, Pinus, bacteria, beta-glucosidase, beta-mannosidase, bioeconomics, biomass, cellulolytic microorganisms, cellulosome, endo-1,4-beta-glucanase, enzymatic hydrolysis, fungi, hydrolysis, kraft pulp, pH, polysaccharides, softwood, sugars, thermal stability, xylanases
- BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: Our 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.