Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization

• Published in: Biotechnology for biofuels Vol. 11; no. 1; p. 220
• Main Authors: Leis, Benedikt, Held, Claudia, Andreeßen, Björn, Liebl, Wolfgang, Graubner, Sigrid, Schulte, Louis-Philipp, Schwarz, Wolfgang H, Zverlov, Vladimir V
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 09.08.2018; BioMed Central; BMC
• Subjects: Adaptation; Advantages; Bacteria; Biodegradation; Biodiesel fuels; Biomass; Biomass energy research; Carbohydrates; Cellulase; Cellulose; Cellulosome; Chemical properties; Clostridium; Clostridium thermocellum; E coli; Endoglucanase; Enzymes; Fungi; Glucosidase; Hydrolysis; Kraft pulp; Microbial enzymes; Microbial genetic engineering; Optimization; pH effects; Physiological aspects; Plant biomass; Polysaccharides; Protein expression; Proteins; Pulp; Saccharides; Screening; Softwood; Softwoods; Substrates; Sugar; Synthetic cellulase complex; System effectiveness; Wood; Xylanase; Screening; Cellulosome; Softwood; Synthetic cellulase complex; Clostridium thermocellum; Cellulose
• ISSN: 1754-6834; 1754-6834
• DOI: 10.1186/s13068-018-1220-y

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 is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass. In this study, 60 cellulosomal components were recombinantly produced in 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. Our results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.