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Background: Increasing the efficiency of enzymatic biomass degradation is crucial for a more economically feasible conversion of abundantly available plant feedstock. Synergistic effects between the enzymes deployed in the hydrolysis of various hemicelluloses have been demonstrated, which can reduce process costs by lowering the amount of enzyme required for the reaction. Xyloglucan is the only major hemicellulose for which no such effects have been described yet. Results: We report the beneficial combination of two enzymes for the degradation of the hemicellulose xyloglucan. The addition of β-galactosidase Bga2B from Clostridium stercorarium to an in vitro hydrolysis reaction of a model xyloglucan substrate increased the enzymatic efficiency of endoglucanase Cel9D from Clostridium thermocellum to up to 22-fold. Furthermore, the total amount of enzyme required for high hydrolysis yields was lowered by nearly 80%. Increased yields were also observed when using a natural complex substrate—tamarind kernel powder. Conclusion: The findings of this study may improve the valorization of feedstocks containing high-xyloglucan amounts. The combination of the endoglucanase Cel9D and the β-galactosidase Bga2B can be used to efficiently produce the heptasaccharide XXXG. The exploitation of one specific oligosaccharide may open up possibilities for the use as a prebiotic or platform chemical in additional reactions. [ABSTRACT FROM AUTHOR]

Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a C. thermocellum cipA mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The in vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases. [ABSTRACT FROM AUTHOR]

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa. [ABSTRACT FROM AUTHOR]

Background: Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. Results: Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. Conclusions: The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] First draft genome sequence of a Clostridium thermocellum strain isolated from a compost treatment site. [•] Identification of genes for carbohydrate-active enzymes and cellulosome components. [•] Possible biotechnological application of C. thermocellum BC1 in thermophilic degradation of cellulosic biomass. [Copyright &y& Elsevier]