Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity

Abstract

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.ResultsAlthough 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0928-4) contains supplementary material, which is available to authorized users.

Cellulose hydrolysis byClostridium thermocellumis agnostic to substrate structural properties in contrast to fungal cellulases

“…As pointed out earlier, 24 out of the over 70 enzymes identified on C. thermocellum's cellulosome are cellulases, along with xylanases, pectinases, and others comprising the rest. 21,24 Model substrates were used in this work to differentiate the impact of cellulose micro-accessibility from that of cellulose macro-accessibility on biological digestion. A similar analysis of deconstruction of more complex lignocellulosic biomass will be valuable to fully understand how cellulose micro-accessibility impacts digestion by fungal enzymes compared to C. thermocellum.…”

“…16 Consolidated bioprocessing (CBP), in contrast, is a simple and effective bioprocess that combines enzyme production, enzymatic hydrolysis, and fermentation by organisms such as C. thermocellum in one step. 4,11,[17][18][19][20] 24 cellulases have been identified out of the more than 70 known enzyme components on the protein scaffoldin CipA of C. thermocellum's cellulosome, which can form cell-enzyme-substrate complexes 19,21,22 as opposed to cell free individual enzyme cocktails produced by T. reesei that typically have only one catalytic unit per protein. 23 This complex, multi-enzyme, multi-functional cellulosome produced by C. thermocellum enhances biomass solubilization compared to fungal enzymes.…”

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Other thermophilic genera also serve as promising consolidated bioprocessing platforms, like H. thermocellum which is notable for its cellulosomes (reviewed in [99]) and cellulolytic efficiency [100], but is subject to feedback inhibition by cellobiose [101]. Prior studies have relieved this feedback inhibition in vitro by exogenous mixing of purified cellulosomes and chimeric native β-glucosidases with cohesion domains [101], or with other thermophilic β-glucosidases [102].…”

Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production

“…Diverse cellulases including an exo-mode cellobiohydrolases (CBH) from Yarrowia lipolytica [32], an endoglucanases (EG) from Clostridium thermocellum DSM1237 [33], a glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 [34], and a CBM from Thermobifida fusca [35] were fused with N-terminal CL7 tags and recombinantly expressed in Escherichia coli. After that, the engineered P. pastoris yeasts were in vitro incubated with the E. coli lysates containing cellulases and CBM, leading to the assembly of minicellulosomes on cell surface.…”