Thermobifida exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Systems and Synthetic Biology 09/2010; 4(3):193-201. DOI: 10.1007/s11693-010-9056-1
Source: PubMed


Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein-protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes' CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.

Download full-text


Available from: David Wilson,
23 Reads
  • Source
    • "The integration into a cellulosome system of cellulases and associated polysaccharide-degrading enzymes produced by anaerobic bacteria can facilitate their synergistic activity. However, it might also cause an opposite effect (anti-proximity) due to conformational restraint and steric clashes between the cellulases [59]. Our understanding of cellulosome architecture and its implications for cellulose hydrolysis is still limited due to the incredible heterogeneity of the cellulosome complex [25]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27-35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.
    Biotechnology for Biofuels 12/2013; 6(1):182. DOI:10.1186/1754-6834-6-182 · 6.04 Impact Factor
  • Source
    • "Interpretation of these results in terms of general saccharification of “crystalline cellulose”, however, suffers from limitations imposed by the relatively low extents of conversion achieved. In terms of action against Avicel, for example, the observed percent-conversion by the most active of the minicellulosomes is under 6% [23]. Given that Avicel may contain as much as 40% amorphous cellulose [24], it is not certain that these constructs, as assayed, have been shown to be truly capable of degrading crystalline cellulose. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Complete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases. Cellulases that are found in Nature vary considerably in their modular diversity and architecture. They include: non-complexed enzymes with single catalytic domains, independent single peptide chains incorporating multiple catalytic modules, and complexed, scaffolded structures, such as the cellulosome. The discovery of the latter two enzyme architectures has led to a generally held hypothesis that these systems take advantage of intramolecular and intermolecular proximity synergies, respectively, to enhance cellulose degradation. We use domain engineering to exploit both of these concepts to improve cellulase activity relative to the activity of mixtures of the separate catalytic domains. We show that engineered minicellulosomes can achieve high levels of cellulose conversion on crystalline cellulose by taking advantage of three types of synergism; (1) a complementary synergy produced by interaction of endo- and exo-cellulases, (2) an intramolecular synergy of multiple catalytic modules in a single gene product (this type of synergism being introduced for the first time to minicellulosomes targeting crystalline cellulose), and (3) an intermolecular proximity synergy from the assembly of these cellulases into larger multi-molecular structures called minicellulosomes. The binary minicellulosome constructed in this study consists of an artificial multicatalytic cellulase (CBM4-Ig-GH9-X11-X12-GH8-Doc) and one cellulase with a single catalytic domain (a modified CelS with the structure CBM4-Ig-GH48-Doc), connected by a non-catalytic scaffoldin protein. The high level endo-exo synergy and intramolecular synergies within the artificial multifunctional cellulase have been combined with an additional proximity-dependent synergy produced by incorporation into a minicellulosome demonstrating high conversion of crystalline cellulose (Avicel). Our minicellulosome is the first engineered enzyme system confirmed by test to be capable of both operating at temperatures as high as 60[degree sign]C and converting over 60 % of crystalline cellulose to fermentable sugars. When compared to previously reported minicellulosomes assembled from cellulases containing only one catalytic module each, our novel minicellulosome demonstrates a method for substantial reduction in the number of peptide chains required, permitting improved heterologous expression of minicellulosomes in microbial hosts. In addition, it has been shown to be capable of substantial conversion of actual crystalline cellulose, as well as of the less-well-ordered and more easily digestible fraction of nominally crystalline cellulose.
    Biotechnology for Biofuels 08/2013; 6(1):126. DOI:10.1186/1754-6834-6-126 · 6.04 Impact Factor
  • Source
    • "The CBM2s of endocellulase Cel6A and exocellulase Cel6B were replaced with dockerins from C. cellulolyticum and C. thermocellum, producing chimeras 6A-c and t-6B. In general, activity was reduced on most substrates; however, surprisingly, t-6B showed about 14-fold higher activity on amorphous cellulose than the native enzyme (Caspi et al., 2006, 2008, 2011). Designer cellulosome harboring cellulases and xylanases was designed using T. fusca endoxylanases Xyn10B and Xyn11A and T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract The process of bioethanol production from biomass comprises pretreatments and enzyme-mediated hydrolysis to convert lignocellulose into fermentable sugars. Because of the recalcitrant character of cellulose, the enzymatic hydrolysis is considered the major challenge in this process to be economically competitive. These technical difficulties highlight the need for the discovery of new enzymes to optimize and lower the cost of current technologies. Microorganisms have developed efficient systems for cellulose degradation. Among cellulolytic microbes, Thermobifida fusca possesses great physiological and cellulolytic characteristics (thermostability, high activity and tolerance to a broad pH range) making it an interesting organism to be studied from an applied perspective. In this review we describe the main enzymes/proteins produced by T.fusca (cellulases, xylanases, mannanase, manosidase, CBM33 and CelR), the effect of substrate on T. fusca proteome, enzyme improvement approaches, synergism between enzymes/proteins and artificial cellulosomes.
    Critical Reviews in Microbiology 03/2013; 40(3). DOI:10.3109/1040841X.2013.776512 · 6.02 Impact Factor
Show more