Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Conversion of Thermobifida fusca free exoglucanases into cellulosomal components: comparative impact on cellulose-degrading activity

Cellulosomes are multi-enzyme complexes produced by certain anaerobic bacteria that exhibit efficient degradation of plant cell wall polysaccharides. To understand their enhanced levels of hydrolysis, we are investigating the effects of converting a free-cellulase system into a cellulosomal one. To achieve this end, we are replacing the cellulose-binding module of the native cellulases, produced by the aerobic bacterium Thermobifida fusca, with a cellulosome-derived dockerin module of established specificity, to allow their incorporation into defined "designer cellulosomes". In this communication, we have attached divergent dockerins to the two exoglucanases produced by T. fusca exoglucanase, Cel6B and Cel48A. The resultant fusion proteins were shown to bind efficiently and specifically to their matching cohesins, and their activities on several different cellulose substrates were compared. The lack of a cellulose-binding module in Cel6B had a deleterious effect on its activity on crystalline substrates. In contrast, the dockerin-bearing family-48 exoglucanase showed increased levels of hydrolytic activity on carboxymethyl cellulose and on both crystalline substrates tested, compared to the wild-type enzyme. The marked difference in the response of the two exoglucanases to incorporation into a cellulosome, suggests that the family-48 cellulase is more appropriate than the family-6 enzyme as a designer cellulosome component.     

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure-function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in 'designer cellulosomes'. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.     

Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.In nature, some anaerobic cellulolytic bacteria produce cellulosomes, which are organized by the action of scaffoldin subunits that usually contain a single carbohydrate-binding module (CBM) and multiple cohesin modules (2, 7, 13, 14, 28, 36). This arrangement allows the integration of several dockerin-containing enzymes into a complex, which is then targeted to the cellulosic substrate by the common CBM. The cellulosomal enzymes then exhibit enhanced synergistic activity, presumably due to their spatial proximity and coordinated interaction. In contrast, the enzyme systems of aerobic bacteria and fungi comprise free (uncomplexed) enzymes, which differ from cellulosomal systems in that many of them contain their own CBM that delivers the individual catalytic module to the surface of the substrate (39, 41, 42).In previous work, we used the designer cellulosome concept (5) to construct unique minicellulosomes of defined content (16, 32, 33). In order to construct designer cellulosomes, chimeric scaffoldins have been prepared which contained two or more cohesins that matched the dockerins of the enzymes (native cellulosomal or dockerin-fused chimeras). Enzymes that contain dockerins that match the specificity of a scaffoldin-borne cohesin can then be selectively integrated into the designer cellulosome at a specified site. Cellulosomal enzymes containing either a native dockerin or a divergent dockerin can be inserted on different sites of a chimeric scaffoldin. Alternatively, a free, noncellulosomal enzyme can be included in designer cellulosomes by replacing its native CBM with a dockerin of choice. In some cases, designer cellulosomes displayed enhanced synergistic activity over the parallel free-enzyme system (15, 17). This increased activity was shown to be a function of both a substrate-targeting effect (contributed by the CBM on the chimeric scaffoldin) and the enzyme proximity effect, thus supporting the initial hypothesis.In recent studies, we have investigated the free-cellulase system of Thermobifida fusca for use in designer cellulosome systems. This aerobic thermophilic cellulolytic bacterium contains a limited set of six free cellulases, each composed of a catalytic module and a crystalline-cellulose binding family 2 CBM (CBM2) module on either the N or C terminus of the protein. T. fusca contains three endoglucanases (Cel5A, Cel6A, and Cel9B), two exocellulases (Cel6B and Cel48A), and one processive endoglucanase (Cel9A). Previously, we converted both family 6 cellulases and the family 48 exoglucanase from the free to the cellulosomal mode of action by replacing their native CBM2s with a dockerin module (11, 12). All three chimeric enzymes exhibited cellulose-degrading activity on both soluble and crystalline substrates. The results indicated that the family 48 exoglucanase appeared to be well adapted to the cellulosomal mode of action, whereas the family 6 exoglucanase is less appropriate for inclusion into cellulosomes. Indeed, family 48 cellulases have been found to be a major component in every native cellulosome thus far described, in contrast to the family 6 cellulases, which have been identified only in free-cellulase systems.An important feature of the free-acting fungal and bacterial cellulases is that they contain a linker segment, often rich in prolines and threonines, that connects the catalytic module to the CBM (37). The role of such flexible linkers is thought to ensure independent action of the adjacent functional modules, thus allowing progressive and efficient hydrolysis of cellulose by the catalytic modules (6, 9, 10, 20, 25-27, 34, 36, 38, 40). The present communication focuses on the effect of linker length and dockerin position (relative to the catalytic module) on enzymatic activity within a designer cellulosome. For this purpose we have employed the highly active family 5 endoglucanase Cel5A from T. fusca (21, 22, 29), which was converted to the cellulosomal mode by replacement of its CBM with a dockerin module. Chimeric dockerin derivatives were prepared on either the N or C terminus of the Cel5A catalytic module, separated by linker segments of different lengths. In most cases, binary designer cellulosomes, comprising the respective Cel5A chimera together with a Cel48A chimera, were shown to be more efficient on crystalline cellulosic substrates than the combination of the wild-type free enzymes.     

Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin

In recent work, we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.     

Exploration of new geometries in cellulosome-like chimeras

In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a "covalent cellulosome" that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.     

Dockerin‐containing protease inhibitor protects key cellulosomal cellulases from proteolysis in Clostridium cellulolyticum

Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin‐containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome‐localized protease inhibitors in Clostridia.     

Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Exploration of New Geometries in Cellulosome-Like Chimeras

In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a “covalent cellulosome” that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.     

In vitro reconstitution of the complete Clostridium thermocellum cellulosome and synergistic activity on crystalline cellulose

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.     

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.     

Comparison of family 9 cellulases from mesophilic and thermophilic bacteria

Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.     

Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes

The isolation of cellulosomes from clostridial sources has been extensively studied; however, the isolation of cellulosomes from facultative soil anaerobes of the family Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylanosome) essentially consists of two complex components: C-I and C-II. This multi-component complex enables Bacillus to degrade a variety of carbonaceous compounds as it is composed of several enzymes, such as cellulases, xylanases and other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium were purified using cellulose affinity chromatography, followed by Sepharose 4B gel filtration chromatography. The objective of this investigation was to establish the effect of sulfate and sulfide on cellulosomal 'cellulase' activity. An increase in sulfide concentration led to a general enhancement of cellulosomal-associated cellulolytic activity, whereas an increase in sulfate concentration resulted in an inhibition of the cellulosome-associated cellulolytic activity.     

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.     

Cellulosomes-structure and ultrastructure

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.