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.     

Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.     

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.     

The cellulosome system of Acetivibrio cellulolyticus includes a novel type of adaptor protein and a cell surface anchoring protein

A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. ScaB cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.     

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.     

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.     

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.     

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Background

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.

Results

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.

Conclusions

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.

     

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.     

Combined Crystal Structure of a Type I Cohesin: MUTATION AND AFFINITY BINDING STUDIES REVEAL STRUCTURAL DETERMINANTS OF COHESIN-DOCKERIN SPECIFICITIES*

Cohesin-dockerin interactions orchestrate the assembly of one of nature''s most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-Å resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at β-strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.     

Architecture of the Bacteroides cellulosolvens cellulosome: description of a cell surface-anchoring scaffoldin and a family 48 cellulase

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.     

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.     

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.     

Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with approximately 20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.     

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.     

Effect of multiple copies of cohesins on cellulase and hemicellulase activities of Clostridium cellulovorans mini-cellulosomes

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpA1), two cohesins (mini-CbpA12), or four cohesins (mini-CbpA1234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and corn fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with corn fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpA1234 (1.8-fold) and then mini-CbpA12 (1.3-fold), and the lowest synergy was observed with mini-CbpA1 (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.     

Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.     

A novel Acetivibrio cellulolyticus anchoring scaffoldin that bears divergent cohesins

Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed. The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here. The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two approximately 30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface.     

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.