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.

     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

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.     

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.     

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.     

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.     

Small Angle X-ray Scattering Analysis of Clostridium thermocellum Cellulosome N-terminal Complexes Reveals a Highly Dynamic Structure

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.     

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.     

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the three-dimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29, 31, 32, 50, 52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, β-xylosidase, α-l-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5, 35, 55). Scaffoldin subunits (nonenzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate (42, 46).In order to evaluate the reasons for the apparent advantage of cellulosomes over free enzymes, it is interesting to compare the properties of the best-characterized free-enzyme systems for degradation of polysaccharides with those of the best-studied cellulosome system. We have initiated a program to convert the free-enzyme system of T. fusca into an artificial designer cellulosome (11-13). The designer cellulosome concept is based on the very high affinity (20, 44) and specific interaction (37, 43, 55) between a cohesin and a dockerin module from the same species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different species, for which matching dockerin-containing enzyme hybrids are prepared, as a platform for promoting synergistic action among enzyme components (5). Free cellulases from the T. fusca system were converted to the cellulosomal mode by replacing their native CBM with a desired dockerin module, and in some cases, the resultant “designer cellulosomes” exhibited enhanced synergistic activity on crystalline cellulosic substrates compared to that of the mixture of wild-type enzymes (11).In this study, we incorporated xylanolytic enzymes into designer cellulosomes and investigated their hydrolytic effects on purified xylans and on a native, complex cellulosic substrate (hatched wheat straw). We focused on T. fusca xylanases 11A and 10B (Xyn11A and Xyn10B), which are the most abundant xylanases produced during growth on xylan (34). Xyn11A and Xyn10B function as endoxylanases (28, 34); Xyn11A contains a C-terminal family 2 CBM that binds both cellulose and xylan, whereas Xyn10B lacks a CBM. In some experiments, one of the previously converted (dockerin-containing) T. fusca endoglucanases, f-5A (11), was also introduced into the designer cellulosomes in order to evaluate cooperation between xylanases and cellulases in hydrolysis of a natural substrate. This study contributes primary information concerning a major feature of cellulosomes that had not been suitably addressed in earlier research: although xylanases are integral components of cellulosomes, their synergistic action in the cellulosome mode has yet to be examined experimentally. The xylan-binding CBM (termed XBM for the purposes of this report) was found to contribute to the activity of the parent Xyn11A enzyme.     

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.     

Resonance assignments of cohesin and dockerin domains from Clostridium acetobutylicum ATCC824

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.     

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.     

Molecular architecture and structural transitions of a Clostridium thermocellum mini-cellulosome

The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (> 10⁹ M^− 1) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis.     

Modeling the self-assembly of the cellulosome enzyme complex

Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.     

Deglycosylation of cellulosomal enzyme enhances cellulosome assembly in Saccharomyces cerevisiae

We have estimated the effects of hyper-mannosylation of dockerin-type cellulase on cellulosome assembly by using Saccharomyces cerevisiae and 44 protein glycosylation mutants, because the heterologous protein displayed on yeast is assumed to be modified by yeast-specific hyper-mannosylation. First, we constructed the yeast strain CtminiCipA, which displays a heterologous scaffolding protein (miniCipA from Clostridium thermocellum) on its cell surface, and glycosylation mutants secreting a dockerin-type cellulase (Cel8Aenz-Cel48Sdoc: a fusion protein of the catalytic domain of C. thermocellum Cel8A and the dockerin domain of C. thermocellum Cel48S). Next, minicellulosomes were assembled by mixing the CtminiCipA strain and the dockerin-type cellulase secreted by each glycosylation mutant. By using an endoglucanase assay and flow cytometric analysis, we showed that some glycosylation mutants enhanced cellulosome assembly; in particular, disruption of glycosylation genes located in the endoplasmic reticulum showed intense enhancement. These findings suggest that inhibition of the core complex or precursor formation in protein glycosylation enhances cellulosome assembly, meaning that absence of glycosylation is more important for cellulosome assembly than reducing the size of the glycochain.     

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.     

Characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi

The assembly into supramolecular complexes of proteins having complementary activities is central to cellular function. One such complex of considerable biological and industrial significance is the plant cell wall-degrading apparatus of anaerobic microorganisms, termed the cellulosome. A central feature of bacterial cellulosomes is a large non-catalytic protein, the scaffoldin, which contains multiple cohesin domains. An array of digestive enzymes is incorporated into the cellulosome through the interaction of the dockerin domains, present in the catalytic subunits, with the cohesin domains that are present in the scaffoldin. By contrast, in anaerobic fungi, such as Piromyces equi, the dockerins of cellulosomal enzymes are often present in tandem copies; however, the identity of the cognate cohesin domains in these organisms is unclear, hindering further biotechnological development of the fungal cellulosome. Here, we characterise the solution structure and function of a double-dockerin construct from the P. equi endoglucanase Cel45A. We show that the two domains are connected by a flexible linker that is short enough to keep the binding sites of the two domains on adjacent surfaces, and allows the double-dockerin construct to bind more tightly to cellulosomes than a single domain and with greater coverage. The double dockerin binds to the GH3 beta-glucosidase component of the fungal cellulosome, which is thereby identified as a potential scaffoldin.     

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.     

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2^nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.     

Insights into Higher-Order Organization of the Cellulosome Revealed by a Dissect-and-Build Approach: Crystal Structure of Interacting Clostridium thermocellum Multimodular Components

Cellulosomes are large, multienzyme, plant cell wall-degrading protein complexes found affixed to the surface of a variety of anaerobic microbes. The core of the cellulosome is a noncatalytic scaffoldin protein, which contains several type-I cohesin modules that bind type-I dockerin-containing enzymatic subunits, a cellulose-binding module, an X module, and a type-II dockerin that interacts with type-II cohesin-containing cell surface proteins. The unique arrangement of the enzymatic subunits in the cellulosome complex, made possible by the scaffoldin subunit, promotes enhanced substrate degradation relative to the enzymes free in solution. Despite representative high-resolution structures of all of the individual modules of the cellulosome, this mechanism of enzymatic synergy remains poorly understood. Consequently, a model of the entire cellulosome and a detailed picture of intermodular contacts will provide more detailed insight into cellulosome activity. Toward this goal, we have solved the structure of a multimodular heterodimeric complex from Clostridium thermocellum composed of the type-II cohesin module of the cell surface protein SdbA bound to a trimodular C-terminal fragment of the scaffoldin subunit CipA to a resolution of 1.95 Å. The linker that connects the ninth type-I cohesin module and the X module has elevated temperature factors, reflecting an inherent flexibility within this region. Interestingly, a novel dimer interface was observed between CipA and a second, symmetry-related CipA molecule within the crystal structure, mediated by contacts between a type-I cohesin and an X module of a symmetry mate, resulting in two intertwined scaffoldins. Sedimentation velocity experiments confirmed that dimerization also occurs in solution. These observations support the intriguing possibility that individual cellulosomes can associate with one another via inter-scaffoldin interactions, which may play a role in the mechanism of action of the complex.