A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates
© Vazana et al.; licensee BioMed Central Ltd. 2013
Received: 23 September 2013
Accepted: 27 November 2013
Published: 16 December 2013
Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.
Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.
The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.
KeywordsCellulosomes Cellulases Multi-enzyme complex Cellulosic biomass Biofuels Clostridium thermocellum
Cellulose is the major component of the plant cell wall and is the most abundant renewable source of carbon and energy on Earth. One of the most potent cellulose-degrading microorganisms is the well-studied bacterium, Clostridium thermocellum. This anaerobic thermophilic cellulolytic bacterium secretes a multi-enzymatic complex called the cellulosome, first discovered in 1983 [1, 2]. Since then, other cellulosomal organisms have been discovered and characterized, which possess an array of cellulosomal architectures [3–16].
Cellulosome architecture is primarily dictated by the non-catalytic scaffoldin subunit. In C. thermocellum the scaffoldin is composed of nine repeating cohesin modules, each of which can bind cohesively to a complementary dockerin-module, borne by a cellulosomal enzyme. The scaffoldin targets the cellulosome complex to the cellulose substrate by virtue of an integral carbohydrate-binding module (CBM) [17–19] and attaches to the bacterial cell wall via an alternative type of cohesin-dockerin interaction [20, 21]. Enhanced synergism among the catalytic units and close association between the cell-bound cellulosome and substrate serve to minimize diffusion of the enzymes and their hydrolytic products, thus providing the bacterium with a competitive advantage over other organisms .
Cellulosome systems in bacteria show great diversity and can be divided into simple and complex systems. Some cellulolytic bacteria, for example, C. thermocellum, Bacteroides cellulosolvens and Acetivibrio cellulolyticus, have a complex cellulosome system. They produce several types of scaffoldins organized in a scaffoldin gene cluster, in which the genes encoding for the major scaffoldins are clustered together on the chromosome and the genes encoding the cellulosomal enzymes are scattered across the chromosome [23, 24]. Another resemblance among these bacteria is that the intermodular linker segments in their primary scaffoldin are relatively long, reaching 20 to 40 residues in length and more, and are rich in proline and threonine residues . In contrast, other clostridial cellulosome-producing bacteria, such as C. cellulovorans, C. cellulolyticum, C. josui, and C. acetobutylicum, have a simple cellulosome system [4, 26–28]. The simple systems contain a single scaffoldin and genes for cellulosomal enzymes are encoded downstream of the scaffoldin gene. The scaffoldins of the simple systems possess markedly shorter linkers than those of the complex systems. For example in C. cellulovorans the linkers of scaffoldin CbpA range between five and eight residues. Interestingly, in some scaffoldins - even those of complex systems - neighboring cohesins may not be separated by linkers at all, such as the first and second or the third and fourth cohesins in ScaB from B. cellulosolvens. At the other extreme are linkers as long as 100 to 700 residues . The lengths and composition of the intermodular linker segments are not arbitrary, and their content and disposition likely play a relevant role in cellulosome function.
The position of the CBM in the scaffoldins also differs between the cellulosome systems. In the simple systems the CBM is invariably positioned at the N-terminus of the scaffoldin and is followed by one or more X 2 modules . In contrast, the complex systems are characterized by an internal CBM, surrounded on both the N- and C-terminal sides by cohesins.
Designer cellulosomes are composed of an artificial chimaeric scaffoldin, and a set of cellulases [20, 30]. The synthetic scaffoldin consists of a CBM module, which targets the entire complex to the cellulosic substrate, together with several cohesin modules, derived from different species, that have divergent specificities. In turn, the cellulases each have a complementary and specific dockerin-module, which binds selectively to one of the divergent cohesins [31–33]. Unlike native cellulosome systems, the use of designer cellulosomes thus enables control over the composition and the positions of the chosen cellulases within a given mini-cellulosome, thereby allowing the production of homogeneous complexes. In this context, the controlled incorporation of cellulases into artificial designer cellulosomes was shown to induce enhanced synergism between cellulases via their targeting to the substrate by the family-3a scaffoldin-borne CBM module, or by the proximity of the cellulases in the complex [34–43]. In this study, we employed a synthetic biology (SynBio) approach by preparing an extensive combinatorial library of appropriate trivalent chimaeric scaffodins in order to address the question of whether enzyme position within the cellulosome complex and/or length of intermodular linker contribute/s to cellulosome performance. The latter scaffoldins enabled precise integration of three different cellulases in distinct spatial and positional arrangements relative to an integral CBM. The resultant designer cellulosomes were examined for activity on recalcitrant microcrystalline cellulose and pretreated cellulose-enriched wheat-straw substrates.
Design and construction of designer-cellulosome subunits
In order to study the importance of the spatial organization of the cellulases in the scaffoldin we used different intermodular linker lengths between the cohesin and the CBM modules, thereby imitating the diversity that exists in nature - long 27 to 35 amino-acid intermodular linkers (simulating the native linkers of C. thermocellum, A. cellulolyticus, B. cellulosolvens and R. flavefaciens), short 5 amino-acid linkers (emulating the linkers of C. cellulovorans, C. cellulolyticum, C. josui, and C. acetobutylicum) or without such linkers (designated no or none) (Figure 1b).
The set of inter-modular linkers used for cloning
The four modules (three divergent cohesin modules and a CBM) could thus be shuffled, resulting in twenty-four different arrangements, each with linkers of three different lengths separating the modules. Therefore, from the basic scaffoldin template, 72 possible combinations could potentially be produced. The cloning of this scaffoldin set, however, proved to be a challenging task, mainly due to the repetitive composition of the intermodular linkers, the length of the DNA encoding the recombinant scaffoldins (approximately 2,100 bp), and the relatively large number of constructs.
Two different approaches were used for cloning: in the first approach, a computer-aided, automated method for combinatorial DNA library design and production was employed for construction and cloning of the scaffoldins that either lacked intermodular linkers or contained short intermodular linkers. The design and synthesis of scaffoldins using this approach were performed using computer-aided methods for specifying, visualizing, planning and executing the actual production of the desired DNA libraries [44, 45]. This approach, however, ultimately proved inadequate for cloning the scaffoldins containing the long intermodular linkers, presumably due to the highly repetitive nature of the DNA sequence of these constructs.
In the end, 56 of the 72 scaffoldins were successfully cloned using the two approaches, and of these, 45 were expressed successfully in an E. coli host-cell system (Figure 3). Special efforts were spent in attempts to complete full sets, although certain sets were eventually abandoned after numerous trials. Specifically, sets 3 and 17 remained unfinished, owing to difficulties in cloning or expression of the residual constructs. The final scaffoldin library used for further study included 14 full sets, consisting of 42 different trivalent chimaeric scaffoldins (Figure 3). In seven of these sets the CBM was located in an internal position: in sets 5, 6, 11, 12, and 18 the CBM occupied the second position, and in sets 4 and 10 the CBM was in the third position. In the remaining seven completed sets, the CBM was located at the extremities: that is, the C-terminal position in set 9 and the N-terminal position in sets 19 to 24. The latter sets with the CBM at the N terminus represented a complete collection of six scaffoldins with the different cohesins located in all possible combinations.
Matching dockerin-bearing cellulases
A general method for the expression and purification of the 42 scaffoldins was designed based on a scheme devised initially for one of the chimaeric scaffoldins (Scaf9S). Interestingly, a heat-treatment step during the purification protocol, which precipitated the bulk of the native E. coli proteins, did not damage the scaffoldin even though two of the cohesin modules, which account for half of the cohesins in the chimaeric scaffoldin (that is, A. cellulolyticus and B. cellulosolvens) were of mesophilic origin. The scaffoldin was further subjected to a two-stage affinity purification procedure, based on its binding to cellulose via the CBM module and to an Ni-NTA column by virtue of its His-tag. This two-step strategy allowed us to purify mostly intact scaffoldins, since almost all of the scaffoldins (apart from scaffoldin 9, 10 and 4) had the CBM module at the N-terminus or relatively close to the N-terminus of the protein, whereas the His-tag was appended to the scaffoldin on the opposite C-terminus part. In this manner we avoided purifying scaffoldins that were proteolytically cleaved during expression, since the truncated forms lost one of the tags that was used for purification. Subsequently, the remaining 41 scaffoldins were expressed and purified accordingly in batch, 2 to 4 scaffoldins at a time.
The purity of the scaffoldins was analyzed using SDS-PAGE. In initial purification experiments, two of the scaffoldins seemed to be truncated, since they migrated as two separate bands on the gel. Scaffoldin ‘9L’ with the following modular composition: Scaf · TBAc showed 72- and 55-kDa bands (data not shown). The 72-kDa band corresponded to that of the intact scaffoldin, and the 55-kDa band was consistent with a scaffoldin without the first C. thermocellum cohesin. Indeed, non-denaturing PAGE of the complex of this scaffoldin showed a shifted band corresponding to the designer-cellulosome complex and a lower band of excess 48S-t, which could not bind to the C. thermocellum that lacked the matching cohesin. The second truncated scaffoldin, Scaf10L (Scaf · TBcA), showed a similar pattern. In order to overcome this problem, we transferred the His-tag of these two scaffoldins from the C to the N terminus. In this manner we could purify the intact scaffoldins, because in the first step the full-length scaffoldin is bound to cellulose via its C-terminal CBM and in the second step via its N-terminal His-tag to the nickel column. Using this approach, all of the purified scaffoldins showed a principal band, which corresponded to the full-length uncleaved protein (Additional file 3: Figure S2). The specificity of the cohesin-dockerin interactions was then verified by affinity-based ELISA. For example, scaffoldin19L was shown to interact with its three matching dockerins C. thermocellum, B. cellulosolvens and A. cellulolyticus but not with the non-matching R. flavefaciens dockerin (Figure 4A).
Analysis of designer-cellulosome assembly
Activity of designer cellulosomes with selected C. thermocellum cellulases
Kinetics, synergy, proximity and targeting of the C. thermocellum enzymes Cel48S, Cel8A and Cel9K
The three enzymes used in this study, Cel48S, Cel9K and Cel8A are major components in the C. thermocellum cellulosome and are up-regulated when C. thermocellum is grown on crystalline cellulose as a carbon source [47–54]. Consequently, we employed as substrates the commonly used commercially available Avicel and a pretreated, cellulose-enriched wheat straw preparation. The final composition of the pretreated wheat straw was determined to be 90% cellulose, 5% hemicellulose and 5% lignin.
The activity of the individual enzymes was tested on both substrates and was compared to that of the combined enzymatic activity in order to determine the apparent synergy between them (Additional file 4: Table S2). The three enzymes displayed 2-fold synergistic activity on pretreated wheat straw but no synergy was observed on Avicel. The overall enhancement in activity of the free enzymes compared to the enzymes bound to a long-linker designer scaffoldin was 1.82-fold for Avicel and 1.93-fold for pretreated wheat straw. The activities of the designer scaffoldins increased with the increase in linker length on both substrates, by 1.4-fold from the no-linker to the long-linker scaffoldin-mediated complex. The proximity effects were determined by comparing the activity of the combined free enzymes to the enzymes with the control scaffoldin, and were 1.17 and 1.14 for Avicel and pretreated wheat straw, respectively. The targeting effect was determined to be 1.56-fold for Avicel and 1.7-fold for pretreated wheat straw by comparing the activities of the enzymes with a designer scaffoldin with the long intermodular linkers, to the activity of the control scaffoldin lacking the CBM (Figure 7).
Effect of spatial organization of the scaffoldin on activity
Next, we tested for cellulose hydrolysis by designer cellulosomes composed of each of the scaffoldins in the library, at a single timepoint (pre-determined by the kinetics assay). For Avicel, activity was tested at 72 h, since shorter incubation times had lower than 5% conversion rates. For pretreated wheat straw the kinetics reaction reached a conversion of about 20% after 3 h; thus longer incubation times were unnecessary. Designer cellulosomes, comprising members of the fourteen different scaffoldin arrangements (sets), were tested on Avicel and pretreated wheat straw for their activities in combination with the three cellulases; for each arrangement we tested three scaffoldins that vary in the length of the intermodular linkers from 0 (no linkers), 5 (short linkers) to an average of 30.5 (27 to 35, long-linker) amino acids, respectively (Figure 1).
All of the designer cellulosomes synthesized in this work acted synergistically and showed substantially higher activities on both substrates compared to those of the combined free dockerin-bearing enzyme systems, whether fitted with matching CBM-fused cohesins (CBM-Cohs) or not. When the three enzymes were in the free state, they degraded Avicel (Figure 8) at a level that produced about 4.0 mM reducing sugars (glucose equivalents); when each enzyme was combined with a matching CBM-Coh for substrate targeting, the observed Avicel-degrading activity increased to about 4.8 mM glucose equivalents. In contrast, the observed activities of the designer cellulosomes ranged from approximately 5.8 to approximately 7.7 mM glucose equivalents after a 72-h reaction period. The lowest value was observed for the designer cellulosome based on the short-linker scaffoldin Scaf · TcAB (Scaf11S, set 11) with an internal CBM, and the highest value was achieved for the designer cellulosome with the long-linker scaffoldin Scaf · cTAB (Scaf21L, set 21). The substantial increase in cellulolytic activity on the microcrystalline cellulose substrate indicates that all of the different combinations of designer cellulosomes exhibited enhanced synergistic activities among the enzymes, irrespective of the position of the enzyme or CBM in the complex.
Equivalent results were observed on the pretreated wheat-straw substrate (Figure 9). The observed activities on this substrate ranged from approximately 2.1 to 3.5 mM reducing sugars following a 3-h reaction period, compared to 1.5 and 2.0 of the free enzymes without and with matching CBM-Cohs, respectively. The lowest value was observed for the no-linker scaffoldin Scaf · cABT (Scaf20N, set 20, N-terminal CBM), and the highest value for the long-linker scaffoldin Scaf · cTAB (Scaf12L, set 12, internal CBM).
The results (Figures 7, 8 and 9) also revealed a trend of increased activity on both substrates, as the intermodular linker length increased from no linkers at all to short 5-residue linkers, and from short to long linkers. Two-way analysis of variance (ANOVA) with interaction was used for statistical verification with length and the 14 scaffoldin arrangements as factors; no interaction was found for either substrate (P = 0.16 for Avicel; P = 0.0595 for pretreated wheat straw), indicating that linker length indeed had a significant effect on activity. The activities exhibited by the long, short and no-linker scaffoldins were thus observed to be significantly different from each other in the majority of the sets for both substrates. Of the 14 sets, 9 exhibited this general trend on Avicel, and 12 of the 14 sets on pretreated wheat straw.
Unlike the apparent dependence of activity on intermodular linker length of the scaffoldins, no clear trend could be discerned for position of the CBM or the cohesins (and consequent position of the respective enzymes used in this study). Designer cellulosomes based on scaffoldins having internal CBMs (Figures 8 and 9, top) exhibited similar levels of activity on the cellulosic substrates when compared to those based on scaffoldins with CBMs at their N or C termini (Figures 8 and 9, bottom). Likewise, the order of the cohesins in the scaffoldin and their nearest neighbor(s) did not have a perceptible effect on cellulolytic activity, using the three particular enzymes selected for this study.
The integration into a cellulosome system of cellulases and associated polysaccharide-degrading enzymes produced by anaerobic bacteria can facilitate their synergistic activity. However, it might also cause an opposite effect (anti-proximity) due to conformational restraint and steric clashes between the cellulases . Our understanding of cellulosome architecture and its implications for cellulose hydrolysis is still limited due to the incredible heterogeneity of the cellulosome complex . However, we are steadily gaining insight into the arrangement of the cellulosome complex and its functional consequences.
It appears that cellulosomal components exhibit diverse strategies that increase the plasticity of the complex and its interaction with its plant cell-wall polysaccharide substrates. This kind of flexibility may have a role in allowing enhanced access of the cellulases to the crystalline-cellulose substrate, which is interwoven together with hemicellulose, pectin and lignin components of the plant cell-wall. For example, the C. thermocellum type-I dockerin module displays internal sequence and structural symmetry that provide a dual mode of binding resulting in two different orientations of the dockerin-borne enzyme [60, 61]. This type of plasticity would thus enable the enzyme to rapidly assume an alternative conformation for interaction with an untouched portion of its complex substrate.
In the past several years, selected studies have addressed the conformational flexibility of the cellulosome and its possible significance to biomass degradation. Early on, it was evident that isolated cellulosomes could attain an overall tight or loose conformation [1, 62]. More recently , ultrastructural studies of a homogeneous mini-cellulosome containing three cohesin modules attached to three matching cellulases suggested that the flexibility of the linkers connecting consecutive cohesin modules could control structural transitions and thus regulate substrate recognition and degradation. In addition, the cellulases were found to be alternately projected from the scaffoldin in numerous directions, as was previously proposed based on the crystal structure of cohesins together with their adjacent linker segments [64, 65]. This property of the scaffoldin can prevent steric clashes between neighboring catalytic modules.
The conformational events that occur upon integration of an enzyme into a cellulosome complex have been studied by examining the interaction between cellulosomal enzymes and cohesins by small-angle x-ray scattering (SAXS) [66, 67]. Using this approach, the linker connecting the catalytic module of the C. cellulolyticum cellulosomal enzyme Cel48F to its dockerin was found to be extended and flexible when the enzyme is in its free form, but acquired a packed and rigid conformation upon binding to the matching cohesin. In another study , crystallographic evidence revealed the flexible nature of an extended intermodular linker of an A. cellulolyticus scaffoldin subunit. Combined SAXS and molecular dynamics studies revealed that the inter-cohesin linkers in binary and ternary complexes are intrinsically disordered, thereby resulting in extensive structural flexibility [66, 68]. In addition, computational biology implementing a coarse-grained model was employed to study self-assembly and flexibility of cellulosomes . In this work, the shape and modularity of the enzyme CbhA, was suggested to account for its tendency to bind more frequently to the scaffoldin due to its flexible nature and multimodularity that allowed a longer residence time around the scaffoldin.
The role of the intermodular linkers was further evaluated in a detailed study  wherein a cohesin dyad was employed for fabrication of hybrid cellulosomes with two model cellulases, Cel48F and Cel9G, derived from the mesophilic bacterium C. cellulolyticum. Several scaffoldin arrangements were used to form mini-cellulosome complexes, each of which contained two cohesins separated by various linker lengths, ranging from 4 to 128 residues, and various linker compositions, but without a CBM. All of the mini-complexes displayed similar activities on Avicel and PASC, and induced a similar 2-fold proximity between the two cellulases. Thus, in this case, the intermodular linker-length did not substantially affect the synergistic action of the two enzymes used in this study.
In the present study, we employed a synthetic biology (SynBio)-based strategy and strove to design a trivalent scaffoldin, which would serve as a basis for establishing a combinatorial library of scaffoldins. This library was then used for functional analysis of the position of the resident module and the length of the linkers between them. In doing so, we thus increased the level of complexity by adding a third cohesin, and hence a third cellulase, together with a supplementary CBM module that targets the scaffoldin to the substrate. This strategy would potentially allow 24 different modular arrangements of the scaffoldin. In addition to the native intermodular linkers, reduced linker length would enable examination of the effects of linker length on CBM-mediated targeting of the complex and enzyme proximity within the complex and their contribution to the overall deconstruction activity on cellulosic substrates. Owing to this broad design, we were able to overcome obstacles of cloning, expression and scaffoldin continuity, which limited the number of scaffoldins included in the final library. We thus succeeded in obtaining complete sets of scaffoldins, which contained all combinations of the desired positions of the modules and incremented lengths of their respective linker segments. Consequently, out of the 24 possible scaffoldin arrangements originally attempted, 14 complete sets were finally obtained, and their respective degradative capacity on cellulosic substrates was compared.
These 14 sets emulate the types of scaffoldin arrangements that occur in nature. For example, in simple cellulosome systems the CBM is positioned at the N terminus of the scaffoldin. We were thus able to express a complete set of six scaffoldins (scaffoldins 19 to 24 in Figure 3) that represent scaffoldins with an N-terminal CBM, with all of the possible permutations of the three cellulases. This scaffoldin set allowed us to determine if the position of a given enzyme in a trivalent designer cellulosome, relative to the other two, would appreciably influence the overall level of activity. An additional group of four scaffoldins carries the same arrangement of cohesins for all of the scaffoldins (T, B, A) with the CBM module at different positions in each of the scaffoldins: that is, a C-terminal position in scaffoldin 9, an internal position in scaffoldins 10 and 12, and N-terminal position in scaffoldin 22. The latter set allowed us to examine whether the position of the CBM would have a significant effect on the degradative capacity of the resultant designer cellulosome. Notably, the position of a C-terminal CBM has yet to be observed in a native multi-modular scaffoldin. The results indicated a lack of preferred scaffoldin arrangement using the specific C. thermocellum cellulases and cellulosic substrates described in this study - including the non-native scaffoldins (scaffoldin set 9, with the CBM at the C terminus) - which were fully functional in their contribution to degradation of cellulosic substrates. These results, however, do not necessarily indicate that other combinations of enzymes will not be affected by their location in a cellulosome. The combinatorial scaffoldin library can therefore be used as a platform to study the activities of other enzymes derived from different glycoside-hydrolase families or from different organisms, as well as other types of enzymes.
The results of the present study demonstrated that the use of native long intermodular linkers (average of 30.5 amino acids) between the scaffoldin-borne modules consistently resulted in higher cellulose-degrading activities. In addition, the designer cellulosome assemblies resulted in enhanced degradation of cellulose, due to the proximity between the enzymes and the targeting effect induced by the CBM3a module of the scaffoldin. The attachment of the CBM to the insoluble substrate may, however, restrict accessibility of the enzymes to the substrate due to attachment of the designer cellulosome on the surface of the cellulose . In this context, the cohesin dyad-based hybrid-cellulosome  was free in the reaction solution and lacked the targeting function. Interestingly, in simple cellulose systems with short intermodular linkers, the CBM is positioned at the N terminus of the scaffoldin together with an X 2 module, so the freedom of movement of enzymes attached to cohesins on the C-terminus of the scaffoldin is less compromised. In contrast, in complex cellulosome systems, with scaffoldins that are characterized by long intermodular linkers, the CBM module is typically positioned in an internal position in the polypeptide chain. The long linkers can thus compensate for the restricted movement induced by the CBM. The flexibility of the intermodular linker segments thus provides cooperativity among the cellulosomal glycoside hydrolases and results in synergy and increased cellulolytic activity . Nevertheless, reduction of the intermodular linker length to 5-residues, or even their complete removal, failed to abolish the observed enhanced activity of the designer cellulosomes, thus underscoring the robust nature of cellulosome structure and architecture.
The different arrangements of the three enzymes on the designer cellulosome and the position of the CBM in the scaffoldin did not have a significant effect on the observed activity. In fact, we should consider that the designer cellulosomes are three-dimensional flexible complexes and not strings of cohesins bound to cellulases. What appears to be a different arrangement of cohesins in the designer scaffoldin would not necessarily result in a markedly different organization of the cellulases, owing to the flexibility or packing of the complex. Indeed, natural cellulosome complexes are intrinsically heterogeneous, since within a given species the cohesins of a given scaffoldin do not exhibit striking differences in specificity for the various dockerin-bearing enzymes, such that individual enzymes can bind to each of the scaffoldin-borne cohesin modules.
A combinatorial library of a relatively simple designer-cellulosome system composed of three enzymes was prepared using a synthetic biology approach for examining the contribution of enzyme position, CBM location and intermodular linker length to cellulose-degrading activity. Longer intermodular scaffoldin linkers were shown to enhance the activity of the complex, whereas the position of the three particular enzymes selected for this study was of no apparent consequence. The high intrinsic flexibility of the intermodular linkers may be a major factor for refinement and optimization of the synergistic activity of the cellulosomal enzymes and may facilitate substrate targeting and binding of the scaffoldins, thereby maximizing the effectiveness of the cellulosome system as a cellulose-degrading machine. The scaffoldin library that we present in this paper can serve as a tool both to study the contribution of specified components of designer cellulosome assemblies and to explore unknown properties of cellulosome architecture, organization and action on plant-derived biomass.
Materials and methods
Cloning of cellulases
The recombinant wild-type family-48 exocellulase, Cel48S (48S-t), was amplified from C. thermocellum ATCC 27405 genomic DNA with the following forward and reverse primers, 5′ CAGTCCATGGGTCCTACAAAGGCACCTAC 3′ and 5′ CGCGAAGCTTTTAATGGTGATGGTGATGGTGG 3′, respectively (NcoI and HindIII restriction sites in boldface), that allow their incorporation into pET28a. Similarly, the recombinant wild-type family-8 endocellulase, Cel8A (8A-t), was cloned from the genomic DNA of C. thermocellum with the following forward and reverse primers, 5′ CAGTCCATGGGTGTGCCTTTTAACACAAA 3′ and 5′ CACGCTCGAGATAAGGTAGGTGGGGTATGC 3′ respectively, (NcoI and XhoI restriction sites in boldface). Likewise, the recombinant wild-type family-9 endocellulase, Cl9K (9K-t), was amplified from the C. thermocellum genomic DNA and cloned into pET28a vector using the restriction-free (RF) method  with the following forward and reverse primers, 5′ GTTTAACTTTAAGAAGGAGATATACCATGGGCCATCACCATCACCATCACTTAGAAGACAAGTCTCCAAAGTTGCCGGAT 3′ and 5′ GAGTGCGGCCGCAAGCTTGTCGACGGAGCTCTTATTTATGTGGCAATACATCTATCTCTTTAAG 3′ respectively, (gene-specific sequences are italicized, plasmid-specific sequences are shown in plain font, His-tag in bold). For the cloning of 9K-a with the divergent dockerin from A. cellulolyticus, the dockerin was amplified from the genomic DNA of A. cellulolyticus and used for the simultaneous insertion of the divergent dockerin and deletion of the wild-type dockerin into the wild-type 9K-t plasmid using the RF cloning method with the following forward and reverse primers, 5′ CTCGATGAAATTGACTTAATAACACCGCCAGGTACCAAATTTATATATGGTGATGTTGATGGTAATG 3′ and 5′ GAGTGCGGCCGCAAGCTTGTCGACGGAGCTCTTATTCTTCTTTCTCTTCAACAGGGAATAAAAATATC 3′ respectively (gene-specific sequences are italicized, plasmid-specific sequences are not). For the cloning of the chimaeric enzyme 8A-b with the C. thermocellum Cel8A catalytic module and a divergent dockerin from B. cellulosolvens, the catalytic module of Cel8A was amplified from C. thermocellum ATCC 27405 genomic DNA with the following forward and reverse primers, 5′ ATTCAACCATGGGTGTGCCTTTTAACACAAAATAC 3′ and 5′ ATATTGCTCGAGTAATGTGGTACCAATGAAGGTGTCGGATTCGACG 3′ respectively (NcoI, KpnI and XhoI restriction sites in boldface). The PCR product was cloned into a pET28a plasmid linearized with NcoI and XhoI restriction enzymes to yield p8A-CD. The dockerin was amplified from B. cellulosolvens genomic DNA with the following forward and reverse primers, 5′ ACTTTAGGTACCTCCAAAAGGCACAGCTAC 3′ and 5′ ATTAATCTCGAGCGCTTTTTGTTCTGCTGG 3′ respectively (KpnI and XhoI restriction sites in boldface). The resultant DNA was cloned into p8A-CD that was linearized with KpnI and XhoI to yield p8A-b.
High-throughput computer-aided cloning of short- and no-linker scaffoldins
The core recursive construction step in this method required four basic enzymatic reactions: phosphorylation, elongation, PCR and Lambda exonucleation, and was performed as previously described by Linshiz et al.  using the primer sets listed in Additional file 5: Table S3.
The PCR product was amplified in order to yield sufficient amounts of DNA for subsequent cloning, by the following upper and lower primers, according to the modules that were located at the 5′ and 3′ of each scaffoldin construct: CtCohA2-Upper: 5′ CGCGAGCCATGGGGTCCGACGGTGTGGTAGTAG 3′, AcCohC3-Upper- 5′ CGCATGCCATGGGATCCGATTTACAGG 3′, CtScaACBM-Upper- 5′ CGACTCCCATGGCAAATACACCGGTATC 3′, BcCohB3-Upper- 5′ CGCAGGCCATGGGTAGTTCACCAGGAAATA 3′, CtCohA2-Lower- 5′ CGCACGCTCGAGTGTTGCATTGCCAACG 3′, AcCohC3-Lower: 5′ GGGCCGCTCGAGACTTGCAATTACCTC 3′, CtScaACBM-Lower- 5′ CTGTCGCTCGAGACTGCCACCGGGTTC 3′ and BcCohB3-Lower- 5′ GGACGGCTCGAGATTAGTTACAGTAATG 3′ (NcoI and XhoI restriction sites in boldface). The amplified product was digested by NcoI and XhoI, and ligated with NcoI-XhoI linearized pET28a vector (Novagene, Madison, WI, USA). Positive clones were selected by colony PCR and verified by sequencing. The approach was also attempted for production of long-linker scaffoldins with negative outcomes.
Restriction-free (RF) cloning of long-linker scaffoldins
The trivalent long-linker scaffoldins were cloned in pET28a expression vector applying the RF method , using a simultaneous multi-component assembly approach. The assembly is described in detail in the Results section. Primer sets were designed for PCR amplification (Additional file 6: Table S4) and subsequent RF reactions were carried out using Phusion polymerase (Thermo Scientific, Hudson, NH, USA).
Expression and purification of cellulases and designer scaffoldins
Escherichia coli BL21 (DE3) cells overproducing pET28a-scaffoldin genes or cellulases were grown at 37°C in Luria-Bertani broth supplemented with 50 μg/ml kanamycin (Sigma-Aldrich Chemical Co, St. Louis, MO, USA) to A600 = 0.8 to 1.0. The cultures were cooled to 16°C, and protein expression was induced by the addition of 0 to 1 mM isopropyl-1-thio-β-D-galactoside - IPTG (Fermentas UAB Vilnius, Lithuania), based on the results of predetermined optimization experiments. The cultures were incubated at 16°C for an additional 16 h, the cells were harvested by centrifugation (3500 g, 15 minutes), resuspendend in Tris-buffered saline (TBS, 137 mM NaCl, 2.7 mM KCl, 25 mM Tris–HCl, pH 7.4) supplemented with 5 mM imidazole (Merck KGaA, Darmstadt, Germany) and disrupted by sonication. The sonicate was heated for 20 minutes to 60°C and centrifuged (20,000 g, 30 minutes). The supernatant fluids were mixed with 4 ml of Ni-NTA beads for 1 h on a 20-ml Econo-pack column for batch purification at 4°C. The column was washed by gravity flow with 100 ml wash buffer (TBS, 50 mM imidazole) and elution was performed with 14 ml of elution buffer (TBS, 250 mM imidazole). For purification of the scaffoldins an additional affinity-purification step was applied: the eluted fractions were incubated in a 50-ml tube with 10 ml PASC (0.75 mg/ml) for 1 h at 4°C to allow binding of the CBM. The matrix was washed three times with TBS, containing 1 M NaCl, and three times with TBS without added salt. The scaffoldin was eluted with 1% triethylamine and neutralized with 1 M 2-(N-Morpholino) ethanesulfonic acid (MES) buffer pH 5. For both scaffoldin and cellulases the buffer was exchanged by dialysis against TBS, and the scaffoldin sample was concentrated using Amicon Ultra 15 ml 50,000 MWCO concentrators (Millipore, Bedford, MA, USA). Protein concentrations were estimated by the absorbance at 280 nm. The extinction coefficient was determined based on the known amino-acid composition of each protein using the ProtParam tool on the EXPASY server (http://www.expasy.org/tools/protparam.html) .
Analysis of cohesin-dockerin specificity
The procedure of Barak et al.  was followed with minor modifications. Maxisorp ELISA plates (Nunc A/S, Roskilde, Denmark) were coated with 1 μg/ml each of the dockerin-containing enzymes 48S-t, 9K-a and 8A-b, and then interacted with 0.1 to 1,000 ng/μl of its matching CBM-cohesin (CBM-Coh T, CBM-Coh A and CBM-Coh B) counterpart. Rabbit-anti-CBM (diluted 1:3,000 in blocking buffer) was used as the primary antibody for detection of the interaction.
For analysis of the chimaeric scaffoldins, Maxisorp ELISA plates were coated with 1 μg/ml of the chimaeric scaffoldin and then interacted with 0.1 to 1,000 ng/μl of matching Xyn-Doc proteins which were prepared as described in Barak et al. . These proteins are composed of xylanase T-6 from Geobacillus stearothermophilus fused to a dockerin module of appropriate specificity. Rabbit anti-xylanase T-6 antibody (diluted 1:10,000 in blocking buffer) was used as primary antibody for detection of the interaction. A secondary antibody preparation of goat-horseradish peroxidase (HRP)-labeled anti-rabbit antibody diluted 1:10,000 was added. The interaction was detected using TMB Substrate-Chromogen (Dako A/S, Glostrup, Denmark), and the reaction was terminated by the addition of 1 M sulfuric acid (H2SO4). Absorbance was measured at 450 nm.
Equimolar concentrations of scaffoldins and matching enzymes (4 to 8 μg each protein) were mixed and added to similar volumes of interaction buffer (TBS with 10 mM calcium chloride (CaCl2) and 0.05% Tween20). Double-distilled water (DDW) was added to a final volume of 30 μl. The proteins were incubated at 37°C for 2 h to allow complex formation. Non-denaturing sample buffer (192 mM glycine, 25 mM Tris) was added, and a total of 15 μl/lane was subjected to PAGE (7.5 to 9.0% acrylamide gels), using a Bio-Rad Laboratories (Hercules, CA, USA) power pack 300. Single components (scaffoldin and enzymes) were used as markers. The remaining 15 μl were used for analysis on SDS-PAGE.
Size-exclusion high performance liquid chromatography (HPLC)
Equimolar protein concentrations (450 picomoles scaffoldin or enzyme) were diluted in 300 μl of loading buffer (TBS, pH 7.4), supplemented with 2 mM of CaCl2. For the formation of designer cellulosome complexes, equimolar concentrations of a scaffoldin and enzymes were incubated at 37°C for 2 h with similar volumes of interaction buffer (TBS with 10 mM CaCl2 and 0.05% Tween20), and loading buffer was added to a final volume of 300 μl. The reactions were injected onto an analytical Superdex 200 HR 10/30 column using an AKTA fast-performance liquid chromatography system (GE Healthcare, Uppsula, Sweden) and loading buffer at a flow rate of 0.5 ml · min-1. Eluted proteins were detected at 280 nm and fractions (0.5 ml) concentrated and analyzed using SDS-PAGE gels.
Preparation of cellulose-enriched (pretreated) wheat straw
Wheat-straw sections were cut with shears into pieces 2 to 5 cm and subsequently ground with a knife mill (Waring Products Inc., Torrington, CT, USA) at 15,000 rpm for 1 minute. The resultant powder was passed through a sieve with mesh =10 to obtain a final biomass powder having an average particle size of 1 to 3 mm. A sample (20 g) of the resultant powder was treated with 85 ml of 5% (v/v) nitric acid for 1 h at 115°C. The acid-treated biomass was washed with DDW and treated further with 150 ml of 1.5% v/v sodium hydroxide (NaOH) for 1 h at 100°C and washed with DDW, yielding a cellulose-enriched substrate.
Determination of wheat-straw substrate chemical composition
The chemical composition of the samples was determined according to the following improvement of the Technical Association of the Pulp and Paper Industry (TAPPI)-method . For hemicellulose content, samples were boiled with 2% HCl for 2 h, washed with DDW and ethanol and dried at 105°C to constant weight (about 2 to 3 h). For cellulose content, samples were boiled with an ethanolic nitric acid (HNO3) solution for 1 h, washed with DDW and ethanol, and dried at 105°C to constant weight (about 2 to 3 h). For lignin content, samples were swollen in 72% H2SO4 at room temperature for 2 h, diluted with DDW to 8 to 10% acid, hydrolyzed with boiling diluted H2SO4 (8 to 10%) for 2 h, washed with DDW and ethanol, and dried at 105°C to constant weight (about 2 to 3 h). Total solid content was determined by drying the samples at 105°C for 2 h.
The hydrolysis reactions were carried out in a total volume of 200 μl, and consisted of reaction buffer (100 mM sodium acetate buffer pH 5.5, 24 mM CaCl2, 4 mM ethylenediaminetetraacetic acid (EDTA)), 0.5 μM of each protein and 2% w/v Avicel (Sigma-Aldrich) or 3.5 g/L pretreated (cellulose-enriched) wheat straw. Prior to the addition of the substrate, each scaffoldin was incubated with equimolar quantities of the three enzymes for 2 h at 37°C with a similar volume of interaction buffer (TBS with 10 mM CaCl2 and 0.05% Tween 20). The reaction was carried out for 24 to 72 h (Avicel) or 3 to 24 h (pretreated wheat straw) at 50°C and terminated by immersion in ice water. The substrate was pelleted by centrifugation at maximum speed (20,800 × g, 10 to 15 minutes), and 100 μl of the supernatant was transferred to a new tube. Dinitrosalycylic acid (DNS, 150 μl) was added, and the samples were boiled for 10 minutes. The absorbance was measured at 540 nm and the reducing sugars were determined according to a glucose calibration curve. Each assay was repeated three times in triplicate.
- CBM and c:
the family 3a carbohydrate binding module of the CipA scaffoldin from C. thermocellum
chimaeric enzyme consisting of the catalytic module of Cel8A from C. thermocellum fused to a divergent dockerin from B. cellulosolvens
chimaeric enzyme consisting of the catalytic module of Cel9K from C. thermocellum fused to a divergent dockerin from A. cellulolyticus
recombinant wild-type cellulosomal enzyme from C. thermocellum consisting of the catalytic module and dockerin
- Coh A:
A. cellulolyticus cohesin C3 module fused to CBM3a from C. thermocellum CipA scaffoldin
- Coh B:
B. cellulosolvens cohesin B3 module fused to CBM3a from C. thermocellum CipA scaffoldin
- Coh T:
C. thermocellum cohesin A2 module fused to CBM3a from C. thermocellum CipA scaffoldin
- no linkers:
Scaffoldin without intermodular linkers
- short linkers:
Scaffoldin with short intermodular linkers
- long linkers:
Scaffoldin with long intermodular linkers
enzyme-linked immunosorbent assay
phosphoric acid-swollen cellulose
polymerase chain reaction
Scaffoldin number 4 without intermodular linkers (the numbers in the terminology change according to the given scaffoldin set)
Scaffoldin number 4 with short intermodular linkers
Scaffoldin number 4 with long intermodular linkers
xylanase T-6 from Geobacillus stearothermophilus fused to a dockerin module of an appropriate specificity.
We are grateful for the assistance of Professor Edna Shechtman (Information Systems Division, Weizmann Institute) in statistical analysis of the results. We thank Rivi Ascarelli-Goell of the Weizmann Sequencing unit for fast and reliable work. This research was supported by a grant from the F Warren Hellman Grant for Alternative Energy Research in Israel in support of alternative energy research in Israel to EAB administered by the Israel Strategic Alternative Energy Foundation (I-SAEF). Additional support was obtained by a grant (No. 24/11) issued to RL by The Sidney E Frank Foundation through the Israel Science Foundation (ISF) and by a grant (No. 1349) to EAB also from the ISF. This research was also supported by the establishment of an Israeli Center of Research Excellence (I-CORE Center No. 152/11, EAB and YS) managed by the Israel Science Foundation, from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, by the Weizmann Institute of Science Alternative Energy Research Initiative (AERI) and the Helmsley Foundation, and a grant to EAB and RL from the Israel Ministry of Science (IMOS). EAB is the incumbent of The Maynard I and Elaine Wishner Chair of Bio-organic Chemistry.
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