Publications

The arsenal of genes that microbes express reflect the way in which they sense their environment. We have previously reported that the rumen microbiome composition and its coding capacity are different in animals having distinct feed efficiency states, even when fed an identical diet. Here, we reveal that many microbial populations belonging to the bacteria and archaea domains show divergent proteome production in function of the feed efficiency state. Thus, proteomic data serve as a strong indicator of host feed efficiency state phenotype, overpowering predictions based on genomic and taxonomic information. We highlight protein production of specific phylogenies associated with each of the feed efficiency states. We also find remarkable plasticity of the proteome both in the individual population and at the community level, driven by niche partitioning and competition. These mechanisms result in protein production patterns that exhibit functional redundancy and checkerboard distribution that are tightly linked to the host feed efficiency phenotype. By linking microbial protein production and the ecological mechanisms that act within the microbiome feed efficiency states, our present work reveals a layer of complexity that bears immense importance to the current global challenges of food security and sustainability.

Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome.
VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers.
We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

Many important proteins undergo pH-dependent conformational changes resulting in "on-off" switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from Clostridium acetobutylicum, which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4.8 and 7.5 with a 180 degrees rotation of the bound dockerin. Combined analysis by nuclear magnetic resonance spectroscopy, crystal structure determination, mutagenesis, and isothermal titration calorimetry elucidates the chemical and structural mechanism of the pH-dependent switching of the binding sites. The pH-dependent dual-binding-site switch not only represents an elegant example of biological regulation but also provides a new approach for developing pH-dependent protein devices and biomaterials beyond an on-off switch for biotechnological applications.

The multi-enzyme cellulosome complex can mediate the valorization of lignocellulosic biomass into soluble sugars that can serve in the production of biofuels and valuable products. A potent bacterial chassis for the production of active cellulosomes displayed on the cell surface is the bacteriumLactobacillus plantarum, a lactic acid bacterium used in many applications. Here, we developed a methodological pipeline to produce improved designer cellulosomes, using a cell-consortium approach, whereby the different components self-assemble on the surface ofL. plantarum.The pipeline served as a vehicle to select and optimize the secretion efficiency of potent designer cellulosome enzyme components, to screen for the most efficient enzymatic combinations and to assess attempts to grow the engineered bacterial cells on wheat straw as a sole carbon source. Using this strategy, we were able to improve the secretion efficiency of the selected enzymes and to secrete a fully functional high-molecular-weight scaffoldin component. The adaptive laboratory process served to increase significantly the enzymatic activity of the most efficient cell consortium. Internal plasmid re-arrangement towards a higher enzymatic performance attested for the suitability of the approach, which suggests that this strategy represents an efficient way for microbes to adapt to changing conditions.

Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass.IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective "first responders." The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from Ruminococcus flavefaciens called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 10(5) pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics.

Background(Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium.ResultsThe current proteome-wide work reveals patterns of protein expression of the various cellulosomal components, and explores the signature of differential expression upon growth of the bacterium on two major carbon sourcescellobiose and microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing components (mainly enzymes), in addition to free enzymatic subunits. The dockerin-bearing components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family members were represented among 102 carbohydrate-degrading enzymes, including the omnipresent, most abundant GH48 exoglucanase. Specific cellulosomal components were found in different molecular-weight fractions associated with cell growth on different carbon sources. Overall, microcrystalline cellulose-derived cellulosomes showed markedly higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different cellulosic and/or hemicellulosic carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by Clostridium thermocellum purified cellulosomes.ConclusionsThe current research demonstrates the involvement of key cellulosomal factors that participate in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies on various carbon sources was revealed. The novel reservoir of cellulolytic components of the cellulosomal degradation machineries may serve as a pool for designing new cellulolytic cocktails for biotechnological purposes.

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin-cohesin interactions persist. Using allatom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as "beads on a string," we propose that cohesincohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σ
Is, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σ
Is in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one σ
I in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different σ
Is, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same σ
I-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by σ
Is to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple σ
Is, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different σ
Is.

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein–protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180 degrees rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wildtype cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome's most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) super-resolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.IMPORTANCE Conversion of plant biomass into soluble sugars is of high interest for production of fermentable industrial materials, such as biofuels. Biofuels are a very attractive alternative to fossil fuels, both for recycling of agricultural wastes and as a source of sustainable energy. Cellulosomes are among the most efficient enzymatic degraders of biomass known to date, due to the incorporation of a multiplicity of enzymes into a potent, multifunctional nanomachine. The intimate association with the bacterial cell surface is inherent in its efficient action on lignocellulosic substrates, although this property has not been properly addressed experimentally. The dramatic increase in cellulosome performance on recalcitrant feedstocks is critical for the design of cost-effective processes for efficient biomass degradation.

The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

Background: (Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature. Results: The organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin-dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin-dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes). Conclusions: The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

The robust plant cell wall polysaccharide-degrading properties of anaerobic bacteria are harnessed within elegant, marcomolecular assemblages called cellulosomes, in which proteins of complementary activities amass on scaffold protein networks. Research efforts have focused and continue to focus on providing detailed mechanistic insights into cellulosomal complex assembly, topology, and function. The accumulated information is expanding our fundamental understanding of the lignocellulosic biomass decomposition process and enhancing the potential of engineered cellulosomal systems for biotechnological purposes. Ongoing biochemical studies continue to reveal unexpected functional diversity within traditional cellulase families. Genomic, proteomic, and functional analyses have uncovered unanticipated cellulosomal proteins that augment the function of the native and designer cellulosomes. In addition, complementary structural and computational methods are continuing to provide much needed insights on the influence of cellulosomal interdomain linker regions on cellulosomal assembly and activity.

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzymeborne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens' cellulosome is assembled from a repertoire of 223 Doccontaining proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative sigma(I) factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum. In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative sigma(I) factors and by the vegetative sigma(A) factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved sigma I and sigma(A)-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted sigma(I)-dependent promoter identified in the present work rather than the previously proposed sigma(A) promoter. With the exception of C. cellulovorans, both sigma(I) and sigma(A) promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5=untranslated regions (5'-TRs).Furthermore, these 5'-Rs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.

Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85 degrees C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

Cohesins and dockerins are complementary interacting protein modules that form stable and highly specific receptor ligand complexes. They play a crucial role in the assembly of cellulose-degrading multi-enzyme complexes called cellulosomes and have potential applicability in several technology areas, including biomass conversion processes. Here, we describe several exceptional properties of cohesin dockerin complexes, including their tenacious biochemical affinity, remarkably high mechanostability and a dual-binding mode of recognition that is contrary to the conventional lock and -key model of receptor-ligand interactions. We focus on structural aspects of the dual mode of cohesin dockerin binding, highlighting recent single-molecule analysis techniques for its explicit characterization.

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable beta-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.

Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities-the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes-4 xylanases and 4 cellulases-thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.IMPORTANCE: Plant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.

Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from scaffoldins and enzyme-borne dockerins, have been characterized recently. In this study, we cloned, expressed and characterized all of the glycoside hydrolases that contain a dockerin module. Among the 25 enzymes, 10 cellulases, 4 xylanases, 3 mannanases, 2 xyloglucanases, 2 arabinofuranosidases, 2 arabinanases and one -glucanase were assessed for their comparative enzymatic activity on their respective substrates. The dockerin specificities of the enzymes were examined by ELISA, and 80 positives out of 525 possible interactions were detected. Our analysis reveals a fine-tuned system for cohesin-dockerin specificity and the importance of diversity among the cohesin-dockerin sequences. Our results imply that cohesin-dockerin pairs are not necessarily assembled at random among the same specificity types, as generally believed for other cellulosome-producing bacteria, but reveal a more organized cellulosome architecture. Moreover, our results highlight the importance of the cellulosome paradigm for cellulose and hemicellulose degradation by R.champanellensis in the human gut.

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 angstrom to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

Dimeric avidins are a newly discovered subgroup of the avidin family that bind biotin with high affinity. Their dimeric configuration is a quaternary substructure of the classical tetrameric avidins which lacks the requirement of the critical Trp that defines the tetramer and dictates the tenacious interaction with biotin. Hoefavidin, derived from the bacterium Hoeflea phototrophica DFL-43(T), is the third characterized member of the dimeric avidin subfamily. Like the other members of this group, hoefavidin is a thermostable protein that contains a disulfide bridge between Cys57 and Cys88, thereby connecting and stabilizing the L3,4 and L5,6 loops. This represents a distinctive characteristic of dimeric avidins that compensates for the lack of Trp and enables their dimeric configuration. The X-ray structure of the intact hoefavidin revealed unique crystal packing generated by an octameric cylindrical structure wherein the C-termini segments of each monomer is introduced into the entrance of the biotin-binding site of an adjacent non-canonical monomer. This anomaly in the protein structure served as a lead toward the design of specific binding peptides. We screened for specific hoefavidin binding peptides derived from the C-terminal region and two peptides were obtained that bind a truncated form of hoefavidin (lacking the last 10 amino acids) with dissociation constants of 10(-5) M. The crystal structure of short hoefavidin complexed with a C-terminal derived peptide revealed the mode of binding. These peptides may form the basis of novel and reversible binders for dimeric avidins.

The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (K-a similar to 10(12) M). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.

Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium. IMPORTANCE Because the reservoir of unsustainable fossil fuels, such as coal, petroleum, and natural gas, is overutilized and continues to contribute to environmental pollution and CO2 emission, the need for appropriate alternative energy sources becomes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost-effective production method must be developed. The cellulosome system of the anaerobic thermophile C. clariflavum comprises a large number of cellulolytic and hemicellulolytic enzymes, which self-assemble in a number of different cellulosome architectures for enhanced cellulosic biomass degradation. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. The findings, together with the thermophilic characteristics of the proteins, render C. clariflavum of great interest for future use in industrial cellulose conversion processes.

Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens. 

Lactic acid bacteria (LAB) have long been used in industrial applications mainly as starters for food fermentation or as biocontrol agents or as probiotics. However, LAB possess several characteristics that render them among the most promising candidates for use in future biorefineries in converting plant-derived biomass either from dedicated crops or from municipal/industrial solid wastes into biofuels and high value-added products. Lactic acid, their main fermentation product, is an attractive building block extensively used by the chemical industry, owing to the potential for production of polylactides as biodegradable and biocompatible plastic alternative to polymers derived from petrochemicals. LA is but one of many high-value compounds which can be produced by LAB fermentation, which also include biofuels such as ethanol and butanol, biodegradable plastic polymers, exopolysaccharides, antimicrobial agents, health-promoting substances and nutraceuticals. Furthermore, several LAB strains have ascertained probiotic properties, and their biomass can be considered a high-value product. The present contribution aims to provide an extensive overview of the main industrial applications of LAB and future perspectives concerning their utilization in biorefineries. Strategies will be described in detail for developing LAB strains with broader substrate metabolic capacity for fermentation of cheaper biomass. (C) 2014 Elsevier Inc. All rights reserved.

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin-and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium-to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin

Background: Lactobacillus plantarum is an attractive candidate for metabolic engineering towards bioprocessing of lignocellulosic biomass to ethanol or polylactic acid, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize the two major polysaccharide constituents of lignocellulolytic biomass (pentoses and hexoses). We recently engineered L. plantarum via separate introduction of a potent cellulase and xylanase, thereby creating two different L. plantarum strains. We used these strains as a combined cell-consortium for synergistic degradation of cellulosic biomass. Results: To optimize enzymatic degradation, we applied the cell-consortium approach to assess the significance of enzyme localization by comparing three enzymatic paradigms prevalent in nature: (i) a secreted enzymes system, (ii) enzymes anchored to the bacterial cell surface and (iii) enzymes integrated into cellulosome complexes. The construction of the three paradigmatic systems involved the division of the production and organization of the enzymes and scaffold proteins into different strains of L. plantarum. The spatial differentiation of the components of the enzymatic systems alleviated the load on the cell machinery of the different bacterial strains. Active designer cellulosomes containing a xylanase and a cellulase were thus assembled on L. plantarum cells by co-culturing three distinct engineered strains of the bacterium: two helper strains for enzyme secretion and one producing only the anchored scaffoldin. Alternatively, the two enzymes were anchored separately to the cell wall. The secreted enzyme consortium appeared to have a slight advantage over the designer cellulosome system in degrading the hypochlorite pretreated wheat straw substrate, and both exhibited significantly higher levels of activity compared to the anchored enzyme consortium. However, the secreted enzymes appeared to be less stable than the enzymes integrated into designer c

Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed "cellulosomes," which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca. The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo-and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture.

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.

The anaerobic, thermophilic, cellulosome-producing bacterium Clostridium thermocellum relies on a variety of carbohydrate-active enzymes in order to efficiently break down complex carbohydrates into utilizable simple sugars. The regulation mechanism of the cellulosomal genes was unknown until recently, when genomic analysis revealed a set of putative operons in C. thermocellum that encode sigma(I) factors (i.e. alternative sigma factors that control specialized regulon activation) and their cognate anti-sigma(I) factor (RsgI). These putative anti-sigma(I)-factor proteins have modules that are believed to be carbohydrate sensors. Three of these modules were crystallized and their three-dimensional structures were solved. The structures show a high overall degree of sequence and structural similarity to the cellulosomal family 3 carbohydrate-binding modules (CBM3s). The structures of the three carbohydrate sensors (RsgI-CBM3s) and a reference CBM3 are compared in the context of the structural determinants for the specificity of cellulose and complex carbohydrate binding. Fine structural variations among the RsgI-CBM3s appear to result in alternative substrate preferences for each of the sensors.

Bacterial cellulosomes are generally believed to assemble at random, like those produced by Clostridiumcellulolyticum. They are composed of one scaffolding protein bearing eight homologous typeI cohesins that bind to any of the typeI dockerins borne by the 62 cellulosomal subunits, thus generating highly heterogeneous complexes. In the present study, the heterogeneity and random assembly of the cellulosomes were evaluated with a simpler model: a miniscaffoldin containing three C.cellulolyticum cohesins and three cellulases of the same bacterium bearing the cognate dockerin (Cel5A, Cel48F, and Cel9G). Surprisingly, rather than the expected randomized integration of enzymes, the assembly of the minicellulosome generated only three distinct types of complex out of the 10 possible combinations, thus indicating preferential integration of enzymes upon binding to the scaffoldin. A hybrid scaffoldin that displays one cohesin from C.cellulolyticum and one from C.thermocellum, thus allowing sequential integration of enzymes, was exploited to further characterize this phenomenon. The initial binding of a given enzyme to the C.thermocellum cohesin was found to influence the type of enzyme that subsequently bound to the C.cellulolyticum cohesin. The preferential integration appears to be related to the length of the inter-cohesin linker. The data indicate that the binding of a cellulosomal enzyme to a cohesin has a direct influence on the dockerin-bearing proteins that will subsequently interact with adjacent cohesins. Thus, despite the general lack of specificity of the cohesin-dockerin interaction within a given species and type, bacterial cellulosomes are not necessarily assembled at random.

The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NAIR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C-and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 angstrom. The structure is a beta-sandwich with a structural Ca2+ ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.

Background: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose. Methodology/Principal Findings: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel. Conclusions/Significance: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

Background: Many bacteria efficiently degrade lignocellulose yet the underpinning genome-wide metabolic and regulatory networks remain elusive. Here we revealed the "cellulose degradome" for the model mesophilic cellulolytic bacterium Clostridium cellulolyticum ATCC 35319, via an integrated analysis of its complete genome, its transcriptomes under glucose, xylose, cellobiose, cellulose, xylan or corn stover and its extracellular proteomes under glucose, cellobiose or cellulose. Results: Proteins for core metabolic functions, environment sensing, gene regulation and polysaccharide metabolism were enriched in the cellulose degradome. Analysis of differentially expressed genes revealed a "core" set of 48 CAZymes required for degrading cellulose-containing substrates as well as an "accessory" set of 76 CAZymes required for specific non-cellulose substrates. Gene co-expression analysis suggested that Carbon Catabolite Repression (CCR) related regulators sense intracellular glycolytic intermediates and control the core CAZymes that mainly include cellulosomal components, whereas 11 sets of Two-Component Systems (TCSs) respond to availability of extracellular soluble sugars and respectively regulate most of the accessory CAZymes and associated transporters. Surprisingly, under glucose alone, the core cellulases were highly expressed at both transcript and protein levels. Furthermore, glucose enhanced cellulolysis in a dose-dependent manner, via inducing cellulase transcription at low concentrations. Conclusion: A molecular model of cellulose degradome in C. cellulolyticum (Ccel) was proposed, which revealed the substrate-specificity of CAZymes and the transcriptional regulation of core cellulases by CCR where the glucose acts as a CCR inhibitor instead of a trigger. These features represent a distinct environment-sensing strategy for competing while collaborating for cellulose utilization, which can be exploited for process and genetic engineering of microbial celluloly

Background: Ruminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in the rumen of herbivores. Bioinformatic analysis of the recently sequenced genome indicated that this bacterium produces one of the most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining of the genome sequence. It is composed of two tandem modules of currently undefined function that share 45% identity and a C-terminal X-dockerin modular dyad. Gaining insight into the diversity, architecture and organization of different types of proteins in the cellulosome system is essential for broadening our understanding of a multi-enzyme complex, considered to be one of the most efficient systems for plant cell wall polysaccharide degradation in nature. Methodology/Principal Findings: Following bioinformatic analysis, the second tandem module of RflaF_05439 was cloned and its selenium-labeled derivative was expressed and crystallized. The crystals belong to space group P21 with unit-cell parameters of alpha = 65.81, b = 60.61, c = 66.13 angstrom, beta = 107.66 degrees and contain two protein molecules in the asymmetric unit. The crystal structure was determined at 1.38-angstrom resolution by X-ray diffraction using the single-wavelength anomalous dispersion (SAD) method and was refined to R-factor and R-free of 0.127 and 0.152 respectively. The protein molecule mainly comprises a beta-sheet flanked by short alpha-helixes, and a globular a-helical domain. The structure was found to be structurally similar to members of the NlpC/P60 superfamily of cysteine peptidases. Conclusions/Significance: The 3D structure of the second repeat of the RflaF_05439 enabled us to propose a role for the currently undefined function of this protein. Its putative function as a cysteine peptidase is inferred from in silico structural homology studies. It is therefore apparent that cellulosomes integrate proteins

The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring proteinprotein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (Delta Delta G) and dissociation constants (K-d) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesindockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious proteinprotein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions. Copyright (c) 2012 John Wiley & Sons, Ltd.

Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides. IMPORTANCE Cellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on t

In Ruminococcus flavefaciens, a predominant fibre-degrading bacterium found in ruminants, cellulosomal proteins are anchored to the bacterial cell wall through a relatively small ScaE scaffoldin which includes a single type III cohesin. The cotton-binding protein CttA consists of two cellulose-binding modules and a C-terminal modular pair (XDoc) comprising an X-module and a contiguous dockerin, which exhibits high affinity towards the ScaE cohesin. Seleno-l-methionine-labelled derivatives of the ScaE cohesin module and the XDoc from CttA have been expressed, copurified and cocrystallized. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 78.7, c = 203.4 angstrom, and the unit cell contains a single cohesinXDoc complex in the asymmetric unit. The diffraction data were phased to 2.0 angstrom resolution using the anomalous signal of the Se atoms.

The crystal structure of the family 3b carbohydrate-binding module (CBM3b) of the cellulosomal multimodular hydrolytic enzyme cellobiohydrolase 9A (Cbh9A) from Clostridium thermocellum has been determined. Cbh9A CBM3b crystallized in space group P4(1) with four molecules in the asymmetric unit and diffracted to a resolution of 2.20 angstrom using synchrotron radiation. The structure was determined by molecular replacement using C. thermocellum Cel9V CBM3b' (PDB entry 2wnx) as a model. The C. thermocellum Cbh9A CBM3b molecule forms a nine-stranded antiparallel beta-sandwich similar to other family 3 carbohydrate-binding modules (CBMs). It has a short planar array of two aromatic residues that are assumed to bind cellulose, yet it lacks the ability to bind cellulose. The molecule contains a shallow groove of unknown function that characterizes other family 3 CBMs with high sequence homology. In addition, it contains a calcium-binding site formed by a group of amino-acid residues that are highly conserved in similar structures. After determination of the three-dimensional structure of Cbh9A CBM3b, the site-specific N126W mutant was produced with the intention of enhancing the cellulose-binding ability of the CBM. Cbh9A CBM3b(N126W) crystallized in space group P4(1)2(1)2, with one molecule in the asymmetric unit. The crystals diffracted to 1.04 angstrom resolution using synchrotron radiation. The structure of Cbh9A CBM3b(N126W) revealed incorporation of the mutated Trp126 into the putative cellulose-binding strip of residues. Cellulose-binding experiments demonstrated the ability of Cbh9A CBM3b(N126W) to bind cellulose owing to the mutation. This is the first report of the engineered conversion of a non-cellulose-binding CBM3 to a binding CBM3 by site-directed mutagenesis. The three-dimensional structure of Cbh9A CBM3b(N126W) provided a structural correlation with cellulose-binding ability, revealing a longer planar array of definitive cellulose-binding residues.

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme beta-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of beta-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused beta-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The beta-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2: 997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85 degrees C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.

beta-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 beta-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 degrees C and 5.5-6, respectively, were determined. The apparent K-m value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k(cat)) was 6.72 s(-1). T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.

The specificity of cohesin dockerin interactions is critically important for the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome). In order to investigate the origins of the observed specificity, a variety of selected amino acid positions at the cohesin dockerin interface can be subjected to mutagenesis, and a library of mutants can be constructed. In this chapter, we describe a protein protein microarray technique based on the high affinity of a carbohydrate-binding module (CBM), attached to mutant cohesins. Using cellulose-coated glass slides, libraries of mutants can be screened for binding to complementary partners. The advantages of this tool are that crude cell lysate can be used without additional purification, and the microarray can be used for screening both large libraries as initial scanning for "positive" plates, and for small libraries, wherein individual colonies are printed on the slide. Since the time-consuming step of purifying proteins can be circumvented, the approach is also appropriate for providing molecular insight into the multicomponent organization of complex cellulosomes.

The carbohydrate-binding module (CBM) of the major scaffoldin subunit ScaA of the cellulosome of Acetivibrio cellulolyticus is classified as a family 3b CBM and binds strongly to cellulose. The CBM3b was overexpressed, purified and crystallized, and its three-dimensional structure was determined. The structure contained a nickel-binding site located at the N-terminal region in addition to a 'classical' CBM3b calcium-binding site. The structure was also determined independently by the SAD method using data collected at the Ni-absorption wavelength of 1.48395 angstrom and even at a wavelength of 0.97625 angstrom in a favourable case. The new scaffoldin-borne CBM3 structure reported here provides clear evidence for the proposition that a family 3b CBM may be accommodated in scaffoldin subunits and functions as the major substrate-binding entity of the cellulosome assembly.

Background: The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA. Principal Findings: As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located. Conclusions: The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens str

The potent cellulose-binding modules of cellulosomal scaffoldin subunits belong to the greater family of carbohydrate-binding modules (CBMs). They have generally been classified as belonging to family 3a on the basis of sequence similarity. They form nine-stranded beta-sandwich structures with jelly-roll topology. The members of this family possess on their surface a planar array of aromatic amino-acid residues (known as the linear strip) that form stacking interactions with the glucose rings of cellulose chains and have a conserved Ca2+-binding site. Intriguingly, the CBM3 from scaffoldin A (ScaA) of Bacteroides cellulosolvens exhibits alterations in sequence that make it more similar to the CBMs of free cellulolytic enzymes, which are classified into CBM family 3b. X-ray structural analysis was undertaken in order to examine the structural consequences of the sequence changes and the consequent family affiliation. The CBM3 crystallized in space group I4(1)22 with one molecule in the asymmetric unit, yielding diffraction to a resolution of 1.83 A using X-ray synchrotron radiation. Compared with the known structures of other scaffoldin-borne CBMs, a sequence insertion and deletion appear to compensate for each other as both contained an aromatic residue that is capable of contributing to cellulose binding; hence, even though there are alterations in the composition and localization of the aromatic residues in the linear strip its binding ability was not compromised. Interestingly, no Ca2+ ions were detected in the conserved calcium-binding site, although the module was properly folded; this suggests that the structural role of Ca2+ is less important than originally supposed. These observations indicate that despite their conserved function the scaffoldin-borne CBMs are more diverse in their sequences and structures than previously assumed.

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.

Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fusca xylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fusca enzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (similar to 2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase-and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an similar to 2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome. IMPORTANCE Global efforts towards alternative energy programs are highlighted by processes for converting plant-derived carbohydrates to biofuels. The major barrier in such pro

Background: The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown. Methodology/Principal Findings: We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca2+-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e. g., cohesin-containing proteins, belonging to the scaffoldins. Conclusions/Significance: This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the p

Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative Sigma factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-Sigma I factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of > 1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-Sigma factors form predicted bicistronic operons, in which the first gene encodes a putative alternative Sigma factor, similar to B. subtilis Sigma I, but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and Sigma I-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.

The cellulosome complex is composed of a conglomerate of subunits, each of which comprises a set of interacting functional modules. Scaffoldin (Sca), a major cellulosomal subunit, is responsible for organizing the cellulolytic subunits into the complex. This is accomplished by the interaction of two complementary classes of modules-a cohesin (Coh) module on the Sca subunit and a dockerin module on each of the enzymatic subunits. Although individual Coh modules from different cellulosomal scaffoldins have been subjected to intensive structural investigation, the Sca subunit in its entirety has not, and there remains a paucity of information on the arrangement and interactions of Cohs within the Sca subunit. In the present work, we describe the crystal structure of a type II Coh dyad from the ScaB "adaptor" Sca of Acetivibrio cellulolyticus. The ScaB Cohs are oriented in an "antiparallel" manner relative to one another, with their dockerin-interacting surfaces (beta-strands 8-3-6-5) facing the same direction-aligned on the same plane. A set of extensive hydrophobic and hydrogen-bond contacts between the Cohs and the short interconnecting linker segment between them stabilizes the modular orientation. This Coh dyad structure provides novel information about Coh-Coh association and arrangement in the Sca and further insight into intermodular linker interactions. Putative structural arrangements of a hexamodular complex, composed of the Coh dyad bound to two X-dockerin modules, were suggested. (C) 2010 Published by Elsevier Ltd.

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

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 angstrom. 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.

The cellulosome is an intriguing multienzyme complex found in cellulolytic bacteria that plays a key role in the degradation of plant cell-wall polysaccharides. In Ruminococcus flavefaciens, a predominant fiber-degrading bacterium found in ruminants, the cellulosome is anchored to the bacterial cell wall through a relatively short ScaE scaffoldin. Determination of the crystal structure of the lone type-III ScaE cohesin from R. flavefaciens (Rf-CohE) was initiated as a part of a structural effort to define cellulosome assembly. The structure was determined at 1.95 angstrom resolution by single-wavelength anomalous diffraction. This is the first detailed description of a crystal structure for a type-III cohesin, and its features were compared with those of the known type-I and type-II cohesin structures. The Rf-CohE module folds into a nine-stranded beta-sandwich with jellyroll topology, typically observed for cohesins, and includes two beta-flaps in the midst of beta-strands 4 and 8, similar to the type-II cohesin structures. However, the presence in Rf-CohE of an additional 13-residue alpha-helix located between beta-strands 8 and 9 represents a dramatic divergence from other known cohesin structures. The prominent alpha-helix is enveloped by an extensive N-terminal loop, not observed in any other known cohesin, which embraces the helix presumably enhancing its stability. A planar surface at the upper portion of the front face of the molecule, bordered by beta-flap 8, exhibits plausible dimensions and exposed amino acid residues to accommodate the dockerin-binding site. Proteins 2009; 77:699-709. (C) 2009 Wiley-Liss, Inc.

Background: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genom

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the mufti-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. Copyright (c) 2008 John Wiley & Sons, Ltd.

The high-affinity cohesin-dockerin interaction was originally discovered as modular components, which mediate the assembly of the various subunits of the multienzyme cellulosome complex that characterizes some cellulolytic bacteria. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive signature of a cellulosome-producing bacterium. Widespread genome sequencing has since revealed a wealth of putative cohesin- and dockerin-containing proteins in Bacteria, Archaea, and in primitive eukaryotes. The newly identified modules appear to serve diverse functions that are clearly distinct from the classical cellulosome archetype, and the vast majority of parent proteins are not predicted glycoside hydrolases. In most cases, only a few such genes have been identified in a given microorganism, which encode proteins containing but a single cohesin and/or dockerin. In some cases, one or the other module appears to be missing from a given species, and in other cases both modules occur within the same protein. This review provides a bioinformatics-based survey of the current status of cohesin- and dockerin-like sequences in species from the Bacteria, Archaea, and Eukarya. Surprisingly, many identified modules and their parent proteins are clearly unrelated to cellulosomes. The cellulosome paradigm may thus be the exception rather than the rule for bacterial, archaeal, and eukaryotic employment of cohesin and dockerin modules.

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the mu-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the mu-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the mu-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens. (c) 2008 Elsevier Ltd. All rights reserved.

Ruminococcus flavefaciens is a vital cellulosome-producing fibrolytic rumen bacterium. The arrangement of the cellulosomal scaffoldin gene cluster (scaC-scaA-scaB-cttA-scaE) is conserved in two R. flavefaciens strains (17 and FD-1). Sequence analysis revealed a high mosaic conservation of the intergenic regions in the two strains that contrasted sharply with the divergence of the structural sca gene sequences. Based on the conserved intergenic regions, we designed PCR primers in order to examine the sca gene cluster in additional R. flavefaciens strains (C94, B34b, C1a and JM1). Using these conserved and/or degenerate primers, the scaC, scaA and scaB genes were amplified in all six strains, while the entire sca gene cluster and the proximal genes cttA and scaE were successfully amplified in four of the strains (17, FD-1, C94 and JM1). The sequencing of scaA and scaC genes in all the strains yielded additional insight into the variability of the structural genes with regard to the number and type of cohesin modules contained in a conserved molecular skeleton. Moreover, the scaC gene, being short and variable, appears to be a promising functional phylotyping target for metagenomic population studies of R. flavefaciens in the rumen as a function of the individual host animal.

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin-dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose-binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM-fused cohesins were immobilized on cellulose-coated glass slides, to which xylanase-fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin-dockein pairs for biotechnological. and nanotechnological. application. Using this approach, extensive cross-species interaction among type-II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.

Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-L-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belong to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 angstrom, beta = 123.4 degrees, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 angstrom using the anomalous signal from the Se atoms.

Cellulosomes are intricate multienzyme systems produced by several cellulolytic bacteria, the first example of which was discovered in the anaerobic thermophilic bacterium, Clostridium thermocellum. Cellulosomes are designed for efficient degradation of plant cell wall polysaccharides, notably cellulose-the most abundant renewable polymer on earth. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture. A full generation of research has elapsed since the original publications that documented the cellulosome concept. In this review, we provide a personal account on the discovery process, while describing how divergent cellulosome systems were identified and investigated, culminating in the collaboration of several labs worldwide to tackle together the challenging field of cellulosome genomics and metagenomics. (c) 2008 The Japan Chemical journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 364-377; 2008: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20160

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.

Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sea cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein (similar to 130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.

Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities - the landfills - are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence inicroscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudosubstrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction. (c) 2006 Elsevier Ltd. All rights reserved.

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.

Sequence extension of the scaffoldin gene cluster from Ruminococcus flavefaciens revealed a new gene (scaE) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in Escherichia coli and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulose-bound Ruminococcus flavefaciens protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in R. flavefaciens differs from those reported thus far for any other cellulosome system.

The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein ( AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin, which account for the observed differences in binding affinities. The increased hyperthermostability can be attributed to the conformation of the critical L3,4 loop and the extensive network of 1 - 3 inter-monomeric interactions. The loop contains a tandem Pro-Gly sequence and an Asp-Arg ion pair that collectively induce rigidity, thus maintaining its closed and ordered conformation in both the apo and biotin-complexed forms. In addition, Tyr115 is present on the AVR4 1 - 3 monomer - monomer interface, which is absent in avidin and streptavidin. The interface tyrosine generates intermonomeric interactions, i.e. a tyrosine - tyrosine pi-pi interaction and a hydrogen bond with Lys92. The resultant network of interactions confers a larger 1 - 3 dimer - dimer contact surface on AVR4, which correlates nicely with its higher thermostability compared with avidin and streptavidin. Several of the proposed thermostability-determining factors were found to play a role in strengthening the tertiary and quaternary integrity of AVR4.

In recent work (Fierobe, H.- P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belai " ch, A., Lamed, R., Shoham, Y., and Belaich, J.- P. ( 2002) J. Biol. Chem. 277, 49621 49630), 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.

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 similar to 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.

The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction. In previous work [J. Biol. Chem. 276 (2001) 9883], we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and high-affinity recognition by the interspecies rival cohesin from C. cellulolyticum. In this report, we show that a single Asp-to-Asn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin. The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogen-bonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain. The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein-protein interaction. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

We recently showed that some of the enzymes underpinning cellulose solubilization by Rundnacoccus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an "X" domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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 (Ce148), 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 Ce148, 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 Ce148 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.

Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h(-1) and 2 to 10 transcripts per cell at a growth rate of 0.35 h(-1) in batch culture. The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at -81 and -50 bp, upstream of the translational start site of the cipA gene. The potential promoters exhibited homology to the known sigma factors sigma(A) and sigma(L) (sigma(54)) of Bacillus subtilis. Transcription from the sigma(L)-Iike promoter was found under all growth conditions, whereas transcription from the sigma(A)-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.

Introduction of enzymatic activity into proteins or other types of polymers by rational design is a major objective in the life sciences. To date, relatively low levels of enzymatic activity could be introduced into antibodies by using transition-state analogues of haptens. In the present study, we identify the structural elements that contribute to the observed hydrolytic activity in egg white avidin, which promote the cleavage of active biotin esters (notably biotinyl p-nitrophenyl ester). The latter elements were then incorporated into bacterial streptavidin via genetic engineering. The streptavidin molecule was thus converted from a protector to an enhancer of hydrolysis of biotin esters. The conversion was accomplished by the combined replacement of a "lid-like loop" (L3,4) and a leucine-to-arginine point mutation in streptavidin. Interestingly, neither of these elements play a direct role in the hydrolytic reaction. The latter features were thus shown to be responsible for enhanced substrate hydrolysis. This work indicates that structural and non-catalytic elements of a protein can be modified to promote the induced fit of a substrate for subsequent interaction with either a catalytic residue or water molecules. This approach complements the conventional design of active sites that involves direct modifications of catalytic residues.

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1-->2 interface was mutated to lysine and asparagine 54 in the 1-->4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K-d similar to10(-15) M of the wild-type tetramer to K-d similar to10(-7) M, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of inter-subunit interactions on the binding of ligands.

Aims: To compare the subcellular distribution of glycanase-related components between wildtype Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. Methods and Results: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. Conclusions: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. Significance and Impact of the Study: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.

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 leach 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.

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine, The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface, Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.

A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens, The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437, CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins, In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II, This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component, The results attest to the emerging diversity of cellulosomes and their component sequences in nature.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex, In a previous article (Pages et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the similar to 70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction.

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin, The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system, A mutant of streptavidin, Trp-120-Lys, was similarly expressed, The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K-d similar to 10(-15) M of the wild-type tetramer down to K-d similar to 10(-8) M for both W --> K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin, The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W --> K mutants were found to form stable dimers in solution, These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization, (C) 1999 Federation of European Biochemical Societies.

The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.

Ligands containing amino or hydroxyl groups were converted to their corresponding activated N-hydroxysuccinimidyl carbamate and carbonate by reaction with disuccinimidyl carbonate (DSC). The latter reagents can be used for the group-specific modification of primary amines as an alternative to the widespread usage of N-hydroxysuccinimide esters. Biotin and 2,4-dinitrophenyl (DNP) derivatives were used as examples to demonstrate the approach. Biotin and DNP were each extended by attaching two different spacer arms, carrying either a hydroxyl group or a primary amine as terminal functions. The latter were then activated via their conversion to N-hydroxysuccinimide carbonates and carbamates, respectively. The usefulness of these reagents for protein modification was investigated. The modified proteins obtained exhibited similar stability and activity characteristics compared to those modified with active N-hydroxysuccinimdyl esters. The activation of hydroxy- or amino-terminating compounds with DSC represents a general method that can be applied to any ligand which contains these functional groups for its covalent coupling to amines. (C) 1999 Elsevier Science B.V. All rights reserved.

Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly, The high pI of avidin (similar to 10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wild-type avidin, Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotin-binding activity. (C) 1998 Federation of European Biochemical Societies.

The structural complexity and rigidity of cellulosic substrates have given rise to a phenomenal diversity of degradative enzymes - the cellulases. Cellulolytic microorganisms produce a wide variety of different catalytic and noncatalytic enzyme modules, which form the cellulases and act synergistically on their substrate. In some microbes, several types of cellulases are organized into an elaborate multifunctional supramolecular complex, known as the cellulosome. A combination of molecular genetic, biochemical, chemical, crystallographic and microscopic techniques are paving the way for new insights into both the structure of cellulose and the mechanisms of its hydrolysis.

Background: The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome. Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex, The cohesin domains are highly conserved in their primary amino acid sequences. These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit. The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome, The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity. Results: We have determined the three-dimensional crystal structure of one of the cohesin domains from C. thermocellum (cohesin 2) at 2.15 Angstrom resolution, The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain. Conclusions: The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues. As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity, Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains.

Chemically modified forms of egg-white avidin and bacterial streptavidin (termed nitro-avidin and nitro-streptavidin, respectively), in which the binding-site tyrosine was nitrated, were used for several biotechnological applications. The fundamental difference between nitro-avidin and the native protein is that interaction of the modified protein with biotin can be reversed under relatively mild conditions. Consequently, nitro-avidin affinity columns or immobilizing matrices can be reused. Three examples are given to demonstrate the possible uses of such columns: (a) biotinylated protein A was attached to a nitro-avidin affinity column, and immunoglobulin was purified directly from whole rabbit serum; (b) biotinylated transferrin was attached to a nitro-streptavidin column, and anti-transferrin was isolated directly from rabbit anti-serum; and (c) biotinylated beta-glucosidase was immobilized onto a nitro-avidin column and used as an enzyme reactor. In each example, the immobilized biotinylated probe could be released selectively from the column and recovered following its utilization. Reusable nitro-avidin thus provides an easy and attractive reversible form of avidin and thereby serves to expand the versatility of avidin-biotin technology. (C) 1996 Academic Press, Inc.

The cellulosome from the anaerobic, thermophilic, cellulolytic bacterium, Clostridium thermocellum, was dissociated under nondenaturing conditions by incubation at 60 degrees C in the combined presence of EDTA and cellulose. This approach differs from previous strategies in that detergents were not necessary for dissociation of the subunits. Selected enzymatic subunits could be isolated with relative ease. One of these is the major cellulosomal cellobiohydrolase, CelS (previously designated subunit S8 or S-s). Both the intact cellulosome complex and the combined dissociated subunits exhibited similar levels of activities on soluble and acid-swollen cellulosic substrates. In contrast, the mixture of the free enzymatic subunits was markedly deficient in its degradation of crystalline cellulose compared with that of the intact cellulosome. The results support the critical role of divalent cations. e.g. calcium, in the stability of the cellulosome complex. In addition, the results reinforce previous indications that the cellulosome undergoes conformational changes upon binding to cellulose. Copyright (C) 1996 Elsevier Science B.V.

Avidin, a positively charged egg-white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS-PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS-PAGE.

A simple procedure for the preparation of deglycosylated avidin is described. Commercially obtained avidin was treated with a mixed microbial culture. The cells were capable of growing on the oligosaccharide residues, but generally ignored the polypeptide portion of the egg white glycoprotein. The resultant deglycosylated avidin retained its biotin-binding characteristics. The major bacterial strain (strain BECH080), responsible for the deglycosylation, was isolated. On the basis of elementary biochemical tests, fatty acid, and phenotypic analyses, the isolate was identified as a strain of Flavobacterium meningosepticum. The primary enzymatic activity that caused the removal of the oligosaccharide residues of avidin appeared to be similar to endoglycosidase F.

The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non-catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose-binding domain (CBD) and nine subunit-binding domains, or cohesin domains, Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits, Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly,

The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component S(s) (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

The crystal structure of the complex formed between the egg-white biotin-binding protein, avidin, and the dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), was determined to a resolution of 2.5 angstrom. The interaction of avidin with the benzoate ring of HABA is essentially identical to that of the complex formed between HABA and streptavidin (the bacterial analogue of the egg-white protein). This interaction emulates the definitive high-affinity interaction of both proteins with the ureido moiety of biotin. The major difference between the avidin- and streptavidin-HABA complexes lies in their interaction with the hydroxypheny) ring of the dye molecule; in avidin, two adjacent amino acid residues (Phe72 and Ser73), which are not present in streptavidin, form additional interactions with this ring. These are suggested to account for the higher affinity of avidin for HABA. The characteristic red shift, which accompanies the interaction of both proteins with the dye, was traced to a proposed charge-transfer complex formed between the hydroxyphenyl ring of HABA and the indole ring of Trp79 in avidin (Trp79 in streptavidin). Comparison of binding site residues of two such similar proteins versus their markedly different affinities for two such different substrates should eventually contribute to a better design of biomimetic reagents and drugs.

The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 angstrom, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.

Streptavidin is a biotin-binding analogue of egg-white avidin which is secreted by the bacterium Streptomyces avidinii. We have recently reported that streptavidin contains an Arg-Tyr-Asp-Ser (RYDS) sequence which exhibits structural homology to the Arg-Gly-Asp-Ser (RGDS) cell adhesion domain of fibronectin and other matrix-associated glycoproteins. Competition studies with RGD peptides indicated that streptavidin binds to cells via this site and that the binding is independent of biotin recognition. Since the RGD-containing peptide has been shown to play a key role in integrin-mediated cell adhesion., we assumed that streptavidin may utilize the RYDS site to bind to immune cells and thereby abrogate their adhesion-dependent functions. We now report that streptavidin modulates several matrix-dependent interactions of immune cells. In this context, immobilized streptavidin was found to support activated human CD4+ T cell adhesion in an RGD-specific, alpha5beta1-dependent manner. In addition, soluble streptavidin (the commercially available or biotin-blocked forms) inhibited T cell adhesion to fibronectin and interfered with its co-stimulatory effect on tumor necrosis factor-alpha secretion by co-cultures of CD4+ T cells and macrophages. These results suggest that streptavidin is a novel example of a bacterial protein which utilizes RGD mimicry to interfere with integrin-mediated immune responses.

The interaction of streptavidin with various cell systems was studied using fluorescent derivatives of the protein. The native unprocessed form of streptavidin bound to cells at low levels and in a nonspecific manner. In contrast, both the truncated "core" streptavidin (the commercially available form) and the biotin-blocked unprocessed protein bound to cells in enhanced levels and in a specific, saturable manner. This suggests that the binding of biotin or cleavage of the terminal portion(s) of the native protein molecule causes conformational changes which lead to the exposure of sites which presumably interact with cell surface receptors. Peptide inhibition studies demonstrated that the majority of binding to cells appears to be dependent on RGD-like specificity, suggesting that the GRYDS sequence of the streptavidin molecule may exhibit such specificity. Indirect immunofluorescence assays revealed that the protein is associated mainly with the cell surface. Moreover, streptavidin was demonstrated to compete with specific monoclonal antibodies to the RGD-binding site on the GpIIbIIIa integrin of activated platelets, thus suggesting that streptavidin may facilitate binding to ubiquitous cell-surface adhesion receptors via RGD mimicry.

The affinity properties displayed by the cellulosome, the multienzyme complex from Clostridium thermocellum, for its insoluble polymeric substrate, cellulose, have previously been employed for its purification by affinity chromatography. In the present communication, a new purification procedure is described which provides five-fold higher yields (> 90%) of the cellulosome with enhanced solubilizing activity (approximately 1.5-fold). The new method is based on the efficient adsorption of the cellulosome onto phosphoric acid-treated substrate (amorphous cellulose) and the subsequent digestion of the carrier/substrate by the adsorbed enzyme complex. The term "affinity digestion" is proposed for such systems in which the affinity matrix is degraded totally by the adsorbed enzyme(s), thus facilitating its recovery.