Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome.

Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome.

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.IMPORTANCE The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of Lactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Lysozyme activity of the Ruminococcus champanellensis cellulosome.

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°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.

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine-tuned system for cohesin-dockerin recognition.

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 ß-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.

Insights into the functionality and stability of designer cellulosomes at elevated temperatures.

Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 °C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 °C. Finally, within this time frame and conditions, the full complex appeared over 50 % more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

Ruminococcal cellulosome systems from rumen to human.

A cellulolytic fiber-degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R. champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin-coding genes that form part of a gene cluster. Representative R. champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin-dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell-bound system and (ii) a simplistic cell-free system composed of a single cohesin-containing scaffoldin. The cell-bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R. champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R. champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.

Cryptic diversity of cellulose-degrading gut bacteria in industrialized humans.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions.

Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.

Carbohydrate Depolymerization by Intricate Cellulosomal Systems.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

Nanoscale resolution of microbial fiber degradation in action.

The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical Clostridium thermocellum system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form 'cellulosome capsules' driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.

Contribution of a xylan-binding module to the degradation of a complex cellulosic substrate by designer cellulosomes.

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and approximately 1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level.

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 bacterium Lactobacillus 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 of L. 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.

Impact of scaffoldin mechanostability on cellulosomal activity.

Lignocellulose is the most abundant renewable carbon source in the biosphere. However, the main bottleneck in its conversion to produce second generation biofuels is the saccharification step: the hydrolysis of lignocellulosic material into soluble fermentable sugars. Some anaerobic bacteria have developed an extracellular multi-enzyme complex called the cellulosome that efficiently degrades cellulosic substrates. Cellulosome complexes rely on enzyme-integrating scaffoldins that are large non-catalytic scaffolding proteins comprising several cohesin modules and additional functional modules that mediate the anchoring of the complex to the cell surface and the specific binding to its cellulosic substrate. It was proposed that mechanical forces may affect the cohesins positioned between the cell- and cellulose-anchoring points in the so-called connecting region. Consequently, the mechanical resistance of cohesins within the scaffoldin is of great importance, both to understand cellulosome function and as a parameter of industrial interest, to better mimic natural complexes through the use of the established designer cellulosome technology. Here we study how the mechanical stability of cohesins in a scaffoldin affects the enzymatic activity of a cellulosome. We found that when a cohesin of low mechanical stability is positioned in the connecting region of a scaffoldin, the activity of the resulting cellulosome is reduced as opposed to a cohesin of higher mechanical stability. This observation directly relates mechanical stability of the scaffoldin-borne cohesins to cellulosome activity and provides a rationale for the design of artificial cellulosomes for industrial applications, by incorporating mechanical stability as a new industrial parameter in the biotechnology toolbox.

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis.

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

Methods for Discovery of Novel Cellulosomal Cellulases Using Genomics and Biochemical Tools.

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.

Colocalization and Disposition of Cellulosomes in Clostridium clariflavum as Revealed by Correlative Superresolution Imaging.

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) superresolution 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.

Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides.

Cellulosomes are multienzyme complexes that are produced by anaerobic cellulolytic bacteria for the degradation of lignocellulosic biomass. They comprise a complex of scaffoldin, which is the structural subunit, and various enzymatic subunits. The intersubunit interactions in these multienzyme complexes are mediated by cohesin and dockerin modules. Cellulosome-producing bacteria have been isolated from a large variety of environments, which reflects their prevalence and the importance of this microbial enzymatic strategy. In a given species, cellulosomes exhibit intrinsic heterogeneity, and between species there is a broad diversity in the composition and configuration of cellulosomes. With the development of modern technologies, such as genomics and proteomics, the full protein content of cellulosomes and their expression levels can now be assessed and the regulatory mechanisms identified. Owing to their highly efficient organization and hydrolytic activity, cellulosomes hold immense potential for application in the degradation of biomass and are the focus of much effort to engineer an ideal microorganism for the conversion of lignocellulose to valuable products, such as biofuels.

Modular Organization of the Thermobifida fusca Exoglucanase Cel6B Impacts Cellulose Hydrolysis and Designer Cellulosome Efficiency.

Cellulose deconstruction can be achieved by three distinct enzymatic paradigms: free enzymes, multifunctional enzymes, and self-assembled, multi-enzyme complexes (cellulosomes). To study their comparative efficiency, the simple and efficient cellulolytic system of the aerobic bacterium, Thermobifida fusca, is developed as an enzymatic model. In previous studies, most of its cellulases are successfully converted to the cellulosomal mode and exhibited high cellulolytic activities, except for Cel6B, a key exoglucanase of the T. fusca enzymatic system. Here, the impact of the modular organization of Cel6B on enzymatic activity is investigated. The position of the cellulose-binding module (CBM), its family and linker segment are shown to affect activity. Surprisingly, exchange of the native family-2 CBM to family-3 generates an increase in Cel6B activity on cellulosic substrates. Conversion of Cel6B to the cellulosomal mode by fusing a cohesin to the catalytic module enables formation of divalent enzyme complexes with dockerin-bearing enzymes. The resultant pseudo-cellulosomes, containing Cel6B combined with endoglucanase Cel5A, exhibits enhanced enzymatic activity, compared to mixtures of wild-type enzymes or bifunctional enzymes, unlike similar pseudo-cellulosomes containing endoglucanase Cel6A or proccessive endoglucanase Cel9A. Insight into the different enzymatic paradigms benefits ongoing development of efficient cellulolytic systems for conversion of plant-derived biomass into valuable sugars. NOVELTY STATEMENT: The protein engineering of the modular arrangement of a key exoglucanase from a highly cellulolytic bacterium, Thermobifida fusca, served to explore and compare three major enzymatic paradigms for cellulose degradation. This approach revealed highly active chimaeric forms of the exoglucanase that act in synergy together with a potent endoglucanase in bifunctional enzymes or divalent pseudo-cellulosome-like complexes. Such engineered enzymes could be further integrated into larger enzymatic complexes, thereby providing a significant step forward towards conversion of the entire T. fusca free cellulolytic system into the cellulosomal modex and the enhanced conversion of cellulosic biomass into soluble sugars.

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions.

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature.

UNLABELLED: 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.