Expression and characterization of spore coat CotH kinases from the cellulosomes of anaerobic fungi (Neocallimastigomycetes).

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.

Self-assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation.

The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization.

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule fluorescence resonance energy transfer and molecular simulations on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules.

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° 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.

A modular approach for dCas9-mediated enzyme cascading via orthogonal bioconjugation.

We report a new modular strategy to assemble dCas9-guided enzyme cascades by employing orthogonal post-translation chemistry. Two orthogonal SpyCatcher and SnoopCatcher pairs were used for the one-pot enzyme bioconjugation onto two different dCas9 proteins to enable their guided assembly onto a DNA scaffold. The resulting two-component cellulosomes exhibited 2.8-fold higher reducing sugar production over unassembled enzymes. This platform retains the high binding affinity afforded by dCas9 proteins for easy control over enzyme assembly while offering the flexibility for both in vivo and in vitro assembly of a wide array of enzyme cascades with minimal optimization.

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.

Cell-surface exposure of a hybrid 3-cohesin scaffoldin allowing the functionalization of Escherichia coli envelope.

Cellulosomes are large plant cell wall degrading complexes secreted by some anaerobic bacteria. They are typically composed of a major scaffolding protein containing multiple receptors called cohesins, which tightly anchor a small complementary module termed dockerin harbored by the cellulosomal enzymes. In the present study, we have successfully cell surface exposed in Escherichia coli a hybrid scaffoldin, Scaf6, fused to the curli protein CsgA, the latter is known to polymerize at the surface of E. coli to form extracellular fibers under stressful environmental conditions. The C-terminal part of the chimera encompasses the hybrid scaffoldin composed of three cohesins from different bacterial origins and a carbohydrate-binding module targeting insoluble cellulose. Using three cellulases hosting the complementary dockerin modules and labeled with different fluorophores, we have shown that the hybrid scaffoldin merged to CsgA is massively exposed at the cell surface of E. coli and that each cohesin module is fully operational. Altogether these data open a new route for a series of biotechnological applications exploiting the cell-surface exposure of CsgA-Scaf6 in various industrial sectors such as vaccines, biocatalysts or bioremediation, simply by grafting the small dockerin module to the desired proteins before incubation with the engineered E. coli.

Isolation and subunit structure of the xylanosome complex produced by Actinotalea fermentans JCM9966.

Xylanosomes, also known as hemicellulosomes, are hemicellulose-degrading nano-scale multienzyme complexes produced by some Firmicutes, Actinobacteria, and Fungi. Here we report the isolation of the MECs produced by Actinotalea fermentas JCM9966, as well as the functional studies and subunit structure revealed by proteomic identifications. The isolated MECs here shows similar particle size with the xylanosomes produced by C. cellulans F16, have several conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions, indicating diverse capability as well as bias in degrading hemicelluloses.

Reconstitution of cellulosome: Research progress and its application in biorefinery.

Lignocellulose, one of the most abundant renewable sources of sugar, can be converted into bioenergy through hydrolysis of cellulose and hemicellulose. Due to its renewability and availability in large quantities, bioenergy is considered as a possible alternative to fossil energy and attracts the attention of the world with increased concerns about environmental protection and energy crisis. The depolymerization of cellulosic substrate to monomer is the rate-limiting step in the bioconversion of lignocellulose by cellulolytic microbes. Cellulosome, a multienzyme complex from anaerobic cellulolytic bacteria, can efficiently degrade the cellulosic substrates. Previous studies have shown that the reconstitution of cellulosome in vitro and its heterologous expression or display on the cell surface can help to solve the low yield problem of cellulosome in cellulolytic bacteria. This paper reviews the research progress in the reconstitution of cellulosome as well as its application in biorefinery, including the construction of cellulosome as well as different methods for cellulosome reconstitution and its surface display. This review will promote the understanding of cellulosome and its reconstitution.

Substrate-Related Factors Affecting Cellulosome-Induced Hydrolysis for Lignocellulose Valorization.

Cellulosomes are an extracellular supramolecular multienzyme complex that can efficiently degrade cellulose and hemicelluloses in plant cell walls. The structural and unique subunit arrangement of cellulosomes can promote its adhesion to the insoluble substrates, thus providing individual microbial cells with a direct competence in the utilization of cellulosic biomass. Significant progress has been achieved in revealing the structures and functions of cellulosomes, but a knowledge gap still exists in understanding the interaction between cellulosome and lignocellulosic substrate for those derived from biorefinery pretreatment of agricultural crops. The cellulosomic saccharification of lignocellulose is affected by various substrate-related physical and chemical factors, including native (untreated) wood lignin content, the extent of lignin and xylan removal by pretreatment, lignin structure, substrate size, and of course substrate pore surface area or substrate accessibility to cellulose. Herein, we summarize the cellulosome structure, substrate-related factors, and regulatory mechanisms in the host cells. We discuss the latest advances in specific strategies of cellulosome-induced hydrolysis, which can function in the reaction kinetics and the overall progress of biorefineries based on lignocellulosic feedstocks.

Exploiting dCas9 fusion proteins for dynamic assembly of synthetic metabolons.

Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σI ) factors that have cognate membrane-associated anti-σI factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σI4 -dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σI3 -dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14A ) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

Artificial scaffolds for enhanced biocatalysis.

Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to colocalize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.

Artificial Cellulosome Complex from the Self-Assembly of Ni-NTA-Functionalized Polymeric Micelles and Cellulases.

Polymer-protein core-shell nanoparticles have been explored for enzyme immobilization. This work reports on the development of functional polymeric micelles for immobilizing His6 -tagged cellulases with controlled spatial orientation of enzymes, resulting in "artificial cellulosomes" for effective cellulose hydrolysis. Poly(styrene)-b-poly(styrene-alt-maleic anhydride) was prepared through one-pot reversible addition-fragmentation chain-transfer polymerization and modified with nitrilotriacetic acid (NTA) to afford an amphiphilic block copolymer. The self-assembled polymer was mixed with a solution of NiSO4 to form Ni-NTA-functionalized micelles, which could successfully capture His6 -tagged cellulases and form hierarchically structured core-shell nanoparticles with cellulases as the corona. Because the anchored enzymes are site-specifically oriented and in close proximity, synergistic catalysis that results in over twofold activity enhancement has been achieved.

Resonance assignments of a cellulosomal double-dockerin from Clostridium thermocellum.

Cellulosomes are highly efficient multienzyme complexes for lignocellulose degradation secreted by some lignocellulolytic bacteria. Cellulosomes are assembled through protein modules named cohesin and dockerin, and multiple cohesin modules in the scaffold protein generally determine the complexity of the cellulosomes. Some cellulosomal proteins contain multiple dockerin modules, which may generate more complex cellulosomal architectures. Genome mining revealed that cellulosomal proteins containing double dockerin modules and a protease module exist in many cellulosome-producing bacteria, and these proteins together with cellulosomal protease inhibitors were proposed to have regulatory roles. However, the structures and functions of these multiple-dockerin proteins in cellulosome have not been reported before. In this paper, we present the NMR chemical shift assignments of the double-dockerin of a cellulosomal protease from Clostridium thermocellum DSM1313. The secondary structures predicted from the chemical shifts agree with the structural arrangement of the tandem dockerin modules. The chemical shift assignments here provide the basis for the structural and functional studies of multiple-dockerin proteins in future.

Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum.

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 all-atom 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 cohesin-cohesin 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.

The spatial proximity effect of beta-glucosidase and cellulosomes on cellulose degradation.

Low-cost saccharification is one of the key bottlenecks hampering the further application of lignocellulosic biomass. Clostridium thermocellum is a naturally ideal cellulose degrading bacterium armed with cellulosomes, which are multienzyme complexes that are capable of efficiently degrading cellulose. However, under controlled condition, the inhibition effect of hydrolysate cellobiose severely restricts the hydrolytic ability of cellulosomes. Although the addition of beta-glucosidase (Bgl) could effectively relieve this inhibition, the spatial proximity effect of Bgl and cellulosomes on cellulose degradation is still unclear. To address this issue, free Bgl from Caldicellulosiruptor sp. F32 (CaBglA), carbohydrate-binding module (CBM) fused CaBglA (CaBglA-CBM) and cellulosomal type II cohesin module (CohII) fused to CaBglA (CaBglA-CohII) were successfully constructed, and their enzymatic activities, binding abilities and saccharification efficiencies were systematically investigated in vitro and in vivo. In vivo, with the adjacency of CaBglA to cellulosomes, the saccharification efficiency of microcrystalline cellulose increased from 40% to 50%. For the pretreated wheat straw, the degradation rate of the combination of cells and the CaBglA-CohII or the CaBglA-CBM was as efficient as that of the free CaBglA (approximately 60%). This study demonstrated that the proximity of CaBglA to cellulosomes had a positive effect on microcrystalline cellulose but not on pretreated wheat straw, which may result from the nonproductive adsorption of lignin and the decreased thermostability of CaBglA-CBM and CaBglA-CohII compared to that of CaBglA. The above results will contribute to the design of cost-effective Bgls for industrial cellulose degradation.

Correlation Between Size and Activity Enhancement of Recombinantly Assembled Cellulosomes.

As multienzyme complexes, cellulosomes hydrolyze cellulosic biomass with high efficiency, which is believed to be attributed to either one or both factors: (1) synergy among the catalytic and substrate-binding entities and (2) the large size of cellulosome complexes. Although the former factor has been extensively documented, the correlation between size and specific activity of cellulosomes is still elusive to date. In this study, primary and secondary scaffoldins with 1, 3, or 5 copies of type I/II cohesin domains were recombinantly synthesized and various cellulosomes carrying 1, 3, 5, 9, 15, or 25 molecules of cellulase mixtures of family 5, 9, and 48 glycoside hydrolases were assembled. In addition, the assembled complex was annexed to cellulose with the aid of a family 3a carbohydrate-binding module (CBM3a). Measuring cellulolytic hydrolysis activities of assembled cellulosomes on crystalline Avicel revealed that higher degree of cellulosome complexity resulted in more efficient cellulose hydrolysis with plateaued synergic effects after the cellulosome size reaches certain degree.

Cellulosome assembly: paradigms are meant to be broken!

Cohesin-Dockerin interactions are at the core of cellulosomal assembly and organization. They are highly specific and form stable complexes, allowing cellulosomes to adopt distinct conformations. Each cellulosomal system seems to have a particular organizational strategy that can vary in complexity according to the nature of its Cohesin-Dockerin interactions. Hence, several efforts have been undertaken to reveal the mechanisms that govern the specificity, affinity and flexibility of these protein-protein interactions. Here we review the most recent studies that have focused on the structural aspects of Cohesin-Dockerin recognition. They reveal an ever-increasing number of subtle intricacies suggesting that cellulosome assembly is more complex than was initially thought.