A cellulosomal yeast reaction system of lignin-degrading enzymes for cellulosic ethanol fermentation.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68 g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/ß/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

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.

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.

Current challenges in designer cellulosome engineering.

Designer cellulosomes (DCs) are engineered multi-enzyme complexes, comprising carbohydrate-active enzymes attached to a common backbone, the scaffoldin, via high-affinity cohesin-dockerin interactions. The use of DCs in the degradation of renewable biomass polymers is a promising approach for biorefineries. Indeed, DCs have shown significant hydrolytic activities due to the enhanced enzyme-substrate proximity and inter-enzyme synergies, but technical hurdles in DC engineering have hindered further progress towards industrial application. The challenge in DC engineering lies in the large diversity of possible building blocks and architectures, resulting in a multivariate and immense design space. Simultaneously, the precise DC composition affects many relevant parameters such as activity, stability, and manufacturability. Since protein engineers face a lack of high-throughput approaches to explore this vast design space, DC engineering may result in an unsatisfying outcome. This review provides a roadmap to guide researchers through the process of DC engineering. Each step, starting from concept to evaluation, is described and provided with its challenges, along with possible solutions, both for DCs that are assembled in vitro or are displayed on the yeast cell surface. KEY POINTS: • Construction of designer cellulosomes is a multi-step process. • Designer cellulosome research deals with multivariate construction challenges. • Boosting designer cellulosome efficiency requires exploring a vast design space.

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.

Advances in consolidated bioprocessing using synthetic cellulosomes.

The primary obstacle impeding the more widespread use of biomass for energy and chemical production is the absence of a low-cost technology for overcoming their recalcitrant nature. It has been shown that the overall cost can be reduced by using a 'consolidated' bioprocessing (CBP) approach, in which enzyme production, biomass hydrolysis, and sugar fermentation can be combined. Cellulosomes are enzyme complexes found in many anaerobic microorganisms that are highly efficient for biomass depolymerization. While initial efforts to display synthetic cellulosomes have been successful, the overall conversion is still low for practical use. This limitation has been partially alleviated by displaying more complex cellulsome structures either via adaptive assembly or by using synthetic consortia. Since synthetic cellulosome nanostructures have also been created using either protein nanoparticles or DNA as a scaffold, there is the potential to tether these nanostructures onto living cells in order to further enhance the overall efficiency.

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode.

Cellulosomes are multi-enzyme complexes produced by specialised micro-organisms. The spatial proximity of synergistically acting enzymes incorporated in these naturally occurring complexes supports the efficient hydrolysis of lignocellulosic biomass. Several functional designer cellulosomes, incorporating naturally non-cellulosomal cellulases, have been constructed and can be used for cellulose saccharification. However, in lignocellulosic biomass, cellulose is tightly intertwined with several hemicelluloses and lignin. One of the most abundant hemicelluloses interacting with cellulose microfibrils is xyloglucan, and degradation of these polymers is crucial for complete saccharification. Yet, designer cellulosome studies focusing on the incorporation of hemicellulases have been limited. Here, we report the conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode. Therefore, we constructed multiple docking enzyme variants of C. japonicus endoxyloglucanase, ß-1,2-galactosidase, α-1,6 xylosidase and ß-1,4-glucosidase, using the combinatorial VersaTile technique dedicated to the design and optimisation of modular proteins. We individually optimised the docking enzymes to degrade the xyloglucan backbone and side chains. Finally, we show that a purified designer xyloglucanosome comprising these docking enzymes was able to release xyloglucan oligosaccharides, galactose, xylose and glucose from tamarind xyloglucan. KEY POINTS: • Construction of xyloglucan-degrading designer cellulosome. • Conversion of free Cellvibrio japonicus enzymes to cellulosomal mode. • Type of linker inserted between dockerin and enzyme module affects docking enzyme activity.

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.

Dissolved xylan inhibits cellulosome-based saccharification by binding to the key cellulosomal component of Clostridium thermocellum.

Polysaccharides derived from lignocellulose are promising sustainable carbon sources. Cellulosome is a supramolecular machine integrating multi-function enzymes for effective lignocellulose bio-saccharification. However, how various non-cellulose components of lignocellulose affect the cellulosomal saccharification is hitherto unclear. This study first investigated the stability and oxygen sensitivity of the cellulosome from Clostridium thermocellum during long-term saccharification process. Then, the differential inhibitory effects of non-cellulose components, including lignin, xylan, and arabinoxylan, on the cellulosome-based saccharification were determined. The results showed that lignin played inhibitory roles by non-productively adsorbing extracellular proteins of C. thermocellum. Differently, arabinoxylan preferred to bind with the cellulosomal components. Almost no adsorption of cellulosomal proteins on solid xylan was detected. Instead, xylan in water-dissolved form interacted with the cellulosomal proteins, especially the key exoglucanase Cel48S, leading to the xylan inhibitory effect. Compared to xylan, xylooligosaccharides influenced the cellulosome activity slightly. Hence, this work demonstrates that the timely hydrolysis or removal of dissolved xylan is important for cellulosome-based lignocellulose saccharification.

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.

Assembling mini-xylanosomes with Clostridium thermocellum XynA, and their properties in lignocellulose deconstruction.

Lignocellulose is a prominent source of carbohydrates to be used in biorefineries. One of the main challenges associated with its use is the low yields obtained during enzymatic hydrolysis, as well as the high cost associate with enzyme acquisition. Despite the great attention in using the fraction composed by hexoses, nowadays, there is a growing interest in enzymatic blends to deconstruct the pentose-rich fraction. Among the organisms studied as a source of enzymes to lignocellulose deconstruction, the anaerobic bacterium Clostridium thermocellum stands out. Most of the remarkable performance of C. thermocellum in degrading cellulose is related to its capacity to assemble enzymes into well-organized enzymatic complexes, cellulosomes. A mini-version of a cellulosome was designed in the present study, using the xylanase XynA and the N-terminus portion of scaffolding protein, mCipA, harboring one CBM3 and two cohesin I domains. The formed mini-xylanosome displayed maximum activity between 60 and 70 °C in a pH range from 6 to 8. Although biochemical properties of complexed/non-complexed enzymes were similar, the formed xylanosome displayed higher hydrolysis at 60 and 70 °C for alkali-treated sugarcane bagasse. Lignocellulose deconstruction using fungal secretome and the mini-xylanosome resulted in higher d-glucose yield, and the addition of the mCipA scaffolding protein enhanced cellulose deconstruction when coupled with fungal enzymes. Results obtained in this study demonstrated that the assembling of xylanases into mini-xylanosomes could improve sugarcane deconstruction, and the mCipA protein can work as a cellulose degradation enhancer.

A novel amylolytic/xylanolytic/cellulolytic multienzyme complex from Clostridium manihotivorum that hydrolyzes polysaccharides in cassava pulp.

Some anaerobic bacteria, particularly Clostridium species, produce extracellular cellulolytic and xylanolytic enzymes as multienzyme complexes (MECs). However, an amylolytic/xylanolytic/cellulolytic multienzyme complex (AXC-MEC) from anaerobic bacteria is rarely found. In this work, the glycoprotein AXC-MEC, composed of subunits of amylolytic, xylanolytic, and cellulolytic enzymes, was isolated from crude extracellular enzyme of the mesophilic anaerobic bacterium Clostridium manihotivorum CT4, grown on cassava pulp, using a milled cassava pulp column and Sephacryl S-500 gel filtration chromatography. The isolated AXC-MEC showed a single band upon native-polyacrylamide gel electrophoresis (native-PAGE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed at least eight protein bands of the multienzyme complex which predominantly exhibited amylolytic enzyme activity, followed by xylanolytic and cellulolytic enzyme activities. The AXC-MEC is highly capable of degrading starch and non-starch polysaccharides present in cassava pulp into glucose and oligosaccharides, without conventional pretreatment. Base on the genomic analysis of C. manihotivorum CT4, we found no evidence of the known structural components of the well-known multienzyme complexes from Clostridium species, cellulosomes such as scaffoldin, cohesin, and dockerin, indicating that AXC-MEC from strain CT4 exhibit a different manner of assembly from the cellulosomes. These results suggest that AXC-MEC from C. manihotivorum CT4 is a new MEC capable of hydrolyzing cassava pulp into value-added products, which will benefit the starch industry. KEY POINTS: • Glycoprotein AXC-MEC was first reported in Clostridium manihotivorum. • Unlike cellulosomes, AXC-MEC consists of amylase, xylanase, and cellulase. • Glucose and oligosaccharides were hydrolysis products from cassava pulp by AXC-MEC.

Solid-Phase Assembly of Multienzyme Systems into Artificial Cellulosomes.

We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.

Coordinated ß-glucosidase activity with the cellulosome is effective for enhanced lignocellulose saccharification.

Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular ß-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.

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.

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.

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

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

Genomic driven factors enhance biocatalyst-related cellulolysis potential in anaerobic digestion.

Anaerobic digestion (AD) is a promising technology to recover bioenergy from biodegradable biomass, including cellulosic wastes. Through a few fractionation/separation techniques, cellulose has demonstrated its potential in AD, but the performance of the process is rather substrate-specific, as cellulolysis bacteria are sensitive to the enzyme-substrate interactions. Cellulosome is a self-assembled enzyme complex with many functionalized modules in the bacteria which has been gradually studied, however the genomic fingerprints of the culture-specific cellulosome in AD are relatively unclear especially under processing conditions. To clarify the key factors affecting the cellulosome induced cellulolysis, this review summarized the most recent publications of AD regarding the fates of cellulose, sources and functional genes of cellulosome, and omics methods for functional analyses. Different processes for organic treatment including applying food grinds in sewer, biomass valorization, cellulose fractionation, microaeration, and enzymatic hydrolysis enhanced fermentation, were highlighted to support the sustainable development of AD technology.

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, 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. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.