Unique Fn3-like biosensor in σ/anti-σ factors for regulatory expression of major cellulosomal scaffoldins in Pseudobacteroides cellulosolvens.

Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti-σ (SigI/RsgI) factors to regulate cellulosomal components for substrate-specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI-RsgI regulons yet discovered.

Phylogenomics and genetic analysis of solvent-producing Clostridium species.

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C.

A cellulosomal double-dockerin module from Clostridium thermocellum shows distinct structural and cohesin-binding features.

Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear.

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.

Structure of the transcription open complex of distinct σ factors.

Bacterial σ factors of the σ-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σ paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.

Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction.

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from , which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis.

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.

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.

Deciphering Cellodextrin and Glucose Uptake in .

Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates.

Metaproteome plasticity sheds light on the ecology of the rumen microbiome and its connection to host traits.

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.

Mapping the deformability of natural and designed cellulosomes in solution.

Background: Natural cellulosome multi-enzyme complexes, their components, and engineered 'designer cellulosomes' (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes, but the modular architecture challenges structure determination and rational design.

Results: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components.

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 system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales.

Combinatorial assembly and optimisation of designer cellulosomes: a galactomannan case study.

Background: 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.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS.

Wilavidin - a novel member of the avidin family that forms unique biotin-binding hexamers.

Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher-order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra-high affinity toward biotin is dependent on their quaternary arrangements.

Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases.

Pullulanases are glycoside hydrolase family 13 (GH13) enzymes that target α1,6 glucosidic linkages within starch and aid in the degradation of the α1,4- and α1,6- linked glucans pullulan, glycogen and amylopectin. The human gut bacterium Ruminococcus bromii synthesizes two extracellular pullulanases, Amy10 and Amy12, that are incorporated into the multiprotein amylosome complex that enables the digestion of granular resistant starch from the diet. Here we provide a comparative biochemical analysis of these pullulanases and the x-ray crystal structures of the wild type and the nucleophile mutant D392A of Amy12 complexed with maltoheptaose and 6-α-D glucosyl-maltotriose.

Coordinated β-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 β-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.

NMR chemical shift assignments of a module of unknown function in the cellulosomal secondary scaffoldin ScaF from Clostridium thermocellum.

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.

Novel clostridial cell-surface hemicellulose-binding CBM3 proteins.

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.

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 , 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.

Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of .

The bacterium KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host.

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.

Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum.

is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that 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).