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).

Directed Evolution of β-Glucosidase A Towards Enhanced Thermostability.

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose.

Unraveling essential cellulosomal components of the () reveals an extensive reservoir of novel catalytic enzymes.

Background: () is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium.

Effects of modification in the white-rot fungus altering substrate preference during biological pretreatment.

Background: During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall.

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

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. 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.

How does cellulosome composition influence deconstruction of lignocellulosic substrates in () DSM 1313?

Background: Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of () the cellulosome, was studied here.

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.

Cellulosomal expansin: functionality and incorporation into the complex.

Background: Expansins are relatively small proteins that lack enzymatic activity and are found in plants and microorganisms. The function of these proteins is to disrupt the plant cell walls by interfering with the non-covalent interchain bonding of the polysaccharides. Expansins were found to be important for plant growth, but they are also expressed by various bacteria known to have interactions with plants.

Clostridium clariflavum: Key Cellulosome Players Are Revealed by Proteomic Analysis.

Unlabelled: Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C.

Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.

Deconstruction of lignocellulose into soluble sugars by native and designer cellulosomes.

Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation.

Immunological complex for enhancement of innate immune response in passive vaccination.

Passive vaccination is used to treat a wide range of infections and cancer. However, this approach has some limitations. An immune complex termed Y-complex was developed to intensify the effect of the passive vaccine.

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome.

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome.

Draft genome sequences for Clostridium thermocellum wild-type strain YS and derived cellulose adhesion-defective mutant strain AD2.

Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain, AD2, played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

High-throughput screening of cohesin mutant libraries on cellulose microarrays.

The specificity of cohesin-dockerin interactions is critically important for the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome). In order to investigate the origins of the observed specificity, a variety of selected amino acid positions at the cohesin-dockerin interface can be subjected to mutagenesis, and a library of mutants can be constructed. In this chapter, we describe a protein-protein microarray technique based on the high affinity of a carbohydrate-binding module (CBM), attached to mutant cohesins.

Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification.

Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules.

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin-dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules.

Versatile protein microarray based on carbohydrate-binding modules.

Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents.

Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping.

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged.

Regulation of the cellulosomal CelS (cel48A) gene of Clostridium thermocellum is growth rate dependent.

Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures.