In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, ...highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca (Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates--a distinction previously reserved for cellulose-degrading enzymes.
Xylanases are important enzymes for our society, due to their variety of industrial applications. Together with cellulases and other glycoside hydrolases, xylanases may also provide cost-effective conversion of plant-derived cellulosic biomass into soluble sugars en route to biofuels as an alternative to fossil fuels. Xylanases are commonly found in multienzyme cellulosome complexes, produced by anaerobic bacteria, which are considered to be among the most efficient systems for degradation of cellulosic biomass. Using a designer cellulosome approach, we have incorporated the entire xylanolytic system of the bacterium Thermobifida fusca into defined artificial cellulosome complexes. The combined action of these designer cellulosomes versus that of the wild-type free xylanase system was then compared. Our data demonstrated that xylanolytic designer cellulosomes displayed enhanced synergistic activities on a natural recalcitrant wheat straw substrate and could thus serve in the development of advanced systems for improved degradation of lignocellulosic material.
The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement ...can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.
The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. ...Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.
The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major ...fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown.
We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca(2+)-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins.
This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.
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.
Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures ...that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose.
The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel.
This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.
Clostridium thermocellum cellulase
9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding ...modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in
Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60–70% of the intact Cel9I endoglucanase activity.
MINT-
6946626:
Cel9I (uniprotkb:
Q02934) and Cel9I (uniprotkb:
Q02934) bind (MI:
0407) by comigration in non-denaturing gel electrophoresis (MI:
0404)
MINT-
6946649:
Cel9I (uniprotkb:
Q02934) and Cel9I (uniprotkb:
Q02934) bind (MI:
0407) by molecular sieving (MI:
0071)
MINT-
6946687:
Cel9I (uniprotkb:
Q02934) and Cel9I (uniprotkb:
Q02934) bind (MI:
0407) by isothermal titration calorimetry (MI:
0065)
MINT-
6946706:
Cel9I (uniprotkb:
Q02934) binds (MI:
0407) to Cel9I (uniprotkb:
Q02934) by pull down (MI:
0096)
Clostridium thermocellum efficiently degrades crystalline cellulose by a high molecular weight protein complex, the cellulosome. The bacterium regulates its cellulosomal genes using a unique ...extracellular biomass‐sensing mechanism that involves alternative sigma factors and extracellular carbohydrate‐binding modules attached to intracellular anti‐sigma domains. In this study, we identified three cellulosomal xylanase genes that are regulated by the σI6/RsgI6 system by utilizingsigI6 andrsgI6 knockout mutants together with primer extension analysis. Our results indicate that cellulosomal genes are expressed from both alternative σI6 and σA vegetative promoters.
Three cellulosomal xylanases are regulated by the SigI6‐RsgI6 biomass sensing system.
Cellulosomal genes are expressed from both alternative sigma and σA promoters.
Disruption of thesigI6 gene affects the solubilization pattern of switchgrass byC. thermocellum.
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