Draft Genome Sequence of the Lignocellulose Decomposer Thermobifida fusca Strain TM51.

Here, we present the complete genome sequence of Thermobifida fusca strain TM51, which was isolated from the hot upper layer of a compost pile in Hungary. T. fusca TM51 is a thermotolerant, aerobic actinomycete with outstanding lignocellulose-decomposing activity

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism

Ruminococcal cellulosome systems from rumen to human

This article is protected by copyright. All rights reserved. The authors appreciate the kind assistance of Miriam Lerner (ImmunArray Ltd. Company, Rehovot, Israel) with experiments involving the MicroGrid II arrayer. This research was supported by a grant (No. 1349) to EAB also from the Israel Science Foundation (ISF) and a grant (No. 24/11) issued to RL by The Sidney E. Frank Foundation also through the ISF. Additional support was obtained from the establishment of an Israeli Center of Research Excellence (I-CORE Center No. 152/11) managed by the Israel Science Foundation, from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, by the Weizmann Institute of Science Alternative Energy Research Initiative (AERI) and the Helmsley Foundation. The authors also appreciate the support of the European Union, Area NMP.2013.1.1-2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530 and an ERA-IB Consortium (EIB.12.022), acronym FiberFuel. HF and SHD acknowledge support from the Scottish Government Food Land and People programme and from BBSRC grant no. BB/L009951/1. In addition, EAB is grateful for a grant from the F. Warren Hellman Grant for Alternative Energy Research in Israel in support of alternative energy research in Israel administered by the Israel Strategic Alternative Energy Foundation (I-SAEF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic ChemistryPeer reviewedPostprin

Paradigmatic status of an endo- and exoglucanase and its effect on crystalline cellulose degradation

BACKGROUND: Microorganisms employ a multiplicity of enzymes to efficiently degrade the composite structure of plant cell wall cellulosic polysaccharides. These remarkable enzyme systems include glycoside hydrolases (cellulases, hemicellulases), polysaccharide lyases, and the carbohydrate esterases. To accomplish this challenging task, several strategies are commonly observed either separately or in combination. These include free enzyme systems, multifunctional enzymes, and multi-enzyme self-assembled designer cellulosome complexes. RESULTS: In order to compare these different paradigms, we employed a synthetic biology approach to convert two different cellulases from the free enzymatic system of the well-studied bacterium, Thermobifida fusca, into bifunctional enzymes with different modular architectures. We then examined their performance compared to those of the combined parental free-enzyme and equivalent designer-cellulosome systems. The results showed that the cellulolytic activity displayed by the different architectures of the bifunctional enzymes was somewhat inferior to that of the wild-type free enzyme system. CONCLUSIONS: The activity exhibited by the designer cellulosome system was equal or superior to that of the free system, presumably reflecting the combined proximity of the enzymes and high flexibility of the designer cellulosome components, thus enabling efficient enzymatic activity of the catalytic modules

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

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

This work was supported in part by the European Union, Area NMP.2013.1.1–2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530, and by the EU Seventh Framework Programme (FP7 2007–2013) under the WallTraC project (Grant Agreement no 263916), and BioStruct-X (grant agreement no 283570). This paper reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. CMGAF is also supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008 and EXPL/BIA-MIC/1176/2012. EAB is also funded by a grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel and by a grant (No. 2013284) from the U.S.-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.Peer reviewedPublisher PD

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine‐tuned system for cohesin‐dockerin recognition

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137458/1/emi13047.pd