scholarly journals Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Katsuaki Hirano ◽  
Masahiro Kurosaki ◽  
Satoshi Nihei ◽  
Hiroki Hasegawa ◽  
Suguru Shinoda ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Biomass ◽  
Crystalline Cellulose ◽  
Enzymatic Diversity

scholarly journals Production of Heterologous and Chimeric Scaffoldins by Clostridium acetobutylicum ATCC 824

2004 ◽  
Vol 186 (1) ◽  
pp. 253-257 ◽  
Author(s):  
S. Perret ◽  
L. Casalot ◽  
H.-P. Fierobe ◽  
C. Tardif ◽  
F. Sabathe ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Crystalline Cellulose ◽  
Truncated Form ◽  
Clostridium Cellulolyticum ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

scholarly journals Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically

2007 ◽  
Vol 268 (2) ◽  
pp. 194-201 ◽  
Author(s):  
Emanuel Berger ◽  
Dong Zhang ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Clostridium Thermocellum ◽  
Crystalline Cellulose

scholarly journals Purification and characterization of endoglucanase Ss from Clostridium thermocellum

1991 ◽  
Vol 279 (1) ◽  
pp. 67-73 ◽  
Author(s):  
U Fauth ◽  
M P M Romaniec ◽  
T Kobayashi ◽  
A L Demain
Keyword(s):  
Clostridium Thermocellum ◽  
Hydrophobic Interaction Chromatography ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Specific Antibodies ◽  
Optimum Ph ◽  
Exchange Adsorption

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

scholarly journals Development of a genome-scale metabolic model of Clostridium thermocellum and its applications for integration of multi-omics datasets and strain design

2020 ◽  
Author(s):  
Sergio Garcia ◽  
R. Adam Thompson ◽  
Richard J. Giannone ◽  
Satyakam Dash ◽  
Costas D. Maranas ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Lignocellulosic Biomass ◽  
Clostridium Thermocellum ◽  
System Analysis ◽  
Plant Biomass ◽  
Metabolic Model ◽  
Efficient Production ◽  
Branch Points ◽  
A Genome ◽  
Genome Scale

AbstractSolving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as catalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade untreated lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

scholarly journals Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 996
Author(s):  
Dung Minh Ha-Tran ◽  
Trinh Thi My Nguyen ◽  
Chieh-Chen Huang
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genetic Material ◽  
Economic Feasibility ◽  
Biofuel Production ◽  
Cellulolytic Bacterium ◽  
Microbial Conversion ◽  
Promising Source

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum

2019 ◽  
Vol 366 (12) ◽  
Author(s):  
Shunsuke Ichikawa ◽  
Satoru Ogawa ◽  
Ayami Nishida ◽  
Yuzuki Kobayashi ◽  
Toshihito Kurosawa ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Activity ◽  
Cellulolytic Bacteria ◽  
Degradation Activity ◽  
Enzyme Complexes

ABSTRACTMembrane vesicles released from bacteria contribute to cell–cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50–300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Predominant secretion of cellobiohydrolases and endo-β-1,4-glucanases in nutrient-limited medium by Aspergillus spp. isolated from subtropical field

2020 ◽  
Vol 168 (3) ◽  
pp. 243-256 ◽  
Author(s):  
May Thin Kyu ◽  
Shunsuke Nishio ◽  
Koki Noda ◽  
Bay Dar ◽  
San San Aye ◽  
...  
Keyword(s):  
Rice Straw ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Industrial Wastes ◽  
Added Value ◽  
Crystalline Cellulose ◽  
Cellulolytic Activity ◽  
Biological Degradation ◽  
Industry Waste ◽  
Degradation Intermediates

Abstract Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

scholarly journals S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

2011 ◽  
Vol 78 (3) ◽  
pp. 768-777 ◽  
Author(s):  
Inci Ozdemir ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly
Keyword(s):  
Cellular Localization ◽  
Catalytic Domain ◽  
Plant Biomass ◽  
Thermophilic Bacteria ◽  
Biochemical Properties ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

Classification of ‘Anaerocellum thermophilum’ strain DSM 6725 as Caldicellulosiruptor bescii sp. nov.

2010 ◽  
Vol 60 (9) ◽  
pp. 2011-2015 ◽  
Author(s):  
Sung-Jae Yang ◽  
Irina Kataeva ◽  
Juergen Wiegel ◽  
Yanbin Yin ◽  
Phuongan Dam ◽  
...  
Keyword(s):  
New Genus ◽  
Plant Biomass ◽  
Hot Spring ◽  
Crystalline Cellulose ◽  
Rrna Gene ◽  
Growth Physiology ◽  
Caldicellulosiruptor Bescii ◽  
Untreated Plant ◽  
The 16S Rrna Gene

The thermophilic, cellulolytic, anaerobic bacterium ‘Anaerocellum thermophilum’ strain Z-1320 was isolated from a hot spring almost two decades ago and deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) as DSM 6725. The organism was classified as representing a new genus, ‘Anaerocellum’, primarily on its growth physiology, cell-wall type and morphology. The results of recent physiological studies and of phylogenetic and genome sequence analyses of strain DSM 6725 of ‘A. thermophilum’ obtained from the DSMZ showed that its properties differed from those originally described for strain Z-1320. In particular, when compared with strain Z-1320, strain DSM 6725 grew at higher temperatures and had an expanded range of growth substrates. Moreover, the 16S rRNA gene sequence of strain DSM 6725 fell within the Caldicellulosiruptor clade. It is therefore suggested that ‘Anaerocellum thermophilum’ should be classified as a member of the genus Caldicellulosiruptor, for which the name Caldicellulosiruptor bescii sp. nov. is proposed (type strain DSM 6725T=ATCC BAA-1888T). C. bescii sp. nov. DSM 6725T is the most thermophilic cellulose-degrading organism known. The strain was able to grow up to 90 °C (pH 7.2) and degraded crystalline cellulose and xylan as well as untreated plant biomass, including potential bioenergy plants such as poplar and switchgrass.

scholarly journals Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Narumon Tangthirasunun ◽  
David Navarro ◽  
Sona Garajova ◽  
Didier Chevret ◽  
Laetitia Chan Ho Tong ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Podospora Anserina ◽  
Cellulosic Biomass ◽  
Striking Difference ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Wild Type ◽  
Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

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