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 Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum

2004 ◽  
Vol 186 (19) ◽  
pp. 6544-6552 ◽  
Author(s):  
Stéphanie Perret ◽  
Anne Bélaich ◽  
Henri-Pierre Fierobe ◽  
Jean-Pierre Bélaich ◽  
Chantal Tardif
Keyword(s):  
Guar Gum ◽  
Specific Activity ◽  
Full Length ◽  
Expression Vectors ◽  
Leader Peptide ◽  
Crystalline Cellulose ◽  
Glycosyl Hydrolases ◽  
Truncated Form ◽  
E Coli ◽  
Clostridium Cellulolyticum

ABSTRACT The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

scholarly journals Heterologous Production, Assembly, and Secretion of a Minicellulosome by Clostridium acetobutylicum ATCC 824

2005 ◽  
Vol 71 (3) ◽  
pp. 1215-1222 ◽  
Author(s):  
Florence Mingardon ◽  
Stéphanie Perret ◽  
Anne Bélaïch ◽  
Chantal Tardif ◽  
Jean-Pierre Bélaïch ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Recombinant Strain ◽  
Large Fraction ◽  
Heterologous Gene ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Heterologous Production ◽  
Suitable Host ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter P thl . The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.

scholarly journals CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose

2003 ◽  
Vol 185 (2) ◽  
pp. 391-398 ◽  
Author(s):  
Rachel Gilad ◽  
Larisa Rabinovich ◽  
Sima Yaron ◽  
Edward A. Bayer ◽  
Raphael Lamed ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Terminal Segment ◽  
Glycoside Hydrolases ◽  
General Nature ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Truncated Form ◽  
Cellulose Substrates ◽  
The Family

ABSTRACT The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

scholarly journals Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

2011 ◽  
Vol 77 (18) ◽  
pp. 6470-6475 ◽  
Author(s):  
Shunichi Nakayama ◽  
Keiji Kiyoshi ◽  
Toshimori Kadokura ◽  
Atsumi Nakazato
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Clostridium Beijerinckii ◽  
Crystalline Cellulose ◽  
Butanol Production ◽  
Content Type ◽  
Coculture System

ABSTRACTWe investigated butanol production from crystalline cellulose by cocultured cellulolyticClostridium thermocellumand the butanol-producing strain,Clostridium saccharoperbutylacetonicum(strain N1-4). Butanol was produced from Avicel cellulose after it was incubated withC. thermocellumfor at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by coculturedClostridium acetobutylicumandClostridium beijerinckiithan by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

Expression of Cloned Homologous Fermentative Genes in Clostridium Acetobutylicum ATCC 824

1992 ◽  
Vol 10 (2) ◽  
pp. 190-195 ◽  
Author(s):  
Lee D. Mermelstein ◽  
Neil E. Welker ◽  
George N. Bennett ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Acetobutylicum Atcc 824

Two stage biodiesel and hydrogen production from molasses by oleaginous fungi and Clostridium acetobutylicum ATCC 824

2014 ◽  
Vol 39 (7) ◽  
pp. 3185-3197 ◽  
Author(s):  
Magdy Mohamed Khalil Bagy ◽  
Mohamed Hemida Abd-Alla ◽  
Fatthy Mohamed Morsy ◽  
Elhagag Ahmed Hassan
Keyword(s):  
Hydrogen Production ◽  
Clostridium Acetobutylicum ◽  
Two Stage ◽  
Oleaginous Fungi ◽  
Clostridium Acetobutylicum Atcc 824

scholarly journals Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose

AMB Express ◽  
2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Felipe Buendia-Kandia ◽  
Emmanuel Rondags ◽  
Xavier Framboisier ◽  
Guillain Mauviel ◽  
Anthony Dufour ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Diauxic Growth ◽  
Clostridium Acetobutylicum Atcc 824

scholarly journals Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

2003 ◽  
Vol 69 (2) ◽  
pp. 869-877 ◽  
Author(s):  
Ana M. López-Contreras ◽  
Aernout A. Martens ◽  
Nora Szijarto ◽  
Hans Mooibroek ◽  
Pieternel A. M. Claassen ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Biochemical Properties ◽  
Open Reading Frames ◽  
Extracellular Medium ◽  
Endoglucanase Activity ◽  
E Coli ◽  
High Affinity ◽  
Clostridial Species ◽  
Reading Frames ◽  
Clostridium Acetobutylicum Atcc 824

ABSTRACT The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.

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