Analysis of a <i>Clostridium josui</i> Cellulase Gene Cluster Containing the <i>man5A</i> Gene and Characterization of Recombinant Man5A

Sakka, Makiko; Goto, Masayuki; Fujino, Takeshi; Fujino, Emi; Karita, Shuichi; Kimura, Takeshi; Sakka, Kazuo

doi:10.1271/bbb.100458

Cited by 20 publications

(11 citation statements)

References 22 publications

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“…For example, Bacillus circulans K-1 ManG displayed highest activity toward konjac glucomannan and moderate activity toward locust bean galactomannan (29). In contrast, C. japonicus Man5A and C. josui Man5A showed the highest activity toward carob or locust bean galactomannan (13,24). These differences in substrate specificities are partially explained by the preference of the subsites for glucose or mannose.…”

Section: Discussionmentioning

confidence: 98%

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

Mizutani

Fernandes²,

Karita

et al. 2012

Appl Environ Microbiol

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eIn general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted ␤-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of ␤-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.

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Section: Discussionmentioning

confidence: 98%

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

Mizutani

Fernandes²,

Karita

et al. 2012

Appl Environ Microbiol

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“…Cellulosome architectures vary greatly among the cellulosome-producing bacterial species, but two global types of architectures of cellulosome systems have been observed, namely simple and complex [ 2 , 7 ]. Simple cellulosomes have so far been observed in mesophilic clostridial species, such as Clostridium cellulovorans , C. cellulolyticum , C. josui, and in the apparently inactive cellulosome of C. acetobutylicum [ 8 , 9 , 10 , 11 ]. The simple cellulosome architecture, includes a single scaffoldin protein, harboring an N-terminal family 3 CBM (CBM3) [ 12 ], and is composed of repeating cohesins and X2 modules [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme

et al. 2017

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The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

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“…Another resemblance among these bacteria is that the intermodular linker segments in their primary scaffoldin are relatively long, reaching 20 to 40 residues in length and more, and are rich in proline and threonine residues [25]. In contrast, other clostridial cellulosome-producing bacteria, such as C. cellulovorans, C. cellulolyticum, C. josui, and C. acetobutylicum, have a simple cellulosome system [4,26-28]. The simple systems contain a single scaffoldin and genes for cellulosomal enzymes are encoded downstream of the scaffoldin gene.…”

Section: Introductionmentioning

confidence: 99%

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Vazana

Barak

Unger

et al. 2013

Biotechnol Biofuels

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BackgroundSelect cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.ResultsUsing a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.ConclusionsThe results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

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Analysis of a Clostridium josui Cellulase Gene Cluster Containing the man5A Gene and Characterization of Recombinant Man5A

Cited by 20 publications

References 22 publications

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase

Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

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