January 2022
·
101 Reads
This page lists the scientific contributions of an author, who either does not have a ResearchGate profile, or has not yet added these contributions to their profile.
It was automatically created by ResearchGate to create a record of this author's body of work. We create such pages to advance our goal of creating and maintaining the most comprehensive scientific repository possible. In doing so, we process publicly available (personal) data relating to the author as a member of the scientific community.
If you're a ResearchGate member, you can follow this page to keep up with this author's work.
If you are this author, and you don't want us to display this page anymore, please let us know.
January 2022
·
101 Reads
November 2021
·
197 Reads
·
9 Citations
The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars.
October 2020
·
76 Reads
·
8 Citations
Ruminiclostridium cellulolyticum is a Gram-positive, mesophilic, anaerobic, cellulolytic, and hemicellulolytic bacterium. The last property qualifies this species as a model species for the study of hemicellulose degradation, import of degradation products, and overall regulation of these phenomena. In this study, we focus on the regulation of xyloglucan dextrin import and intracellular degradation and show that the two components of the two-component regulation system XygSR are essential for growth on xyloglucan and that the response regulator XygR regulates the transcription of genes involved in the extracellular degradation of the polysaccharide, the import of degradation products, and their intracellular degradation.
December 2019
·
24 Reads
·
2 Citations
Cellulosomes are complex nanomachines produced by cellulolytic anaerobic bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum). Cellulosomes are composed of a scaffoldin protein displaying several cohesin modules on which enzymatic components can bind to through their dockerin module. Although cellulosomes have been studied for decades, very little is known about the dynamics of complex assembly. We have investigated the ability of some dockerin‐bearing enzymes to chase the catalytic subunits already bound onto a miniscaffoldin displaying a single cohesin. The stability of the preassembled enzyme–scaffoldin complex appears to depend on the nature of the dockerin, and we have identified a key position in the dockerin sequence that is involved in the stability of the complex with the cohesin. Depending on the residue occupying this position, the dockerin can establish with the cohesin partner either a nearly irreversible or a reversible interaction, independently of the catalytic domain associated with the dockerin. Site‐directed mutagenesis of this residue can convert a dockerin able to form a highly stable complex with the miniscaffoldin into a reversible complex forming one and vice versa. We also show that refunctionalization can occur with natural purified cellulosomes. Altogether, our results shed light on the dynamics of cellulosomes, especially their capacity to be remodeled even after their assembly is ‘achieved’, suggesting an unforeseen adaptability of their enzymatic composition over time.
June 2019
·
343 Reads
·
12 Citations
Biotechnology for Biofuels
Background: The α-l-arabinofuranosidases (α-l-ABFs) are exoenzymes involved in the hydrolysis of α-l-arabinosyl linkages in plant cell wall polysaccharides. They play a crucial role in the degradation of arabinoxylan and arabinan and they are used in many biotechnological applications. Analysis of the genome of R. cellulolyticum showed that putative cellulosomal α-l-ABFs are exclusively encoded by the xyl-doc gene cluster, a large 32-kb gene cluster. Indeed, among the 14 Xyl-Doc enzymes encoded by this gene cluster, 6 are predicted to be α-l-ABFs belonging to the CAZyme families GH43 and GH62. Results: The biochemical characterization of these six Xyl-Doc enzymes revealed that four of them are α-l-ABFs. GH4316-1229 (RcAbf43A) which belongs to the subfamily 16 of the GH43, encoded by the gene at locus Ccel_1229, has a low specific activity on natural substrates and can cleave off arabinose decorations located at arabinoxylan chain extremities. GH4310-1233 (RcAbf43Ad2,3), the product of the gene at locus Ccel_1233, belonging to subfamily 10 of the GH43, can convert the double arabinose decorations present on arabinoxylan into single O2- or O3-linked decorations with high velocity (kcat = 16.6 ± 0.6 s-1). This enzyme acts in synergy with GH62-1234 (RcAbf62Am2,3), the product of the gene at locus Ccel_1234, a GH62 α-l-ABF which hydrolyzes α-(1 → 3) or α-(1 → 2)-arabinosyl linkages present on polysaccharides and arabinoxylooligosaccharides monodecorated. Finally, a bifunctional enzyme, GH62-CE6-1240 (RcAbf62Bm2,3Axe6), encoded by the gene at locus Ccel_1240, which contains a GH62-α-l-ABF module and a carbohydrate esterase (CE6) module, catalyzes deacylation of plant cell wall polymers and cleavage of arabinosyl mono-substitutions. These enzymes are also active on arabinan, a component of the type I rhamnogalacturonan, showing their involvement in pectin degradation. Conclusion: Arabinofuranosyl decorations on arabinoxylan and pectin strongly inhibit the action of xylan-degrading enzymes and pectinases. α-l-ABFs encoded by the xyl-doc gene cluster of R. cellulolyticum can remove all the decorations present in the backbone of arabinoxylan and arabinan, act synergistically, and, thus, play a crucial role in the degradation of plant cell wall polysaccharides.
December 2017
·
62 Reads
·
2 Citations
Ruminiclostridium cellulolyticum produces extracellular cellulosomes which contain inter alia numerous Family-9 Glycoside Hydrolases, including the inactive Cel9V. The latter shares the same organization and 79% sequence identity with the active cellulase Cel9E. Nevertheless two aromatic residues, and a four-residue stretch putatively critical for the activity are missing in Cel9V. Introduction of one Trytophan and the four-residue stretch restored some weak activity in Cel9V, whereas the replacement of its catalytic domain by that of Cel9E generated a fully active cellulase. Altogether our data indicate that a series of mutations in the catalytic domain of Cel9V lead to an essentially inactive cellulase. This article is protected by copyright. All rights reserved.
December 2015
·
278 Reads
·
66 Citations
Biotechnology for Biofuels
Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L. phytofermentans secretes in the environment some cellulose-degrading enzymes as free enzymes. In the present study, the major cellulase from L. phytofermentans was introduced as a free enzyme or as a cellulosomal component in R. cellulolyticum to improve its cellulolytic capacities. The gene at locus Cphy_3367 encoding the major cellulase Cel9A from L. phytofermentans and an engineered gene coding for a modified enzyme harboring a R. cellulolyticum C-terminal dockerin were cloned in an expression vector. After electrotransformation of R. cellulolyticum, both forms of Cel9A were found to be secreted by the corresponding recombinant strains. On minimal medium containing microcrystalline cellulose as the sole source of carbon, the strain secreting the free Cel9A started to grow sooner and consumed cellulose faster than the strain producing the cellulosomal form of Cel9A, or the control strain carrying an empty expression vector. All strains reached the same final cell density but the strain producing the cellulosomal form of Cel9A was unable to completely consume the available cellulose even after an extended cultivation time, conversely to the two other strains. Analyses of their cellulosomes showed that the engineered form of Cel9A bearing a dockerin was successfully incorporated in the complexes, but its integration induced an important release of regular cellulosomal components such as the major cellulase Cel48F, which severely impaired the activity of the complexes on cellulose. In contrast, the cellulosomes synthesized by the control and the free Cel9A-secreting strains displayed similar composition and activity. Finally, the most cellulolytic strain secreting free Cel9A, was also characterized by an early production of lactate, acetate and ethanol as compared to the control strain. Our study shows that the cellulolytic capacity of R. cellulolyticum can be augmented by supplementing the cellulosomes with a free cellulase originating from L. phytofermentans, whereas integration of the heterologous enzyme in the cellulosomes is rather unfavorable.
August 2013
·
88 Reads
·
32 Citations
Bacterial cellulosomes are generally believed to assemble at random like those produced by Clostridium cellulolyticum. They are composed of one scaffolding protein displaying 8 homologous type I cohesins that bind to any of the type I dockerins borne by the 62 cellulosomal subunits, thus generating highly heterogeneous complexes. In the present study the heterogeneity and random assembly of the cellulosomes were evaluated using a simpler model: a miniscaffoldin containing three C. cellulolyticum cohesins and three cellulases of the same bacterium bearing the cognate dockerin (Cel5A, Cel48F and Cel9G). Surprisingly, in lieu of the anticipated randomized integration of enzymes, the assembly of the minicellulosome generated only three distinct types of complex out of the ten possible combinations, thus indicating preferential integration of enzymes upon binding to the scaffoldin. A hybrid scaffoldin that displays one cohesin from C. cellulolyticum and one from C. thermocellum thus allowing sequential integration of enzymes was exploited to further characterize this phenomenon. The initial binding of a given enzyme onto the C. thermocellum cohesin was found to influence the type of enzyme which is bound subsequently onto the C. cellulolyticum cohesin. The preferential integration appears to be related to the length of the inter-cohesin linker. The data indicate that the binding of a cellulosomal enzyme onto a cohesin has direct influence on the dockerin-bearing proteins that will subsequently interact with adjacent cohesins. Thus, despite the general lack of specificity of the cohesin/dockerin interaction within a given species and type, bacterial cellulosomes are not necessarily assembled at random. This article is protected by copyright. All rights reserved.
July 2013
·
888 Reads
·
18 Citations
Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.
July 2013
·
19 Reads
Amino-acid sequence alignment of HYR modules identified in HycP. Sequences alignment has been performed using ClustalW2. Stars and grey box indicate identical residues; double dot, strongly similar residues; simple dot, weakly similar residues. Sequence of HYR modules were delimited and numbered as shown in figure 1A. (TIF)
... Ruminiclostridium cellulolyticum and Ruminococcus flavefaciens are the model bacteria to produce cellulosomes and efficiently degrade cellulose from plant cells (Kampik et al., 2021;Yeoman et al., 2021), with significantly increased abundance, supporting the improvement of digestive function of plant fibers in the gut after combined intervention. Sutterella and Acidaminoccus were the two consistently increased genus in both probiotics and combined group in our study. ...
November 2021
... qPCR was performed on cDNA synthesized from mRNA as previously described [30]. For each point, technical duplicates and biological triplicates were performed. ...
October 2020
... As a consequence, the cellulosomes produced display a higher amount of scaffoldins compared with those expressed in presence of oligosaccharides, exposing up to 56 different active GHs on complex substrate. In this context, research on cellulosomes from Ruminiclostridium cellulolyticum has revealed that the conformation of the two domains in dockerins involved in the interaction with cohesins allows the refunctionalization of the cellulosome, and so the exchange between catalytic domains as an adaptation to heterogeneous plant cell wall substrates in active degradation [21]. Furthermore, spatial expression and exposition of cellulosomes on the cell surface varies on substrate used for growth, as exemplified by the cellulosomes produced by C. clariflavum [22]. ...
December 2019
... This versatile bacterium is capable to grow on AX, cellulose, xyloglucan and more complex natural substrates like wheat straw. On one hand, this organism produces extracellular multienzymatic complexes called cellulosomes for the degradation of plant cell wall polysaccharides; while, on the other hand, it has the capacity to import and intracellularly break down large AXOS of up to 6 monosaccharides through a system known as Xua (xylan utilization-associated genes) [14,[23][24][25][26]. The Xua system is encoded by a cluster of 13 genes (Fig. 1) and is important for the growth of the strain on arabinoxylan [14]. ...
June 2019
Biotechnology for Biofuels
... The genera in branch (a) decreased in relative abundance during all four fermentations. Ruminiclostridium, Herbinix, and Tepidimicrobium are strictly anaerobic, cellulosic hemicellulolytic bacteria (Koeck et al., 2016;Phitsuwan et al., 2010;Vita et al., 2018), probably derived from cattle manure, and gradually died out during the high-temperature aerobic fermentation. Bacillus of Firmicutes was the dominant genus in the middle stage of fermentation. ...
December 2017
... These artificial nanodevices comprise a chimaeric scaffoldin with cohesin modules of divergent specificity, usually derived from different bacteria, and allow the controlled incorporation of recombinant matching dockerin-bearing enzymes that might benefit from enzyme proximity (Fig. 2). The designer cellulosome concept is based on the high affinity and specific interaction between a cohesin and a dockerin [20]. ...
December 1997
Proteins Structure Function and Bioinformatics
... 38,39 Furthermore, autumn-enriched taxa included Ruminococcaceae UCG-010, Bacillus, Paenibacillus, Pseudomonas, and Ruminiclostridium, which have enhanced cellulose and hemicellulose digestion capabilities, via either secreted free enzymes or extracellular multi-enzyme structures called cellulosomes. 33,[40][41][42][43][44] Interestingly, the seasonal changes in the Alpine ibex gut microbiome taxonomy would explain the corresponding shifts in the overall gut metabolome layout, as well as in the measured profiles of SCFAs. In particular, the higher levels of acetic acid in autumn may be due to the prevalence, in this season, of some well-known acetate producers such as Ruminococcaceae UCG-010, 33 Ruminiclostridium, 40 Bacillus, 45 and Paenibacillus. ...
December 2015
Biotechnology for Biofuels
... The structure of the type I cohesin is composed of a nine-stranded flattened β-barrel with jelly-roll topology. Type I dockerins contain a pair of 22-residue segment that is organized into an F-hand calcium-binding motif and an α-helix (Pages et al., 1997). Each segment displays an identical overall structure as reflected by the internal twofold symmetry in the dockerin (Carvalho et al., 2003;Chen et al., 2014), and generally presents two similar Cohbinding interfaces supporting a dual-binding mode (Carvalho et al., 2007;Flint et al., 2008). ...
December 1997
Proteins Structure Function and Bioinformatics
... The way in which the different dockering bearing enzymes are assembled with their cohesin counterparts are of great importance. Far from being purely the result of chance, studies developing cellulosome chimeras have demonstrated that the distribution of the different catalytic domains is in fact driven by the length of the intercohesin-linker segments of the scaffoldin rather than by the cohesin-dockerin interactions [19]. This observation suggests that the spatial proximity between enzymes is relevant to increase synergy and how it affects also the overall conformational flexibility. ...
August 2013
... The ClosTron method was used to construct mutants as previously described, using specific primers [27,28]. The mutant strains interrupted in the gene located at the locus Ccel_1250 with group II intron was called MTLx-uaS. ...
July 2013