In vitro and in vivo exploration of the cellobiose and cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism

International audienceBackground: In anaerobic cellulolytic microorganisms , cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes. Their action releases cellobiose and longer cellodex-trins which are imported and further degraded in the cytosol to fuel the cells. In Ruminiclostridium cellulolyticum, an anaerobic and cellulolytic mesophilic bacteria, three cellodextrin phosphorylases named CdpA, CdpB, and CdpC, were identified in addition to the cellobiose phosphorylase (CbpA) previously characterized. The present study aimed at characterizing them, exploring their implication during growth on cellulose to better understand the lifestyle of cellulolytic bacteria on such substrate. Results: The three cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for cellodextrins of different lengths and present different k cat values. CdpC is the most active enzyme before CdpA, and CdpB is weakly active. Modeling studies revealed that a mutation of a conserved histidine residue in the phosphate ion-binding pocket in CdpB and CdpC might explain their activity-level differences. The genes encoding these enzymes are scattered over the chromosome of R. cellulolyticum and only the expression of the gene encoding the cellobiose phosphorylase and the gene cdpA is induced during cellulose growth. Characterization of four independent mutants constructed in R. cellulolyticum for each of the cellobiose and cellodextrin phosphorylases encoding genes indicated that only the cellobiose phosphorylase is essential for growth on cellulose. Conclusions: Unexpectedly, the cellobiose phosphorylase but not the cellodextrin phosphorylases is essential for the growth of the model bacterium on cellulose. This suggests that the bacterium adopts a "short" dextrin strategy to grow on cellulose, even though the use of long cellodextrins might be more energy-saving. Our results suggest marked differences in the cellulose catabolism developed among cellulolytic bacteria, which is a result that might impact the design of future engineered strains for biomass-to-biofuel conversion

6. Biomasse : filière ligno-cellulosique, filière bois

L’énergie issue de la biomasse représente actuellement 77 % des énergies renouvelables et 10 % (1 200 millions de tonnes équivalent pétrole* – Mtep – par an) de l’énergie totale produite au niveau mondial. Selon l’agence internationale d’énergie (l’AIE), la biomasse reste sous-utilisée et une augmentation de la proportion, (25 - 33 % de la demande totale en 2050), sans impact supplémentaire sur l’environnement est envisageable. En France, le Grenelle de l’environnement a fixé la production à ..

Le système de dégradation des parois végétales de Clostridium cellulolyticum (diversité et adaptation au substrat)

Clostridium cellulolyticum est une bactérie anaérobie capable de dégrader les parois végétales grâce à la sécrétion d enzymes libres de complexes multienzymatiques et appelés cellulosomes. Les enzymes, libres ou cellulosomales, sont dotées d activités variées afin de dégrader efficacement les différents polymères des parois végétales (cellulose, hémicelluloses, pectines). Au début de ma thèse, seulement seize gènes étaient identifiés. Ils codent essentiellement pour des cellulases ; impliquées dans l hydrolyse de la cellulose. Mon travail de thèse a consisté à appréhender la diversité enzymatique du système de dégradation. Des électrophorèses bidimensionnelles ont révélé une soixantaine de protéines dont trente cellulosomales. Une méthode innovante a permis d identifier sept gènes jusqu alors inconnus dont quatre gènes appartenant à un regroupement de quatorze gènes spécialisés dans la dégradation des hémicelluloses. Au cours de ma thèse, la mise à disposition de la séquence du génome a permis de réaliser l inventaire complet des composants du système de dégradation. L identification par spectrométrie de masse des composants sécrétés dans différentes conditions de culture, alliée à ces données de séquence, ont révélé que C. cellulolyticum module la composition de ses cellulosomes en fonction du substrat utilisé. L expression du regroupement de gènes spécialisés dans la dégradation des hémicellulose est notamment induite lors d une croissance sur paille. Un dernier aspect de mon travail était de construire un outil de mutagénèse aléatoire fonctionnel chez C. cellulolyticum afin d identifier les composants essentiels du système. Parmi les outils testés, un premier, dérivant du transposon Tn1545 de Streptococcus pneumoniae, permet l insertion aléatoire et monocopie dans le génome de C. cellulolyticum alors que le second, dérivant de la séquence d insertion ISS1 de Lactococcus lactis, peut être utilisé comme outil d insertion ciblé dans le génome.Clostridium cellulolyticum is an anaerobic bacterium able to degrade plant cell wall (cellulose, hemicelluloses, pectins) through the secretion of multienzymatic complexes, named cellulosomes, and free enzymes. These enzymes, cellulosomal or free, have various enzymatic activities to efficiently degrade the miscellaneous polymers of plant cell wall. At the beginning of my PhD, only 16 genes encoding cellulases were identified. Cellulases are implicated in the cellulose hydrolysis. My PhD aim consisted in highlighting the enzymatic diversity of this degradation system. 2D-electrophoresis revealed about sixty proteins, among them, thirty are cellulosomal. Using an innovative PCR method we identified seven genes belonging to the system. Among them, four belong to a fourteen gene cluster specialized in the hemicellulose degradation. During my PhD, released of the C. cellulolyticum genome enabled the establishment of the complete repertory of degradation system components. Identification of components, by mass spectrometry, secreted during growth on different substrates, highlighted that C. cellulolyticum could modulate the cellulosome composition according to the growth substrate. In particular, the expression of the gene cluster involves in hemicellulose degradation is induced when straw is the growth substrate. Another aspect of my work was to develop a tool to perform random insertional mutagenesis in C. cellulolyticum. A Tn1545 derivative allowed random and monocopie mutagenesis in genome whereas the use of ISS1 insertion sequence from Lactococcus lactis, allow monocopie integration of genes in the genome.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Cell‐surface exposure of a hybrid 3‐cohesin scaffoldin allowing the functionalization of Escherichia coli envelope

International audienceCellulosomes are large plant cell wall degrading complexes secreted by some anaerobic bacteria. They are typically composed of a major scaffolding protein containing multiple receptors called cohesins, which tightly anchor a small complementary module termed dockerin harbored by the cellulosomal enzymes. In the present study, we have successfully cell surface exposed in Escherichia coli a hybrid scaffoldin, Scaf6, fused to the curli protein CsgA, the latter is known to polymerize at the surface of E. coli to form extracellular fibers under stressful environmental conditions. The C‐terminal part of the chimera encompasses the hybrid scaffoldin composed of three cohesins from different bacterial origins and a carbohydrate‐binding module targeting insoluble cellulose. Using three cellulases hosting the complementary dockerin modules and labeled with different fluorophores, we have shown that the hybrid scaffoldin merged to CsgA is massively exposed at the cell surface of E. coli and that each cohesin module is fully operational. Altogether these data open a new route for a series of biotechnological applications exploiting the cell‐surface exposure of CsgA‐Scaf6 in various industrial sectors such as vaccines, biocatalysts or bioremediation, simply by grafting the small dockerin module to the desired proteins before incubation with the engineered E. coli

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

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

Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis

International audienceIn bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. in Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDp-GlcnAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity

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

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

Cel5I, a SLH-Containing Glycoside Hydrolase: Characterization and Investigation on Its Role in Ruminiclostridium cellulolyticum

International audienceRuminiclostridium cellulolyticum (Clostridium cellulolyticum) is a mesophilic cellulolytic anaerobic bacterium that produces a multi-enzymatic system composed of cellulosomes and non-cellulosomal enzymes to degrade plant cell wall polysaccharides. We characterized one of the non-cellulosomal enzymes, Cel5I, composed of a Family-5 Glycoside Hydrolase catalytic module (GH5), a tandem of Family-17 and -28 Carbohydrate Binding Modules (CBM), and three S-layer homologous (SLH) modules, where the latter are expected to anchor the protein on the cell surface. Cel5I is the only putative endoglucanase targeting the cell surface as well as the only putative protein in R. cellulolyticum containing CBM17 and/or CBM28 modules. We characterized different recombinant structural variants from Cel5I. We showed that Cel5I has an affinity for insoluble cellulosic substrates through its CBMs, that it is the most active endoglucanase on crystalline cellulose of R. cellulolyticum characterized to date and mostly localized in the cell envelope of R. cellulolyticum. Its role in vivo was analyzed using a R. cellulolyticum cel5I mutant strain. Absence of Cel5I in the cell envelope did not lead to a significant variation of the phenotype compared to the wild type strain. Neither in terms of cell binding to cellulose, nor for its growth on crystalline cellulose, thus indicating that the protein has a rather subtle role in tested conditions. Cel5I might be more important in a natural environment, at low concentration of degradable glucose polymers, where its role might be to generate higher concentration of short cellodextrins close to the cell surface, facilitating their uptake or for signalization purpose

Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass (Retraction of vol 79, pg 867, 2013)

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