Biochemical characterization of a SusD-like protein involved in β-1,3-glucan utilization by an uncultured cow rumen .

In ruminants, the rumen is a specialized stomach that is adapted to the breakdown of plant-derived complex polysaccharides through the coordinated activities of a diverse microbial community. Bacteroidota is a major phylum in this bovine rumen microbiota. They contain several clusters of genes called polysaccharide utilization loci (PULs) that encode proteins working in concert to capture, degrade, and transport polysaccharides.

O-Mucin-degrading carbohydrate-active enzymes and their possible implication in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are modern diseases, with incidence rising around the world. They are associated with perturbation of the intestinal microbiota, and with alteration and crossing of the mucus barrier by the commensal bacteria that feed on it. In the process of mucus catabolism and invasion by gut bacteria, carbohydrate-active enzymes (CAZymes) play a critical role since mucus is mainly made up by O- and N-glycans.

Structural and Biochemical Characterization of a Nonbinding SusD-Like Protein Involved in Xylooligosaccharide Utilization by an Uncultured Human Gut Strain.

In the human gut microbiota, break down dietary and endogenous glycosides through highly specific polysaccharide utilization loci (PULs). PULs encode a variety of sensor regulators, binding proteins, transporters, and carbohydrate-active enzymes (CAZymes). Surface glycan-binding proteins (SGBPs) are essential for the efficient capture of the glycosides present on the cell surface, providing with a competitive advantage in colonizing their habitats.

Identification of Glycoside Transporters From the Human Gut Microbiome.

Transport is a crucial step in the metabolism of glycosides by bacteria, which is itself key for microbiota function and equilibrium. However, most transport proteins are function-unknown or only predicted, limiting our understanding of how bacteria utilize glycosides. Here, we present an activity-based screening method to identify functional glycoside transporters from microbiomes.

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases.

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies.

MINTIA: a metagenomic INserT integrated assembly and annotation tool.

The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes.

Anomeric Retention of Carbohydrates in Multistage Cyclic Ion Mobility (IMS): De Novo Structural Elucidation of Enzymatically Produced Mannosides.

Carbohydrates are complex structures that still challenge analysts today because of their different levels of isomerism, notably the anomerism of the glycosidic bond. It has been shown recently that anomerism is preserved upon gas-phase fragmentation and that high-resolution ion mobility (IMS) can distinguish anomers. However, these concepts have yet to be applied to complex biological products.

Crystal and molecular structure of V-amylose complexed with ibuprofen.

Rectangular V-amylose single crystals were prepared by adding racemic ibuprofen to hot dilute aqueous solutions of native and enzymatically-synthesized amylose. The lamellar thickness increased with increasing degree of polymerization of amylose and reached a plateau at about 7 nm, consistent with a chain-folding mechanism. The CP/MAS NMR spectrum as well as base-plane electron and powder X-ray diffraction patterns recorded from hydrated specimens were similar to those of V-amylose complexed with propan-2-ol.

α-Galactosidase and Sucrose-Kinase Relationships in a Bi-functional AgaSK Enzyme Produced by the Human Gut Symbiont E1.

Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the E1 symbiont-well-known for various benefit-is able to produce an original AgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively.

Investigating host-microbiome interactions by droplet based microfluidics.

Background: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota.

Results: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries.

Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function.

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes.

A catalog of microbial genes from the bovine rumen unveils a specialized and diverse biomass-degrading environment.

Background: The rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals.

Harvesting of Prebiotic Fructooligosaccharides by Nonbeneficial Human Gut Bacteria.

Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization.

Investigating Host Microbiota Relationships Through Functional Metagenomics.

The human Intestinal mucus is formed by glycoproteins, the O- and N-linked glycans which constitute a crucial source of carbon for commensal gut bacteria, especially when deprived of dietary glycans of plant origin. In recent years, a dozen carbohydrate-active enzymes from cultivated mucin degraders have been characterized. But yet, considering the fact that uncultured species predominate in the human gut microbiota, these biochemical data are far from exhaustive.

Sucrose 6-phosphate phosphorylase: a novel insight in the human gut microbiome.

The human gut microbiome plays an essential role in maintaining human health including in degradation of dietary fibres and carbohydrates further used as nutrients by both the host and the gut bacteria. Previously, we identified a polysaccharide utilization loci (PUL) involved in sucrose and raffinose family oligosaccharide (RFO) metabolism from one of the most common Firmicutes present in individuals, Ruminococcus gnavus E1. One of the enzymes encoded by this PUL was annotated as a putative sucrose phosphate phosphorylase (RgSPP).

In Vitro Synthesis and Crystallization of β-1,4-Mannan.

In vitro polymerization of β-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the β-anomer. Whatever the approach used to date-whether chemical, or enzymatic with glycosynthases and mannosyltransferases-pure β-1,4-mannans have never been synthesized in vitro. This has limited attempts to investigate their role in the production of plant and algal cell walls, in which they are highly abundant.

Highly Promiscuous Oxidases Discovered in the Bovine Rumen Microbiome.

The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated.

Two new gene clusters involved in the degradation of plant cell wall from the fecal microbiota of Tunisian dromedary.

Dromedaries are capable of digesting plant cell wall with high content of lignocellulose of poor digestibility. Consequently, their intestinal microbiota can be a source of novel carbohydrate-active enzymes (CAZymes). To the best of our knowledge, no data are available describing the biochemical analysis of enzymes in dromedary intestinal microbiota.

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening.

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells.

Macromolecular structure and film properties of enzymatically-engineered high molar mass dextrans.

New α(1→2) or α(1→3) branched dextrans with high molar masses and controlled architecture were synthesized using a dextransucrase and branching sucrases. Their molecular structure, solubility, conformation, film-forming ability, as well as their thermal and mechanical properties were determined. These new dextrans present structures with low densities from 9,500 to 14,000gm in HO/DMSO medium, their molar mass, size and dispersity increase with increasing branching degree (weight-average molar mass up to 10gmol and radius of gyration around 500nm).

Discovery of carbamate degrading enzymes by functional metagenomics.

Bioremediation of pollutants is a major concern worldwide, leading to the research of new processes to break down and recycle xenobiotics and environment contaminating polymers. Among them, carbamates have a very broad spectrum of uses, such as toxinogenic pesticides or elastomers. In this study, we mined the bovine rumen microbiome for carbamate degrading enzymes.

Biochemical characterization of Arabidopsis thaliana starch branching enzyme 2.2 reveals an enzymatic positive cooperativity.

Starch Branching Enzymes (SBE) catalyze the formation of α(1 → 6) branching points on starch polymers: amylopectin and amylose. SBEs are classified in two groups named type 1 and 2. Both types are present in the entire plant kingdom except in some species such as Arabidopsis thaliana that expresses two type 2 SBEs: BE2.

Mannoside recognition and degradation by bacteria.

Mannosides constitute a vast group of glycans widely distributed in nature. Produced by almost all organisms, these carbohydrates are involved in numerous cellular processes, such as cell structuration, protein maturation and signalling, mediation of protein-protein interactions and cell recognition. The ubiquitous presence of mannosides in the environment means they are a reliable source of carbon and energy for bacteria, which have developed complex strategies to harvest them.

Functional characterization of a gene locus from an uncultured gut Bacteroides conferring xylo-oligosaccharides utilization to Escherichia coli.

In prominent gut Bacteroides strains, sophisticated strategies have been evolved to achieve the complete degradation of dietary polysaccharides such as xylan, which is one of the major components of the plant cell wall. Polysaccharide Utilization Loci (PULs) consist of gene clusters encoding different proteins with a vast arsenal of functions, including carbohydrate binding, transport and hydrolysis. Transport is often attributed to TonB-dependent transporters, although major facilitator superfamily (MFS) transporters have also been identified in some PULs.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial ecosystems.

Background: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans.

Results: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database.