Discovering novel carbohydrate-active enzymes

Carbohydrate-active enzymes (CAZymes) mediate the assembly and breakdown of the multitude of complex carbohydrate structures that are found in nature. Because CAZymes find numerous applications in health and in industry, the discovery of novel enzymes is of wide interest. A sequence-based family classification of these enzymes was initiated over 25 years ago, and is continuously updated in the CAZy database (www.cazy.org). While the number of sequences of CAZymes continues to grow exponentially, the number of experimentally characterized enzymes grows at a much lower pace. Strikingly, the number of families keeps on growing at a steady rate, suggesting that many more CAZyme families are to be discovered. Traditionally CAZymes were identified (i) by serendipity, (ii) by screening against a particular substrate or (iii) by direct similarity to a previously characterized CAZyme. We will present various alternative methods that we have used recently to discover novel CAZymes and novel CAZyme families, including the module walk, the exploitation of genetic context and the systematic exploration of sequence space. Please click Additional Files below to see the full abstract

Possible adsorption sites of cellulases on crystalline cellulose

AbstractThe possible adsorption sites of cellulases on crystalline cellulose were investigated by molecular graphic representation of a crystal of cellulose and estimation of the accessibility of the various glycosidic bonds to enzymatic attack. The results show that only certain glycosidic bonds of certain surface cellulose chains are susceptible to enzymatic hydrolysis. These preferential sites correlate well with previous electron microscopy observations of the adsorption sites of 1,4-β-D-glucan cellobiohydrolase I (CBHI) from Trichoderma reesei on Valonia cellulose

The mucin-degradation strategy of Ruminococcus gnavus:The importance of intramolecular trans-sialidases

We previously identified and characterized an intramolecular trans-sialidase (IT-sialidase) in the gut symbiont Ruminococcus gnavus ATCC 29149, which is associated to the ability of the strain to grow on mucins. In this work we have obtained and analyzed the draft genome sequence of another R. gnavus mucin-degrader, ATCC 35913, isolated from a healthy individual. Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans. R. gnavus ATCC 35913 also encodes a predicted IT-sialidase and harbors a Nan cluster dedicated to sialic acid utilization. We showed that the Nan cluster was upregulated when the strains were grown in presence of mucin. In addition we demonstrated that both R. gnavus strains were able to grow on 2,7-anyhydro-Neu5Ac, the IT-sialidase transglycosylation product, as a sole carbon source. Taken together these data further support the hypothesis that IT-sialidase expressing gut microbes, provide commensal bacteria such as R. gnavus with a nutritional competitive advantage, by accessing and transforming a source of nutrient to their own benefit

Viewing the human microbiome through three-dimensional glasses: integrating structural and functional studies to better define the properties of myriad carbohydrate-active enzymes

Metagenomics has unleashed a deluge of sequencing data describing the organismal, genetic, and transcriptional diversity of the human microbiome. To better understand the precise functions of the myriad proteins encoded by the microbiome, including carbohydrate-active enzymes, it will be critical to combine structural studies with functional analyses

Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp

Fungi are important for biomass degradation processes in mangrove forests. Given the presence of sea water in these ecosystems, mangrove fungi are adapted to high salinity. Here we isolate Pestalotiopsis sp. NCi6, a halotolerant and lignocellulolytic mangrove fungus of the order Xylariales. We study its lignocellulolytic enzymes and analyse the effects of salinity on its secretomes. De novo transcriptome sequencing and assembly indicate that this fungus possesses of over 400 putative lignocellulolytic enzymes, including a large fraction involved in lignin degradation. Proteomic analyses of the secretomes suggest that the presence of salt modifies lignocellulolytic enzyme composition, with an increase in the secretion of xylanases and cellulases and a decrease in the production of oxidases. As a result, cellulose and hemicellulose hydrolysis is enhanced but lignin breakdown is reduced. This study highlights the adaptation to salt of mangrove fungi and their potential for biotechnological applications

Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

<p>Abstract</p> <p>Background</p> <p>Spore-forming <it>Bacilli </it>are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI)-tract of animals. Isolates of various <it>Bacillus </it>species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms.</p> <p>Results</p> <p>We report the annotation of carbohydrate active enzymes (CAZymes) of two pigmented <it>Bacilli </it>isolated from the human GI-tract and belonging to the <it>Bacillus indicus </it>and <it>B. firmus </it>species. A high number of glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both <it>Bacilli</it>. Experimental results on biofilm formation confirmed genomic data on the potentials of <it>B. indicus </it>HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both <it>Bacilli </it>to degrade mammalian glycans.</p> <p>Conclusions</p> <p>CAZy analyses of the genomes of the two pigmented <it>Bacilli</it>, compared to other <it>Bacillus </it>species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented <it>Bacilli </it>are adapted to the intestinal environment and are suited to grow in and colonize the human gut.</p

Rapid divergence of genome architectures following the origin of an ectomycorrhizal symbiosis in the genus <i>Amanita</i>

Fungi are evolutionary shape shifters and adapt quickly to new environments. Ectomycorrhizal (EM) symbioses are mutualistic associations between fungi and plants and have evolved repeatedly and independently across the fungal tree of life, suggesting lineages frequently reconfigure genome content to take advantage of open ecological niches. To date analyses of genomic mechanisms facilitating EM symbioses have involved comparisons of distantly related species, but here, we use the genomes of three EM and two asymbiotic (AS) fungi from the genus Amanita as well as an AS outgroup to study genome evolution following a single origin of symbiosis. Our aim was to identify the defining features of EM genomes, but our analyses suggest no clear differentiation of genome size, gene repertoire size, or transposable element content between EM and AS species. Phylogenetic inference of gene gains and losses suggests the transition to symbiosis was dominated by the loss of plant cell wall decomposition genes, a confirmation of previous findings. However, the same dynamic defines the AS species A. inopinata, suggesting loss is not strictly associated with origin of symbiosis. Gene expansions in the common ancestor of EM Amanita were modest, but lineage specific and large gene family expansions are found in two of the three EM extant species. Even closely related EM genomes appear to share few common features. The genetic toolkit required for symbiosis appears already encoded in the genomes of saprotrophic species, and this dynamic may explain the pervasive, recurrent evolution of ectomycorrhizal associations

The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics

The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families. These families are created based on experimentally characterized proteins and are populated by sequences from public databases with significant similarity. Protein biochemical information is continuously curated based on the available literature and structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification (i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties. This resource has been available for over 10 years to the scientific community, contributing to information dissemination and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/

The human gut microbe <i>Bacteroides thetaiotaomicron</i> encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of yet uncharacterized GAG-inducible genes in its genome and that of related species however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron or Bt) and ability to degrade the GAGs CS, dermatan sulfate (DS) and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac) as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologues were also detected in several other HGM species and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM

Niche differentiation and evolution of the wood decay machinery in the invasive fungus Serpula lacrymans

Abstract Ecological niche breadth and the mechanisms facilitating its evolution are fundamental to understanding adaptation to changing environments, persistence of generalist and specialist lineages and the formation of new species. Woody substrates are structurally complex resources utilized by organisms with specialized decay machinery. Wood-decaying fungi represent ideal model systems to study evolution of niche breadth, as they vary greatly in their host range and preferred decay stage of the substrate. In order to dissect the genetic basis for niche specialization in the invasive brown rot fungus Serpula lacrymans , we used phenotyping and integrative analysis of phylogenomic and transcriptomic data to compare this species to wild relatives in the Serpulaceae with a range of specialist to generalist decay strategies. Our results indicate specialist species have rewired regulatory networks active during wood decay towards decreased reliance on enzymatic machinery, and therefore nitrogen-intensive decay components. This shift was likely accompanied with adaptation to a narrow tree line habitat and switch to a pioneer decomposer strategy, both requiring rapid colonization of a nitrogen-limited substrate. Among substrate specialists with narrow niches, we also found evidence for pathways facilitating reversal to generalism, highlighting how evolution may move along different axes of niche space