Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.

Probiotic dosing of Ruminococcus flavefaciens affects rumen microbiome structure and function in reindeer.

Highly cellulolytic bacterial species such as Ruminococcus flavefaciens are regarded essential for the microbial breakdown of cellulose in the rumen. We have investigated the effect of ruminal dosing of R. flavefaciens strain 8/94-32 during realimentation of starved reindeer (males, n = 3). Microbiome function measured as in situ digestion of cellulose and food pellets (percent DMD; dry matter disappearance) decreased after probiotic dosing. Microbial community analyses (>100,000 16S rDNA gene sequences for 27 samples) demonstrated that ruminal dosing influenced the microbiome structure; reflected by increased phylogenetic distances from background samples (unweighted UniFrac analysis) and reduced species diversity and evenness. Despite the inability to detect strain 8/94-32 post-dosing, the relative abundance of its affiliate family Ruminococcaceae remained consistent throughout the trial, whilst a dominant peak in the genus Prevotella and decline in uncharacterized Bacteroidetes (uBacNR) were observed in treatment samples. No clear relationships were observed between the relative abundance of Ruminococcaceae, Prevotella and uBacNR with cellulose DMD; however, Prevotella (negative) and uBacNR (positive) exhibited relationships with pellet DMD. These unexpected effects of ruminal dosing of a cellulolytic bacterium on digestibility are relevant for other studies on rumen manipulation.

Functional metagenomics reveals abundant polysaccharide-degrading gene clusters and cellobiose utilization pathways within gut microbiota of a wood-feeding higher termite.

Plant cell-wall polysaccharides constitute the most abundant but recalcitrant organic carbon source in nature. Microbes residing in the digestive tract of herbivorous bilaterians are particularly efficient at depolymerizing polysaccharides into fermentable sugars and play a significant support role towards their host's lifestyle. Here, we combine large-scale functional screening of fosmid libraries, shotgun sequencing, and biochemical assays to interrogate the gut microbiota of the wood-feeding "higher" termite Globitermes brachycerastes. A number of putative polysaccharide utilization gene clusters were identified with multiple fibrolytic genes. Our large-scale functional screening of 50,000 fosmid clones resulted in 464 clones demonstrating plant polysaccharide-degrading activities, including 267 endoglucanase-, 24 exoglucanase-, 72 ß-glucosidase-, and 101 endoxylanase-positive clones. We sequenced 173 functionally active clones and identified ~219 genes encoding putative carbohydrate-active enzymes (CAZymes) targeting cellulose, hemicellulose and pectin. Further analyses revealed that 68 of 154 contigs encode one or more CAZyme, which includes 35 examples of putative saccharolytic operons, suggesting that clustering of CAZymes is common in termite gut microbial inhabitants. Biochemical characterization of a representative xylanase cluster demonstrated that constituent enzymes exhibited complementary physicochemical properties and saccharolytic capabilities. Furthermore, diverse cellobiose-metabolizing enzymes include ß-glucosidases, cellobiose phosphorylases, and phopho-6-ß-glucosidases were identified and functionally verified, indicating that the termite gut micro-ecosystem utilizes diverse metabolic pathways to interconnect hydrolysis and central metabolism. Collectively, these results provide an in-depth view of the adaptation and digestive strategies employed by gut microbiota within this tiny-yet-efficient host-associated ecosystem.

From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus.

Microbial communities that degrade lignocellulosic biomass are typified by high levels of species- and strain-level complexity, as well as synergistic interactions between both cellulolytic and non-cellulolytic microorganisms. Coprothermobacter proteolyticus frequently dominates thermophilic, lignocellulose-degrading communities with wide geographical distribution, which is in contrast to reports that it ferments proteinaceous substrates and is incapable of polysaccharide hydrolysis. Here we deconvolute a highly efficient cellulose-degrading consortium (SEM1b) that is co-dominated by Clostridium (Ruminiclostridium) thermocellum and multiple heterogenic strains affiliated to C. proteolyticus. Metagenomic analysis of SEM1b recovered metagenome-assembled genomes (MAGs) for each constituent population, whereas in parallel two novel strains of C. proteolyticus were successfully isolated and sequenced. Annotation of all C. proteolyticus genotypes (two strains and one MAG) revealed their genetic acquisition of carbohydrate-active enzymes (CAZymes), presumably derived from horizontal gene transfer (HGT) events involving polysaccharide-degrading Firmicutes or Thermotogae-affiliated populations that are historically co-located. HGT material included a saccharolytic operon, from which a CAZyme was biochemically characterized and demonstrated hydrolysis of multiple hemicellulose polysaccharides. Finally, temporal genome-resolved metatranscriptomic analysis of SEM1b revealed expression of C. proteolyticus CAZymes at different SEM1b life stages as well as co-expression of CAZymes from multiple SEM1b populations, inferring deeper microbial interactions that are dedicated toward community degradation of cellulose and hemicellulose. We show that C. proteolyticus, a ubiquitous population, consists of closely related strains that have adapted via HGT to presumably degrade both oligo- and longer polysaccharides present in decaying plants and microbial cell walls, thus explaining its dominance in thermophilic anaerobic digesters on a global scale.

Discovery and characterization of a thermostable two-domain GH6 endoglucanase from a compost metagenome.

Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.

Metagenomics and CAZyme Discovery.

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems using common culture-dependent techniques restricts access to potentially novel cellulolytic bacteria and beneficial enzymes. The development of molecular-based culture-independent methods such as metagenomics enables researchers to study microbial communities directly from environmental samples, and presents a platform from which enzymes of interest can be sourced. We outline key methodological stages that are required as well as describe specific protocols that are currently used for metagenomic projects dedicated to CAZyme discovery.

Omics-based interpretation of synergism in a soil-derived cellulose-degrading microbial community.

Reaching a comprehensive understanding of how nature solves the problem of degrading recalcitrant biomass may eventually allow development of more efficient biorefining processes. Here we interpret genomic and proteomic information generated from a cellulolytic microbial consortium (termed F1RT) enriched from soil. Analyses of reconstructed bacterial draft genomes from all seven uncultured phylotypes in F1RT indicate that its constituent microbes cooperate in both cellulose-degrading and other important metabolic processes. Support for cellulolytic inter-species cooperation came from the discovery of F1RT microbes that encode and express complimentary enzymatic inventories that include both extracellular cellulosomes and secreted free-enzyme systems. Metabolic reconstruction of the seven F1RT phylotypes predicted a wider genomic rationale as to how this particular community functions as well as possible reasons as to why biomass conversion in nature relies on a structured and cooperative microbial community.

Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.