The complex carbohydrates of terrestrial and marine biomass represent a rich nutrient source for free-living and mutualistic microbes alike. The enzymatic saccharification of these diverse substrates is of critical importance for fueling a variety of complex microbial communities, including marine, soil, ruminant, and monogastric microbiota. Consequently, highly specific carbohydrate-active enzymes, recognition proteins, and transporters are enriched in the genomes of certain species and are of critical importance in competitive environments. In Bacteroidetes bacteria, these systems are organized as polysaccharide utilization loci (PULs), which are strictly regulated, colocalized gene clusters that encode enzyme and protein ensembles required for the saccharification of complex carbohydrates. This review provides historical perspectives and summarizes key findings in the study of these systems, highlighting a critical shift from sequence-based PUL discovery to systems-based analyses combining reverse genetics, biochemistry, enzymology, and structural biology to precisely illuminate the molecular mechanisms underpinning PUL function. The ecological implications of dynamic PUL deployment by key species in the human gastrointestinal tract are explored, as well as the wider distribution of these systems in other gut, terrestrial, and marine environments.

A genetic locus from the gut symbiont Bacteroides ovatus is identified and described that encodes a cohort of enzymes and carbohydrate-binding proteins necessary for the metabolism of xyloglucans—a predominant component of dietary fibre. We can derive energy from dietary fibre thanks largely to the ability of the gut microbiota to digest and ferment complex polysaccharides. The mechanism of degradation of one ubiquitous family of branched plant cell wall polysaccharides, the xyloglucans, has been unclear. Here Harry Brumer and colleagues report the identification and molecular characterization of an archetypal genetic locus from the gut symbiont Bacteroides ovatus encoding enzymes dedicated to xyloglucan utilization. The ability to utilize xyloglucan is shown not to be universal among the Bacteroidetes but rather is carried out by a few specialists. The importance of a niche species of bacteria in the utilization of such an abundant form of dietary fibre is of relevance to the design of dietary strategies for the treatment of intestinal disease and highlights the need for careful strain selection and monitoring for achieving a healthy, balanced microbiota. A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed ‘dietary fibre’, from the cell walls of diverse fruits and vegetables1. Owing to the paucity of alimentary enzymes encoded by the human genome2, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut3,4. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides5,6 whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear1,7,8. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health9,10,11,12.

Glycoside Hydrolase Family 16 (GH16) comprises a large and taxonomically diverse family of glycosidases and transglycosidases that adopt a common beta-jelly-roll fold and are active on a range of terrestrial and marine polysaccharides. Presently, broadly insightful sequence-function correlations in GH16 are hindered by a lack of a systematic subfamily structure. To fill this gap, we have used a highly scalable protein Sequence Similarity Network (SSN) analysis to delineate nearly 23,000 GH16 sequences into 23 robust subfamilies, which are strongly supported by Hidden Markov Model (HMM) and Maximum Likelihood (ML) molecular phylogenetic analyses. Subsequent evaluation of over 40 experimental three-dimensional structures has highlighted key tertiary structural differences, predominantly manifested in active-site loops, which dictate substrate specificity across the GH16 evolutionary landscape. As for other large GH families (i.e. GH5, GH13, and GH43), this new subfamily classification provides a roadmap for functional glycogenomics that will guide future bioinformatics and experimental structure-function analyses. The GH16 subfamily classification is publicly available in the CAZy database via URL www.cazy.org/GH16.html. The SSN workflow used here is available via URL https://github.com/ahvdk/SSNpipe/.

Microbial utilization of complex polysaccharides is a major driving force in shaping the composition of the human gut microbiota. There is a growing appreciation that finely tuned polysaccharide utilization loci enable ubiquitous gut Bacteroidetes to thrive on the plethora of complex polysaccharides that constitute "dietary fiber." Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs) are a key family of plant cell wall polysaccharides with recognized health benefits but whose mechanism of utilization has remained unclear. Here, we provide molecular insight into the function of an archetypal MLG utilization locus (MLGUL) through a combination of biochemistry, enzymology, structural biology, and microbiology. Comparative genomics coupled with growth studies demonstrated further that syntenic MLGULs serve as genetic markers for MLG catabolism across commensal gut bacteria. In turn, we surveyed human gut metagenomes to reveal that MLGULs are ubiquitous in human populations globally, which underscores the importance of gut microbial metabolism of MLG as a common cereal polysaccharide.

Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.