Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses.

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria.

Gut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as "medium grower" and "high grower." Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and "omics" approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem. Video abstract.

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

Effect of ammonia fiber expansion-treated wheat straw and a recombinant fibrolytic enzyme on rumen microbiota and fermentation parameters, total tract digestibility, and performance of lambs.

The objective of this study was to evaluate the effect of ammonia fiber expansion (AFEX)-treated wheat straw pellets and a recombinant fibrolytic enzyme on the rumen microbiome, rumen fermentation parameters, total tract diet digestibility, and performance of lambs. Eight rumen cannulated wethers and 60 lambs (n = 15 per diet, 8 rams and 7 ewes) were used in a replicated 4 × 4 Latin square design digestibility study and a complete randomized growth performance study, respectively. Four treatment diets were arranged in a 2 × 2 factorial structure with AFEX wheat straw (0% or 30% AFEX straw pellets on a dietary DM basis replacing alfalfa hay pellets) and fibrolytic enzyme (with or without XYL10C, a ß-1,4-xylanase, from Aspergillus niger) as main factors. Enzyme was applied at 100 mg/kg of diet DM, 22 h before feeding. Rumen bacteria diversity Pielou evenness decreased (P = 0.05) with AFEX compared with the control diet and increased (P < 0.01) with enzyme. Enzyme increased (P ≤ 0.02) the relative abundancies of Prevotellaceae UCG-004, Christensenellaceae R-7 group, Saccharofermentans, and uncultured Kiritimatiellaeota. Total protozoa counts were greater (P ≤ 0.04) in the rumen of lambs fed AFEX compared with control, with enzyme reducing (P ≤ 0.05) protozoa counts for both diets. Digestibility of DM did not differ (P > 0.10) among diets, but digestibility of CP was reduced (P = 0.001), and digestibility of NDF and ADF increased (P < 0.05) as AFEX replaced alfalfa. Compared with control, AFEX promoted greater DMI (P = 0.003) and improved ADG up to 42 d on feed (P = 0.03), but not (P = 0.51) over the full ~94-d experiment. Consequently, overall G:F was reduced (P = 0.04) for AFEX when compared with control (0.188 vs. 0.199), but days on feed were lower (P = 0.04) for AFEX (97 vs. 91 d). Enzyme improved DMI of AFEX up to day 70 (P = 0.01), but did not affect DMI of the control diet. Enzyme addition improved ADG of lambs fed both diets in the first 28 d (P = 0.02), but not over the entire feeding period (P ≥ 10). As a result, G:F was improved with enzyme for the first 28 d (P = 0.04), but not overall (P = 0.45). This study shows that AFEX-treated wheat straw can replace alfalfa hay with no loss in lamb growth performance. Additionally, the enzyme XYL10C altered the rumen microbiome and improved G:F in the first month of the feeding.

Analysis of Active Site Architecture and Reaction Product Linkage Chemistry Reveals a Conserved Cleavage Substrate for an Endo-alpha-mannanase within Diverse Yeast Mannans.

Yeast α-mannan (YM) is a densely branched N-linked glycan that decorates the surface of yeast cell walls. Owing to the high degree of branching, cleavage of the backbone of YM appears to rely on the coupled action of side-chain-cleaving enzymes. Upon examining the genome sequences of bovine-adapted Bacteroides thetaiotaomicron strains, isolated for their ability to degrade YM, we have identified a tandem pair of genes inserted into an orphan pathway predicted to be involved in YM metabolism. Here, we investigated the activity of one of these enzymes, a predicted endo-mannanase from glycoside hydrolase (GH) family 76 (BtGH76-MD40). Purified recombinant BtGH76-MD40 displayed activity on structurally distinct YMs from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Linkage analysis of released oligosaccharide products from S. cerevisiae and S. pombe mannan determined BtGH76-MD40 targets a specific linkage that is conserved in structurally diverse YM substrates. In addition, using two differential derivatization methods, we have shown that there is an absolute requirement for undecorated d-mannopyranose in the -1 subsite. Determination of the BtGH76-MD40 X-ray crystal structure and structural superimposition and molecular docking of a branched alpha-mannopentatose substrate supported these findings. In contrast, BtGH76-MD40 can accommodate extended side chains in the +1 and -2 subsites, highlighting that a single alpha-1,6-mannosyl residue is a prerequisite for activity, and cleavage occurs at the reducing end of the undecorated monosaccharide. Collectively these results demonstrate how acquisition of new enzymes within extant pathways contributes to the functional abilities of saccharolytic bacteria persisting in complex digestive ecosystems.

Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron.

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Identification of novel enzymes to enhance the ruminal digestion of barley straw.

Crude enzyme extracts typically contain a broad spectrum of enzyme activities, most of which are redundant to those naturally produced by the rumen microbiome. Identification of enzyme activities that are synergistic to those produced by the rumen microbiome could enable formulation of enzyme cocktails that improve fiber digestion in ruminants. Compared to untreated barley straw, Viscozyme® increased gas production, dry matter digestion (P < 0.01) and volatile fatty acid production (P < 0.001) in ruminal batch cultures. Fractionation of Viscozyme® by Blue Native PAGE and analyses using a microassay and mass-spectrometry revealed a GH74 endoglucanase, GH71 α-1,3-glucanase, GH5 mannanase, GH7 cellobiohydrolase, GH28 pectinase, and esterases from Viscozyme® contributed to enhanced saccharification of barley straw by rumen mix enzymes. Grouping of these identified activities with their carbohydrate active enzymes (CAZy) counterparts enabled selection of similar CAZymes for downstream production and screening. Mining of these specific activities from other biological systems could lead to high value enzyme formulations for ruminants.

SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.

BACKGROUND: Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. RESULTS: SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. CONCLUSIONS: Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates.

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, ß1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and ß1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both ß-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

An Improved Kinetic Assay for the Characterization of Metal-Dependent Pectate Lyases.

Pectate lyases are a subset of polysaccharide lyases (PLs) that specifically utilize a metal dependent ß-elimination mechanism to cleave glyosidic bonds in homogalacturonan (HG; α-D-1,4-galacturonic acid). Most commonly, PLs harness calcium for catalysis; however, some PL families (e.g., PL2 and PL22) display preferences for transitional metals. Deploying alternative metals during ß-elimination is correlated with signature coordination pocket chemistry, and is reflective of the evolution, functional specialization, and cellular location of PL activity. Here we describe an optimized method for the analysis of metal-dependent polysaccharide lyases (PLs). We use an endolytic PL2 from Yersinia enterocolitica (YePL2A) as example to demonstrate how altering the catalytic metal within the reaction can modulate PL kinetics.

Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the ß-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus.

A novel non-canonical forkhead-associated (FHA) domain-binding interface mediates the interaction between Rad53 and Dbf4 proteins.

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase.

Dbf4 is a conserved eukaryotic protein that functions as the regulatory subunit of the Dbf4-dependent kinase (DDK) complex. DDK plays essential roles in DNA replication initiation and checkpoint activation. During the replication checkpoint, Saccharomyces cerevisiae Dbf4 is phosphorylated in a Rad53-dependent manner, and this, in turn, inhibits initiation of replication at late origins. We have determined the minimal region of Dbf4 required for the interaction with the checkpoint kinase Rad53 and solved its crystal structure. The core of this fragment of Dbf4 folds as a BRCT domain, but it includes an additional N-terminal helix unique to Dbf4. Mutation of the residues that anchor this helix to the domain core abolish the interaction between Dbf4 and Rad53, indicating that this helix is an integral element of the domain. The structure also reveals that previously characterized Dbf4 mutants with checkpoint phenotypes destabilize the domain, indicating that its structural integrity is essential for the interaction with Rad53. Collectively, these results allow us to propose a model for the association between Dbf4 and Rad53.

The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress.

The Dbf4/Cdc7 kinase (DDK) plays an essential role in stimulating DNA replication by phosphorylating subunits of the Mcm2-7 helicase complex at origins. This kinase complex is itself phosphorylated and removed from chromatin in a Rad53-dependent manner when an S phase checkpoint is triggered. Comparison of Dbf4 sequence across a variety of eukaryotic species has revealed three conserved regions that have been termed motifs N, M and C. The most highly conserved of the three, motif C, encodes a zinc finger, which are known to mediate protein-protein and protein-DNA interactions. Mutation of conserved motif C cysteines and histidines disrupted the association of Dbf4 with ARS1 origin DNA and Mcm2, but not other known ligands including Cdc7, Rad53 or the origin recognition complex subunit Orc2. Furthermore, these mutations impaired the ability of Dbf4 to phosphorylate Mcm2. Budding yeast strains for which the single genomic DBF4 copy was replaced with these motif C mutant alleles were compromised for entry into and progression through S phase, indicating that the observed weakening of the Mcm2 interaction prevents DDK from efficiently stimulating the initiation of DNA replication. Following initiation, Mcm2-7 migrates with the replication fork. Interestingly, the motif C mutants were sensitive to long-term, but not short-term exposure to the genotoxic agents hydroxyurea and methyl methanesulfonate. These results support a model whereby DDK interaction with Mcm2 is important to stabilize and/or restart replication forks during conditions where a prolonged S-phase checkpoint is triggered.