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Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]