CAZyme Profile Research Articles

Tailored microbial consortium producing hydrolytic enzyme cocktail for maximum saccharification of wheat straw

The potential of hydrolytic enzyme cocktail obtained from designed bacterial consortium WSh-1 comprising Bacillus subtilis CRN 16, Paenibacillus dendritiformis CRN 18, Niallia circulans CRN 24, Serratia marscens CRN 29, and Streptomyces sp. CRN 30, was investigated for maximum saccharification. Activity was further enhanced to 1.01 U/ml from 0.82 U/ml by supplementing growth medium with biotin and cellobiose as a cofactor and inducer. Through kinetic analysis, the enzyme cocktail showed a high wheat straw affinity with Michaelis-Menten constant (Km) of 0.68 µmol/L and a deconstruction rate (Vmax) of 4.5 U/ml/min. The statistical optimization of critical parameters increased saccharification to 89 %. The optimized process in a 5-L lab-scale bioreactor yielded 501 mg/g of reducing sugar from NaOH-pretreated wheat straw. Lastly, genomic insights revealed unique abundant oligosaccharide deconstruction enzymes with the most diverse CAZyme profile. The consortium-mediated enzyme cocktails offer broader versatility with efficiency for the economical and sustainable valorization of lignocellulosic waste.

Comparative Rumen Metagenome and CAZyme Profiles in Cattle and Buffaloes: Implications for Methane Yield and Rumen Fermentation on a Common Diet.

A study was undertaken to compare the rumen microbial community composition, methane yield, rumen fermentation, and CAZyme profiles between cattle and buffaloes. The primary aim of this study was to ascertain the impact of the host species on the above when diet and environmental factors are fixed. A total of 43 phyla, 200 orders, 458 families, and 1722 microbial genera were identified in the study. Bacteroidetes was the most prominent bacterial phylum and constituted >1/3rd of the ruminal microbiota; however, their abundances were comparable between cattle and buffaloes. Firmicutes were the second most abundant bacteria, found to be negatively correlated with the Bacteroidetes. The abundances of Firmicutes as well as the F/B ratio were not different between the two host species. In this study, archaea affiliated with the nine phyla were identified, with Euryarchaeota being the most prominent. Like bacterial phyla, the abundances of Euryarchaeota methanogens were also similar between the cattle and buffaloes. At the order level, Methanobacteriales dominated the archaea. Methanogens from the Methanosarcinales, Methanococcales, Methanomicrobiales, and Methanomassiliicoccales groups were also identified, but at a lower frequency. Methanobrevibacter was the most prevalent genus of methanogens, accounting for approximately three percent of the rumen metagenome. However, their distribution was not different between the two host species. CAZymes affiliated with five classes, namely CBM, CE, GH, GT, and PL, were identified in the metagenome, where the GH class was the most abundant and constituted ~70% of the total CAZymes. The protozoal numbers, including Entodiniomorphs and Holotrichs, were also comparable between the cattle and buffaloes. Results from the study did not reveal any significant difference in feed intake, nutrient digestibility, and rumen fermentation between cattle and buffaloes fed on the same diet. As methane yield due to the similar diet composition, feed ingredients, rumen fermentation, and microbiota composition did not vary, these results indicate that the microbiota community structure and methane emissions are under the direct influence of the diet and environment, and the host species may play only a minor role until the productivity does not vary. More studies are warranted to investigate the effect of different diets and environments on microbiota composition and methane yield. Further, the impact of variable productivity on both the cattle and buffaloes when the diet and environmental factors are fixed needs to be ascertained.

Bioconversion of citrus waste by long-term DMSO-cryopreserved rumen fluid to volatile fatty acids and biogas is feasible: A microbiome perspective

Preserving rumen fluid as the inoculum for anaerobic digestion of food waste is necessary when access to animal donors or slaughterhouses is limited. This study aims to compare two preservation methods relative to fresh ruminal inoculum: (1) cryoprotected with 5% dimethyl sulfoxide (DMSO) and stored at −20 °C and (2) frozen at −20 °C, both for 6 months. The fermentation activity of different inoculum was evaluated by rumen-based in vitro anaerobic fermentation tests (volatile fatty acids, biomass digestibility, and gas production). Citrus pomace was used as the substrate during a 96-h fermentation. The maximum volatile fatty acids, methane production, and citrus pomace digestibility from fresh rumen fluid were not significantly different from rumen fluid preserved with DMSO. Metagenome analysis revealed a significant difference in the rumen microbial composition and functions between fresh rumen fluid and frozen inoculum without DMSO. Storage of rumen fluid using −20 °C with DMSO demonstrated the less difference compared with fresh rumen fluid in microbial alpha diversity and taxa composition. The hierarchical clustering tree of CAZymes showed that DMSO cryoprotected fluid was clustered much closer to the fresh rumen fluid, showing more similarity in CAZyme profiles than frozen rumen fluid. The abundance of functional genes associated with carbohydrate metabolism and methane metabolism did not differ between fresh rumen fluid and the DMSO–20 °C, whereas the abundance of key functional genes significantly decreased in frozen rumen fluid. These findings suggest that using rumen liquid preserved using DMSO at −20 °C for 180 days is a feasible alternative to fresh rumen fluid. This would reduce the need for laboratories to maintain animal donors and/or reduce the frequency of collecting rumen fluid from slaughterhouses.

Biocatalytic potential of Pseudolycoriella CAZymes (Sciaroidea, Diptera) in degrading plant and fungal cell wall polysaccharides

Soil biota has a crucial impact on soil ecology, global climate changes, and effective crop management and studying the diverse ecological roles of dipteran larvae deepens the understanding of soil food webs. A multi-omics study of Pseudolycoriella hygida comb. nov. (Diptera: Sciaroidea: Sciaridae) aimed to characterize carbohydrate-active enzymes (CAZymes) for litter degradation in this species. Manual curation of 17,881 predicted proteins in the Psl. hygida genome identified 137 secreted CAZymes, of which 33 are present in the saliva proteome, and broadly confirmed by saliva CAZyme catalytic profiling against plant cell wallpolysaccharides and pNP-glycosyl substrates. Comparisons with two other sciarid species and the outgroup Lucilia cuprina (Diptera: Calliphoridae) identified 42 CAZyme families defining a sciarid CAZyme profile. The litter-degrading potential of sciarids corroborates their significant role as decomposers, yields insights to the evolution of insect feeding habits, and highlights the importance of insects as a source of biotechnologically relevant enzymes.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Integrative functional analysis uncovers metabolic differences between Candida species.

Candida species are a dominant constituent of the human mycobiome and associated with the development of several diseases. Understanding the Candida species metabolism could provide key insights into their ability to cause pathogenesis. Here, we have developed the BioFung database, providing an efficient annotation of protein-encoding genes. Along, with BioFung, using carbohydrate-active enzyme (CAZymes) analysis, we have uncovered core and accessory features across Candida species demonstrating plasticity, adaption to the environment and acquired features. We show a greater importance of amino acid metabolism, as functional analysis revealed that all Candida species can employ amino acid metabolism. However, metabolomics revealed that only a specific cluster of species (AGAu species—C. albicans, C. glabrata and C. auris) utilised amino acid metabolism including arginine, cysteine, and methionine metabolism potentially improving their competitive fitness in pathogenesis. We further identified critical metabolic pathways in the AGAu cluster with biomarkers and anti-fungal target potential in the CAZyme profile, polyamine, choline and fatty acid biosynthesis pathways. This study, combining genomic analysis, and validation with gene expression and metabolomics, highlights the metabolic diversity with AGAu species that underlies their remarkable ability to dominate they mycobiome and cause disease.

Carbohydrate Active Enzyme Profiles of Gut Microbiomes From Low and Adequate Fiber Consumers in a Healthy U.S. Cohort

ObjectivesCarbohydrate structures are incredibly diverse, but humans possess only about a dozen endogenous enzymes to break down carbohydrates in the diet. Microbial enzymes, produced by the gut microbiota, can degrade and ferment what human bodies cannot. We studied the microbial genes for enzymes that degrade carbohydrates in the microbiota of healthy adult men and women in the USDA Nutritional Phenotyping Study. Individual carbohydrate active enzyme (CAZyme) gene abundances and CAZyme family diversity metrics were compared between groups of adequate and low fiber consumers in the study. We hypothesized that the CAZyme profiles of these different fiber consumption groups would differ, with adequate fiber consumers showing higher CAZyme family diversity and a different suite of CAZyme genes than the low fiber consumers.MethodsThe USDA Nutritional Phenotyping Study is a cross-sectional observational trial designed to evaluate dietary and other factors associated with metabolic health. Participants completed dietary recalls with ASA24 and a Block 2014 Food frequency questionnaire and submitted stool samples for shotgun metagenomics and fecal pH measurements. Adequate (14g/1000kcal) and low fiber consumers (lowest quartile) were identified using both dietary instruments. DNA was extracted from fecal samples and sequenced. Shotgun metagenomes (n = 290) were analyzed and mapped to the CAZy database. DESeq2 was used to compare CAZyme gene abundances. Plant substrate CAZyme family diversity using the Chao1 and Shannon metrics were tested for association with fecal pH by regression model.ResultsPlant substrate CAZyme family Chao1 and Shannon diversity metrics were negatively associated with fecal pH (p = 0.0009 and p = 0.0008, respectively) but were not significantly different between the consumers of adequate (n = 47) and low fiber (n = 34). However, the abundance of 28 genes for CAZyme glycoside hydrolases differed significantly between adequate and low fiber groups.ConclusionsTaken together, the enzymatic capacity of the gut microbiome may only be partly explained by habitual or recent consumption of dietary fiber.Funding SourcesUSDA ARS 2032–51,530-026–00D.

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover.

A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost.

Metagenomics reveals differences in microbial composition and metabolic functions in the rumen of dairy cows with different residual feed intake

BackgroundRumen microbial composition and functions have vital roles in feed digestion and fermentation and are linked to feed efficiency in cattle. This study selected Holstein cows, which are high in both milk protein content and milk yield, to analyse the relationship between the rumen microbiota and residual feed intake (RFI). Eighteen multiparous lactating cows were divided into low RFI (LRFI, high efficiency, n = 9) and high RFI (HRFI, low efficiency, n = 9) groups to investigate the differences in microbial composition and functions.ResultsThe relative abundances of butyrate producers, including the Clostridium, Butyrivibrio, Eubacterium and Blautia genera, were higher in HRFI cows than in LRFI cows (P < 0.05). Four carbohydrate metabolic pathways (glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism, and butanoate metabolism) and one energy metabolism pathway (methane metabolism), were more abundant in HRFI animals (P < 0.05). Quorum sensing and DNA replication pathways were more abundant in HRFI cows. For CAZyme profiles, 14 out of 19 genes encoding carbohydrates-deconstructing enzymes were more abundant in HRFI cows (P < 0.05). Seven Lachnospiraceae species associated with carbohydrate metabolism and quorum sensing may contribute to the difference in feed efficiency. Moreover, the LRFI cows had lower abundances of Methanosphaera (P < 0.01), Methanobrevibacter ruminantium (P = 0.09) and methanogenesis functions (P = 0.04).ConclusionsThe rumen microbiota of low-efficiency cows has stronger abilities to degrade carbohydrates and produce methane, and quorum sensing pathways could also be associated with differences in feed efficiency. This study provides a deeper understanding of the microbial ecology of dairy cows with different feed efficiencies and highlights the possibility of modulating the rumen microbiome or microbial functions to improve the feed efficiency of dairy cows.

Oral and Gut Microbial Carbohydrate-Active Enzymes Landscape in Health and Disease.

Inter-individual variability in the microbial gene complement encoding for carbohydrate-active enzymes (CAZymes) can profoundly regulate how the host interacts with diverse carbohydrate sources thereby influencing host health. CAZy-typing, characterizing the microbiota-associated CAZyme-coding genes within a host individual, can be a useful tool to predict carbohydrate pools that the host can metabolize, or identify which CAZyme families are underrepresented requiring supplementation via microbiota transplantation or probiotics. CAZy-typing, moreover, provides a novel framework to search for disease biomarkers. As a proof of concept, we used publicly available metagenomes (935) representing 310 type strain bacterial genomes to establish the link between disease status and CAZymes in the oral and gut microbial ecosystem. The abundance and distribution of 220 recovered CAZyme families in saliva and stool samples from patients with colorectal cancer, rheumatoid arthritis, and type 1 diabetes were compared with healthy subjects. Based on the multivariate discriminant analysis, the disease phenotype did not alter the CAZyme profile suggesting a functional conservation in carbohydrate metabolism in a disease state. When disease and healthy CAZyme profiles were contrasted in differential analysis, CAZyme markers that were underrepresented in type 1 diabetes (15), colorectal cancer (12), and rheumatoid arthritis (5) were identified. Of interest, are the glycosyltransferase which can catalyze the synthesis of glycoconjugates including lipopolysaccharides with the potential to trigger inflammation, a common feature in many diseases. Our analysis has also confirmed the expansive carbohydrate metabolism in the gut as evidenced by the overrepresentation of CAZyme families in the gut compared to the oral site. Nevertheless, each site exhibited specific CAZyme markers. Taken together, our analysis provides an insight into the CAZyme landscape in health and disease and has demonstrated the diversity in carbohydrate metabolism in host-microbiota which can be a sound basis for optimizing the selection of pre, pro, and syn-biotic candidate products.

Characterizing rumen microbiota and CAZyme profile of Indian dromedary camel (Camelus dromedarius) in response to different roughages

In dromedary camels, which are pseudo-ruminants, rumen or C1 section of stomach is the main compartment involved in fiber degradation, as in true ruminants. However, as camels are adapted to the harsh and scarce grazing conditions of desert, their ruminal microbiota makes an interesting target of study. The present study was undertaken to generate the rumen microbial profile of Indian camel using 16S rRNA amplicon and shotgun metagenomics. The camels were fed three diets differing in the source of roughage. The comparative metagenomic analysis revealed greater proportions of significant differences between two fractions of rumen content followed by diet associated differences. Significant differences were also observed in the rumen microbiota collected at different time-points of the feeding trial. However, fraction related differences were more highlighted as compared to diet dependent changes in microbial profile from shotgun metagenomics data. Further, 16 genera were identified as part of the core rumen microbiome of Indian camels. Moreover, glycoside hydrolases were observed to be the most abundant among all Carbohydrate-Active enzymes and were dominated by GH2, GH3, GH13 and GH43. In all, this study describes the camel rumen microbiota under different dietary conditions with focus on taxonomic, functional, and Carbohydrate-Active enzymes profiles.

New Method for Identifying Fungal Kingdom Enzyme Hotspots from Genome Sequences.

Fungal genome sequencing data represent an enormous pool of information for enzyme discovery. Here, we report a new approach to identify and quantitatively compare biomass-degrading capacity and diversity of fungal genomes via integrated function-family annotation of carbohydrate-active enzymes (CAZymes) encoded by the genomes. Based on analyses of 1932 fungal genomes the most potent hotspots of fungal biomass processing CAZymes are identified and ranked according to substrate degradation capacity. The analysis is achieved by a new bioinformatics approach, Conserved Unique Peptide Patterns (CUPP), providing for CAZyme-family annotation and robust prediction of molecular function followed by conversion of the CUPP output to lists of integrated “Function;Family” (e.g., EC 3.2.1.4;GH5) enzyme observations. An EC-function found in several protein families counts as different observations. Summing up such observations allows for ranking of all analyzed genome sequenced fungal species according to richness in CAZyme function diversity and degrading capacity. Identifying fungal CAZyme hotspots provides for identification of fungal species richest in cellulolytic, xylanolytic, pectinolytic, and lignin modifying enzymes. The fungal enzyme hotspots are found in fungi having very different lifestyle, ecology, physiology and substrate/host affinity. Surprisingly, most CAZyme hotspots are found in enzymatically understudied and unexploited species. In contrast, the most well-known fungal enzyme producers, from where many industrially exploited enzymes are derived, are ranking unexpectedly low. The results contribute to elucidating the evolution of fungal substrate-digestive CAZyme profiles, ecophysiology, and habitat adaptations, and expand the knowledge base for novel and improved biomass resource utilization.

Fungal secretome profile categorization of CAZymes by function and family corresponds to fungal phylogeny and taxonomy: Example Aspergillus and Penicillium

Fungi secrete an array of carbohydrate-active enzymes (CAZymes), reflecting their specialized habitat-related substrate utilization. Despite its importance for fitness, enzyme secretome composition is not used in fungal classification, since an overarching relationship between CAZyme profiles and fungal phylogeny/taxonomy has not been established. For 465 Ascomycota and Basidiomycota genomes, we predicted CAZyme-secretomes, using a new peptide-based annotation method, Conserved-Unique-Peptide-Patterns, enabling functional prediction directly from sequence. We categorized each enzyme according to CAZy-family and predicted molecular function, hereby obtaining a list of “EC-Function;CAZy-Family” observations. These “Function;Family”-based secretome profiles were compared, using a Yule-dissimilarity scoring algorithm, giving equal consideration to the presence and absence of individual observations. Assessment of “Function;Family” enzyme profile relatedness (EPR) across 465 genomes partitioned Ascomycota from Basidiomycota placing Aspergillus and Penicillium among the Ascomycota. Analogously, we calculated CAZyme “Function;Family” profile-similarities among 95 Aspergillus and Penicillium species to form an alignment-free, EPR-based dendrogram. This revealed a stunning congruence between EPR categorization and phylogenetic/taxonomic grouping of the Aspergilli and Penicillia. Our analysis suggests EPR grouping of fungi to be defined both by “shared presence“ and “shared absence” of CAZyme “Function;Family” observations. This finding indicates that CAZymes-secretome evolution is an integral part of fungal speciation, supporting integration of cladogenesis and anagenesis.

Proteomic enzyme analysis of the marine fungus Paradendryphiella salina reveals alginate lyase as a minimal adaptation strategy for brown algae degradation

We set out to investigate the genetic adaptations of the marine fungus Paradendryphiella salina CBS112865 for degradation of brown macroalgae. We performed whole genome and transcriptome sequencing and shotgun proteomic analysis of the secretome of P. salina grown on three species of brown algae and under carbon limitation. Genome comparison with closely related terrestrial fungi revealed that P. salina had a similar but reduced CAZyme profile relative to the terrestrial fungi except for the presence of three putative alginate lyases from Polysaccharide Lyase (PL) family 7 and a putative PL8 with similarity to ascomycete chondroitin AC lyases. Phylogenetic and homology analyses place the PL7 sequences amongst mannuronic acid specific PL7 proteins from marine bacteria. Recombinant expression, purification and characterization of one of the PL7 genes confirmed the specificity. Proteomic analysis of the P. salina secretome when growing on brown algae, revealed the PL7 and PL8 enzymes abundantly secreted together with enzymes necessary for degradation of laminarin, cellulose, lipids and peptides. Our findings indicate that the basic CAZyme repertoire of saprobic and plant pathogenic ascomycetes, with the addition of PL7 alginate lyases, provide P. salina with sufficient enzymatic capabilities to degrade several types of brown algae polysaccharides.

Comprehensive Analysis of Carbohydrate-Active Enzymes from the Filamentous Fungus Scytalidium candidum 3C.

Complete enzymatic degradation of plant polysaccharides is a result of combined action of various carbohydrate-active enzymes (CAZymes). In this paper, we demonstrate the potential of the filamentous fungus Scytalidium candidum 3C for processing of plant biomass. Structural annotation of the improved assembly of S. candidum 3C genome and functional annotation of CAZymes revealed putative gene sequences encoding such proteins. A total of 190 CAZyme-encoding genes were identified, including 104 glycoside hydrolases, 52 glycosyltransferases, 28 oxidative enzymes, and 6 carbohydrate esterases. In addition, 14 carbohydrate-binding modules were found. Glycoside hydrolases secreted during the growth of S. candidum 3C in three media were analyzed with a variety of substrates. Mass spectrometry analysis of the fungal culture liquid revealed the presence of peptides identical to 36 glycoside hydrolases, three proteins without known enzymatic function belonging to the same group of families, and 11 oxidative enzymes. The activity of endo-hemicellulases was determined using specially synthesized substrates in which the glycosidic bond between monosaccharide residues was replaced by a thio-linkage. During analysis of the CAZyme profile of S. candidum 3C, four β-xylanases from the GH10 family and two β-glucanases from the GH7 and GH55 families were detected, partially purified, and identified.

Comparative Metagenomics of Cellulose- and Poplar Hydrolysate-Degrading Microcosms from Gut Microflora of the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus) after Long-Term Enrichment

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization.

Ninety-nine de novo assembled genomes from the moose (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation.

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet.

Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied in vitro, knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes in vivo. The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria (Prevotella, Ruminocccus and Fibrobacter), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them.

Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and associated biotechnological applications.

Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome.

MotivationCarbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits.ResultsThis study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as 'CAZotypes', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition.