Carbohydrate-active Enzyme Families Research Articles

Constrained Catalytic Itinerary of a Retaining 3,6-Anhydro-D-Galactosidase, a Key Enzyme in Red Algal Cell Wall Degradation.

The marine Bacteroidota Zobellia galactanivorans has a polysaccharide utilization locus dedicated to the catabolism of the red algal cell wall galactan carrageenan and its unique and industrially important α-3,6-anhydro-D-galactose (ADG) monosaccharide. Here we present the first analysis of the specific molecular interactions that the exo-(α-1,3)-3,6-anhydro-D-galactosidase ZgGH129 uses to cope with the strict steric restrictions imposed by its bicyclic ADG substrate - which is ring flipped relative to D-galactose. Crystallographic snapshots of key catalytic states obtained with the natural substrate and novel chemical tools designed to mimic species along the reaction coordinate, together with quantum mechanics/molecular mechanics (QM/MM) metadynamics methods and kinetic studies, demonstrate a retaining mechanism where the second step is rate limiting. The conformational landscape of the constrained 3,6-anhydro-D-galactopyranose ring proceeds through enzyme glycosylation B1,4→[E4]≠→E4/1C4 and deglycosylation E4/1C4→[E4]≠→B1,4 itineraries limited to the Southern Hemisphere of the Cremer-Pople sphere. These results demonstrate the conformational changes throughout catalysis in a non-standard, sterically restrained, bicyclic monosaccharide, and provide a molecular framework for mechanism-based inhibitor design for anhydro-type carbohydrate-processing enzymes and for future applications involving carrageenan degradation. In addition, our study provides a rare example of distinct niche-based conformational itineraries within the same carbohydrate-active enzyme family.

Random forest machine-learning algorithm classifies white- and brown-rot fungi according to the number of the genes encoding Carbohydrate-Active enZyme families.

Wood-rotting fungi are categorized as either white- or brown-rot modes based on the coloration of decomposed wood. The process of classification can be influenced by human biases. The random forest machine learning algorithm effectively distinguishes between white- and brown-rot fungi based on the presence of Carbohydrate-Active enZyme genes. These findings not only aid in the classification of wood-rotting fungi but also facilitate the identification of the enzymes responsible for degrading woody biomass.

Metagenomic insights into inhibition of soil microbial carbon metabolism by phosphorus limitation during vegetation succession.

There is growing awareness of the need for regenerative practices in the fight against biodiversity loss and climate change. Yet, we lack a mechanistic understanding of how microbial community composition and functioning are likely to change alongside transition from high-density tillage to large-scale vegetation restoration. Here, we investigated the functional dynamics of microbial communities following a complete vegetation successional chronosequence in a subtropical zone, Southwestern China, using shotgun metagenomics approaches. The contents of total soil phosphorus (P), available P, litter P, and microbial biomass P decreased significantly during vegetation succession, indicating that P is the most critical limiting nutrient. The abundance of genes related to P-uptake and transport, inorganic P-solubilization, organic P-mineralization, and P-starvation response regulation significantly increased with successional time, indicating an increased microbial "mining" for P under P limitation. Multi-analysis demonstrated microbial P limitation strongly inhibits carbon (C) catabolism potential, resulting in a significant decrease in carbohydrate-active enzyme family gene abundances. Nevertheless, over successional time, microorganisms increased investment in genes involved in degradation-resistant compounds (lignin and its aromatic compounds) to acquire P resources in the litter. Our study provides functional gene-level insights into how P limitation during vegetation succession in subtropical regions inhibits soil microbial C metabolic processes, thereby advancing our understanding of belowground C cycling and microbial metabolic feedback during forest restoration.

High-yield synthesis of 2-O-α-D-glucosyl-D-glycerate by a bifunctional glycoside phosphorylase.

Osmolytes are produced by various microorganisms as a defense mechanism to protect cells and macromolecules from damage caused by external stresses in harsh environments. Due to their useful stabilizing properties, these molecules are applied as active ingredients in a wide range of cosmetics and healthcare products. The metabolic pathways and biocatalytic syntheses of glycosidic osmolytes such as 2-O-α-D-glucosyl-D-glycerate often involve the action of a glycoside phosphorylase. Here, we report the discovery of a glucosylglycerate phosphorylase from carbohydrate-active enzyme family GH13 that is also active on sucrose, which contrasts the strict specificity of known glucosylglycerate phosphorylases that can only use α-D-glucose 1-phosphate as glycosyl donor in transglycosylation reactions. The novel enzyme can be distinguished from other phosphorylases from the same family by the presence of an atypical conserved sequence motif at specificity-determining positions in the active site. The promiscuity of the sucrose-active glucosylglycerate phosphorylase can be exploited for the high-yielding and rapid synthesis of 2-O-α-D-glucosyl-D-glycerate from sucrose and D-glycerate. KEY POINTS: • A Xylanimonas protaetiae glycoside phosphorylase can use both d-glycerate and fructose as glucosyl acceptor with high catalytic efficiency • Biocatalytic synthesis of the osmolyte 2-O-α-d-glucosyl-d-glycerate • Positions in the active site of GH13 phosphorylases act as convenient specificity fingerprints.

Genome and transcriptome reveal lithophilic adaptation of Cladophialophora brunneola, a new rock-inhabiting fungus

ABSTRACT Rock-inhabiting fungi (RIF) are slow-growing microorganisms that inhabit rocks and exhibit exceptional stress tolerance owing to their thick melanised cell walls. This study reports the identification of a novel rock-inhabiting fungus, Cladophialophora brunneola sp. nov. which was isolated from a karst landform in Guizhou, China, using a combination of morphological and phylogenetic analyses. The genome of C. brunneola was sequenced and assembled, with a total size of approximately 33.8 Mb, encoding 14,168 proteins and yielding an N50 length of 1.88 Mb. C. brunneola possessed a larger proportion of species-specific genes, and phylogenomic analysis positioned it in an early diverged lineage within Chaetothyriales. In comparison to non-RIF, C. brunneola displayed reduction in carbohydrate-active enzyme families (CAZymes) and secondary metabolite biosynthetic gene clusters (BGCs). Transcriptome analysis conducted under PEG-induced drought stress revealed elevated expression levels of genes associated with melanin synthesis pathways, cell wall biosynthesis, and lipid metabolism. This study contributes to our understanding of the genomic evolution and polyextremotolerance exhibited by rock-inhabiting fungi.

Phylogenetic systematics of Butyrivibrio and Pseudobutyrivibrio pure culture and metagenomically assembled genomes suggest existence of 59 genera and 75 species, alongside possession of open pangenomes with an abundance of carbohydrate-active enzyme family isoforms

Phylogenetic systematics of Butyrivibrio and Pseudobutyrivibrio pure culture and metagenomically assembled genomes suggest existence of 59 genera and 75 species, alongside possession of open pangenomes with an abundance of carbohydrate-active enzyme family isoforms

Metatranscriptomics Unravel Composition, Drivers, and Functions of the Active Microorganisms in Light-Flavor Liquor Fermentation.

ABSTRACTThe microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor.IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.

Phylogenetic systematics of Butyrivibrio and Pseudobutyrivibrio genomes illustrate vast taxonomic diversity, open genomes and an abundance of carbohydrate-active enzyme family isoforms.

Butyrivibrio and Pseudobutyrivibrio dominate in anaerobic gastrointestinal microbiomes, particularly the rumen, where they play a key role in harvesting dietary energy. Within these genera, five rumen species have been classified ( Butyrivibrio fibrisolvens , Butyrivibrio hungatei , Butyrivibrio proteoclasticus , Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans ) and more recently an additional Butyrivibrio sp. group was added. Given the recent increase in available genomes, we re-investigated the phylogenetic systematics and evolution of Butyrivibrio and Pseudobutyrivibrio . Across 71 genomes, we show using 16S rDNA and 40 gene marker phylogenetic trees that the current six species designations ( P. ruminis , P. xylanivorans , B. fibrisolvens , Butyrivibrio sp., B. hungatei and B. proteclasticus) are found. However, pangenome analysis showed vast genomic variation and a high abundance of accessory genes (91.50–99.34 %), compared with core genes (0.66–8.50 %), within these six taxonomic groups, suggesting incorrectly assigned taxonomy. Subsequent pangenome accessory genomes under varying core gene cut-offs (%) and average nucleotide identity (ANI) analysis suggest the existence of 42 species within 32 genera. Pangenome analysis of those that still group within B. fibrisolvens , B. hungatei and P. ruminis , based on revised ANI phylogeny, also showed possession of very open genomes, illustrating the diversity that exists even within these groups. All strains of both Butyrivibrio and Pseudobutyrivibrio also shared a broad range of clusters of orthologous genes (COGs) (870), indicating recent evolution from a common ancestor. We also demonstrate that the carbohydrate-active enzymes (CAZymes) predominantly belong to glycosyl hydrolase (GH)2, 3, 5, 13 and 43, with numerous within family isoforms apparent, likely facilitating metabolic plasticity and resilience under dietary perturbations. This study provides a major advancement in our functional and evolutionary understanding of these important anaerobic bacteria.

Stereoselective synthesis of a 4-\u237a-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

Larvae of longhorned beetles (Coleoptera; Cerambycidae) have evolved a diverse and phylogenetically conserved array of plant cell wall degrading enzymes

AbstractLonghorned beetles (Cerambycidae) are the most diverse group of predominantly wood‐feeding (xylophagous) insects on Earth. Larvae of most species feed within tissues of plants made up of large amounts of plant cell wall (PCW), which is notoriously difficult to digest. To efficiently access nutrients from their food source, cerambycid larvae have to deconstruct PCW polysaccharides – such as cellulose, hemicelluloses and pectin – requiring them to possess a diversity of plant cell wall degrading enzymes (PCWDEs) in their digestive tract. Genomic data for Cerambycidae are mostly limited to notorious forest pests and are lacking for most of the taxonomic groups. Consequently, our understanding of the distribution and evolution of cerambycid PCWDEs is quite limited. We addressed the numbers, kinds and evolution of cerambycid PCWDEs by surveying larval midgut transcriptomes from 23 species representing six of the eight recognized subfamilies of Cerambycidae and each with very diverse host types (i.e., gymnosperms, angiosperms, xylem, phloem, fresh or dead plant tissues). Using these data, we identified 340 new putative PCWDEs belonging to ten carbohydrate active enzyme families, including two gene families (GH7 and GH53) not previously reported from insects. The remarkably wide range of PCWDEs expressed by Cerambycidae should allow them to break down most PCW polysaccharides. Moreover, the observed distribution of PCWDEs encoded in cerambycid genomes agreed more with phylogenetic relationship of the species studied than with the taxonomic origin or quality of the host plant tissues.

Agricultural Soil Management Practices Differentially Shape the Bacterial and Fungal Microbiome of Sorghum bicolor.

Soils play important roles in biological productivity. While past work suggests that microbes affect soil health and respond to agricultural practices, it is not well known how soil management shapes crop host microbiomes. To elucidate the impact of management on microbial composition and function in the sorghum microbiome, we performed 16S rRNA gene and ITS2 amplicon sequencing and metatranscriptomics on soil and root samples collected from a site in California's San Joaquin Valley that is under long-term cultivation with 1) standard (ST) or no tilling (NT) and 2) cover-cropping (CC) or leaving the field fallow (NO). Our results revealed that microbial diversity, composition, and function change across tillage and cover type, with a heightened response in fungal communities, versus bacterial. Surprisingly, ST harbored greater microbial alpha diversity than NT, indicating that tillage may open niche spaces for broad colonization. Across management regimes, we observed class-level taxonomic level shifts. Additionally, we found significant functional restructuring across treatments, including enrichment for microbial lipid and carbohydrate transport and metabolism and cell motility with NT. Differences in carbon cycling were also observed, with increased prevalence of glycosyltransferase and glycoside hydrolase carbohydrate active enzyme families with CC. Lastly, treatment significantly influenced arbuscular mycorrhizal fungi, which had the greatest prevalence and activity under ST, suggesting that soil practices mediate known beneficial plant-microbe relationships. Collectively, our results demonstrate how agronomic practices impact critical interactions within the plant microbiome and inform future efforts to configure trait-associated microbiomes in crops.Importance While numerous studies show that farming practices can influence the soil microbiome, there are often conflicting results on how microbial diversity and activity respond to treatment. In addition, there is very little work published on how the corresponding crop plant microbiome is impacted. With bacteria and fungi known to critically affect soil health and plant growth, we concurrently compared how the practices of no and standard tillage, in combination with either cover-cropping or fallow fields, shape soil and plant-associated microbiomes between the two classifications. In determining not only the response to treatment in microbial diversity and composition, but for activity as well, this work demonstrates the significance of agronomic practice in modulating plant-microbe interactions, as well as encourages future work on the mechanisms involved in community assemblages supporting similar crop outcomes.

Life at the Frozen Limit: Microbial Carbon Metabolism Across a Late Pleistocene Permafrost Chronosequence.

Permafrost is an extreme habitat yet it hosts microbial populations that remain active over millennia. Using permafrost collected from a Pleistocene chronosequence (19 to 33 ka), we hypothesized that the functional genetic potential of microbial communities in permafrost would reflect microbial strategies to metabolize permafrost soluble organic matter (OM) in situ over geologic time. We also hypothesized that changes in the metagenome across the chronosequence would correlate with shifts in carbon chemistry, permafrost age, and paleoclimate at the time of permafrost formation. We combined high-resolution characterization of water-soluble OM by Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS), quantification of organic anions in permafrost water extracts, and metagenomic sequencing to better understand the relationships between the molecular-level composition of potentially bioavailable OM, the microbial community, and permafrost age. Both age and paleoclimate had marked effects on both the molecular composition of dissolved OM and the microbial community. The relative abundance of genes associated with hydrogenotrophic methanogenesis, carbohydrate active enzyme families, nominal oxidation state of carbon (NOSC), and number of identifiable molecular formulae significantly decreased with increasing age. In contrast, genes associated with fermentation of short chain fatty acids (SCFAs), the concentration of SCFAs and ammonium all significantly increased with age. We present a conceptual model of microbial metabolism in permafrost based on fermentation of OM and the buildup of organic acids that helps to explain the unique chemistry of ancient permafrost soils. These findings imply long-term in situ microbial turnover of ancient permafrost OM and that this pooled biolabile OM could prime ancient permafrost soils for a larger and more rapid microbial response to thaw compared to younger permafrost soils.

The Organic Degradation and Potential Microbial Function in a 15-Day Sewage Sludge Biodrying

To meet the challenge of increased sludge generation, shortening of biodrying periods are required. This study assesses a shortened sewage sludge biodrying period of 15 days. The fundamental physicochemical properties of samples from different phases were determined, the functional groups were identified using infrared spectroscopy and the biodrying associated microbial functions were annotated against gene databases. After a 15-day biodrying period, the moisture content, readily degradable carbohydrate, lignocellulose and protein levels were significantly reduced. The distinct attenuation of peaks identified by infrared spectroscopy, indicates that the degradation of most lipids, proteins and polysaccharides in the biodrying matrix had reached equilibration on Day 15 and following this biosynthesis may result in an increased polysaccharide content. However, the biodrying matrix on Day 20 was only partially matured. Firmicutes, Actinobacteria and Proteobacteria were the most ecologically dominant phyla. These ecologically dominant microorganisms were also functionally dominant in biodrying associated metabolic pathways (glycolysis, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism), as well as containing carbohydrate-active enzyme families. The modified 15-day biodrying period reduced the treatment time and achieved a competent biodrying result without increasing the operating costs. The 15-day treatment would increase the rate of existing systems or decrease the capital cost of new systems.

Potential correlation between carbohydrate-active enzyme family 48 expressed by gut microbiota and the expression of intestinal epithelial AMP-activated protein kinase β.

The expression of the carbohydrate-active enzyme family and related genes is known to be influenced by the response of intestinal microbiota to dietary changes. However, it is uncertain whether this is caused by variation in the intestinal microecology. In this study, metabolite analysis, 16S rDNA sequencing, metagenomics, and Western blotting were employed to investigate the effects of dietary intervention on the composition of gut microbiota and microbiota-mediated changes. The results showed that compared with the low fiber-fed group, the fiber diet-fed mice displayed a shift in gut microbiota composition to contain more members of phylum Bacteroidetes, accompanied by higher proportions of Akkermansia and typical probiotic Bifidobacterium. Moreover, correlations were found between microbial genes coding for carbohydrate-binding module family 48 (CBM48) and intestinal epithelial expression levels of AMPK β. This finding provides new insight for elucidating the contribution of dietary intervention through AMPK regulation linked to the microbial carbohydrate-binding family. PRACTICAL APPLICATIONS: The relationship suggested by these data will provide theoretical and applied foundations for the development of potential intervention targeting the interaction between gut microbiota and host health, particularly the use of dietary fiber as a medically relevant food. Additionally, a better understanding of the interactions between gut microbiota and intestinal epithelial will inform the development of gut microbiota intervention as a health-promoting procedure.

CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.

Metagenomics Reveals Seasonal Functional Adaptation of the Gut Microbiome to Host Feeding and Fasting in the Chinese Alligator.

As a natural hibernator, the Chinese alligator (Alligator sinensis) is an ideal and intriguing model to investigate changes in microbial community structure and function caused by hibernation. In this study, we used 16S rRNA profiling and metagenomic analysis to compare the composition, diversity, and functional capacity in the gut microbiome of hibernating vs. active Chinese alligators. Our results show that gut microbial communities undergo seasonal restructuring in response to seasonal cycles of feeding and fasting in the Chinese alligator, but this animal harbors a core gut microbial community primarily dominated by Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes across the gut regions. During hibernation, there is an increase in the abundance of bacterial taxa (e.g., the genus Bacteroides) that can degrade host mucin glycans, which allows adaptation to winter fasting. This is accompanied by the enrichment of mucin oligosaccharide-degrading enzyme and carbohydrate-active enzyme families. In contrast, during the active phase (feeding), active Chinese alligators exhibit a carnivore gut microbiome dominated by Fusobacteria, and there is an increase in the relative abundance of bacteria (e.g., Cetobacterium somerae) with known proteolytic and amino acids-fermentating functions that improve host protein-rich food digestion efficiency. In addition, seasonal variations in the expression of β-defensins play a protective role in intestinal immunity. These findings provide insights into the functional adaptations of host–gut microbe symbioses to seasonal dietary shifts to maintain gut homeostasis and health, especially in extreme physiological states.

Genome expansion by allopolyploidization in the fungal strain Coniochaeta 2T2.1 and its exceptional lignocellulolytic machinery

BackgroundParticular species of the genus Coniochaeta (Sordariomycetes) exhibit great potential for bioabatement of furanic compounds and have been identified as an underexplored source of novel lignocellulolytic enzymes, especially Coniochaeta ligniaria. However, there is a lack of information about their genomic features and metabolic capabilities. Here, we report the first in-depth genome/transcriptome survey of a Coniochaeta species (strain 2T2.1).ResultsThe genome of Coniochaeta sp. strain 2T2.1 has a size of 74.53 Mbp and contains 24,735 protein-encoding genes. Interestingly, we detected a genome expansion event, resulting ~ 98% of the assembly being duplicated with 91.9% average nucleotide identity between the duplicated regions. The lack of gene loss, as well as the high divergence and strong genome-wide signatures of purifying selection between copies indicates that this is likely a recent duplication, which arose through hybridization between two related Coniochaeta-like species (allopolyploidization). Phylogenomic analysis revealed that 2T2.1 is related Coniochaeta sp. PMI546 and Lecythophora sp. AK0013, which both occur endophytically. Based on carbohydrate-active enzyme (CAZy) annotation, we observed that even after in silico removal of its duplicated content, the 2T2.1 genome contains exceptional lignocellulolytic machinery. Moreover, transcriptomic data reveal the overexpression of proteins affiliated to CAZy families GH11, GH10 (endoxylanases), CE5, CE1 (xylan esterases), GH62, GH51 (α-l-arabinofuranosidases), GH12, GH7 (cellulases), and AA9 (lytic polysaccharide monoxygenases) when the fungus was grown on wheat straw compared with glucose as the sole carbon source.ConclusionsWe provide data that suggest that a recent hybridization between the genomes of related species may have given rise to Coniochaeta sp. 2T2.1. Moreover, our results reveal that the degradation of arabinoxylan, xyloglucan and cellulose are key metabolic processes in strain 2T2.1 growing on wheat straw. Different genes for key lignocellulolytic enzymes were identified, which can be starting points for production, characterization and/or supplementation of enzyme cocktails used in saccharification of agricultural residues. Our findings represent first steps that enable a better understanding of the reticulate evolution and “eco-enzymology” of lignocellulolytic Coniochaeta species.

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.

Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases

Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated ∼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149. Characterization identified cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, and a β-1,3-glucan phosphorylase. To demonstrate the versatility of the screening method, varying substrate combinations were used to identify GP activity from families GH13, GH65, GH112, and GH130 in addition to GH94 and GH149. These pilot screen and substrate versatility results provide a screening paradigm platform for recovering diverse GPs from uncultivated microbial communities acting on different substrates with considerable potential to unravel previously unknown degradative pathways within microbiomes.

Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43.

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-β-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-β-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related β-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the β-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.