Glucomannan (Konjac; Low Viscosity)

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40°C to 70°C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut.

The rise of functional diversity through gene duplication contributed to the adaption of organisms to various environments. Here we investigate the evolution of putative cellulases of the subfamily 2 of glycoside hydrolase family 5 (GH5_2) in the Cerambycidae (longhorned beetles), a megadiverse assemblage of mostly xylophagous beetles. Cerambycidae originally acquired GH5_2 from a bacterial donor through horizontal gene transfer (HGT), and extant species harbor multiple copies that arose from gene duplication. We ask how these digestive enzymes contributed to the ability of these beetles to feed on wood. We analyzed 113 GH5_2, including the functional characterization of 52 of them, derived from 25 species covering most subfamilies of Cerambycidae. Ancestral gene duplications led to five well-defined groups with distinct substrate specificity, allowing these beetles to break down, in addition to cellulose, polysaccharides that are abundant in plant cell walls (PCWs), namely, xyloglucan, xylan, and mannans. Resurrecting the ancestral enzyme originally acquired by HGT, we show it was a cellulase that was able to break down glucomannan and xylan. Finally, recent gene duplications further expanded the catalytic repertoire of cerambycid GH5_2, giving rise to enzymes that favor transglycosylation over hydrolysis. We suggest that HGT and gene duplication, which shaped the evolution of GH5_2, played a central role in the ability of cerambycid beetles to use a PCW-rich diet and may have contributed to their successful radiation.

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual β-glucan/xyloglucan hydrolase activity. Molecular phylogeny indicates that EG16 members comprise a sister clade with a deep evolutionary relationship to the widely studied apoplastic xyloglucan endo-transglycosylases/hydrolases (XTH). A cross-genome survey indicated that EG16 members occur as a single ortholog across species and are widespread in early diverging plants, including the non-vascular bryophytes, for which functional data were previously lacking. Remarkably, enzymological characterization of an EG16 ortholog from the model moss Physcomitrella patens (PpEG16) revealed that EG16 activity and sequence/structure are highly conserved across 500 million years of plant evolution, vis-à-vis orthologs from grapevine and poplar. Ex vivo biomechanical assays demonstrated that the application of EG16 gene products caused abrupt breakage of etiolated hypocotyls rather than slow extension, thereby indicating a mode-of-action distinct from endogenous expansins and microbial endo-glucanases. The biochemical data presented here will inform future genomic, genetic, and physiological studies of EG16 enzymes.

Talaromyces cellulolyticus is a promising fungus for providing a cellulase preparation suitable for the hydrolysis of lignocellulosic material, although its mannan-degrading activities are insufficient. In the present study, three core mannanolytic enzymes, including glycosyl hydrolase family 5-7 (GH5-7) β-mannanase (Man5A), GH27 α-galactosidase, and GH2 β-mannosidase, were purified from a culture supernatant of T. cellulolyticus grown with glucomannan, and the corresponding genes were identified based on their genomic sequences. Transcriptional analysis revealed that these genes were specifically induced by glucomannan. Two types of Man5A products, Man5A1 and Man5A2, were found as major proteins in the mannanolytic system. Man5A1 was devoid of a family 1 carbohydrate-binding module (CBM1) at the N-terminus, whereas Man5A2 was devoid of both CBM1 and Ser/Thr-rich linker region. The physicochemical and catalytic properties of both Man5A1 and Man5A2 were identical to those of recombinant Man5A (rMan5A) possessing CBM1, except for the cellulose-binding ability. Man5A CBM1 had little effect on mannan hydrolysis of pretreated Hinoki cypress. The results suggest that an improvement in Man5A CBM1 along with the augmentation of identified mannanolytic enzyme components would aid in efficient hydrolysis of softwood using T. cellulolyticus cellulase preparation.

Lignocellulosic biomass is receiving growing interest as a renewable source of biofuels, chemicals and materials. Lignocellulosic polymers and cellulose nanocrystals (CNCs) present high added-value potential in the nanocomposite field, but some issues have to be solved before large-scale applications. Among them, the interaction between polymers at the nanoscale and the effect of the external parameters on the mechanical properties have to be more precisely investigated. The present study aims at evaluating how the relative humidity affects the reduced Young’s modulus of lignocellulosic films prepared with crystalline cellulose, glucomannan, xylan and lignin and how relative humidity changes their nanoscale adhesion properties with CNCs. Using atomic force microscopy and force volume experiments with CNC-functionalized levers, increasing the relative humidity is shown to decrease the Young’s modulus values of the different films and promote their adhesion forces with CNCs. In particular, CNCs more strongly interact with glucomannan and lignin than xylan, and in the case of lignin, the oxidation of the film promotes strong variations in the adhesion force. Such results allow to better understand the lignocellulosic film properties at the nanoscale, which should lead to an improvement in the production of new highly added-value composites.

Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomona species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-L-arabinofuranosidases and GH95 alpha-L-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select Bacteroides.

The rate-limiting step in cutaneous wound healing, namely, the transition from inflammation to cell proliferation, depends on the high plasticity of macrophages to prevent inflammation in the wound tissues in a timely manner. Thus, strategies that reprogram inflammatory macrophages may improve the healing of poor wounds, particularly in the aged skin of individuals with diabetes or other chronic diseases. As shown in our previous study, KGM-modified SiO₂ nanoparticles (KSiNPs) effectively activate macrophages to differentiate into the M2-type phenotype by inducing mannose receptor (MR) clustering on the cell surface. Here, we assess whether KSiNPs accelerate wound healing following acute or chronic skin injury. Using a full-thickness excision model in either diabetic mice or healthy mice, the wounds treated with KSiNPs displayed a dramatically increased closure rate and collagen production, along with decreased inflammation and increased angiogenesis in the regenerating tissues. Furthermore, KSiNPs induced the formation of M2-like macrophages by clustering MR on the cells. Accordingly, the cytokines produced by the KSiNP-treated macrophages were capable of inducing fibroblast proliferation and subsequent secretion of extracellular matrix (ECM). Based on these results, KSiNPs display great potential as an effective therapeutic approach for cutaneous wounds by effectively suppressing excessive or persistent inflammation and fibrosis.

Background: The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked D-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity. Results: The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-D-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well. Conclusions: Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

Woody biomass is a sustainable and virtually unlimited source of hemicellulosic polysaccharides. The predominant hemicelluloses in softwood and hardwood are galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), respectively. Based on the structure similarity with common dietary fibers, GGM and AGX may be postulated to have prebiotic properties, conferring a health benefit on the host through specific modulation of the gut microbiota. In this study, we evaluated the prebiotic potential of acetylated GGM (AcGGM) and highly acetylated AGX (AcAGX) obtained from Norwegian lignocellulosic feedstocks in vitro. In pure culture, both substrates selectively promoted the growth of Bifidobacterium, Lactobacillus, and Bacteroides species in a manner consistent with the presence of genetic loci for the utilization of -manno-oligosaccharides/-mannans and xylo-oligosaccharides/ xylans. The prebiotic potential of AcGGM and AcAGX was further assessed in a pH controlled batch culture fermentation system inoculated with healthy adult human feces. Results were compared with those obtained with a commercial fructooligosaccharide (FOS) mixture. Similarly to FOS, both substrates significantly increased (P < 0.05) the Bifidobacterium population. Other bacterial groups enumerated were unaffected with the exception of an increase in the growth of members of the Bacteroides-Prevotella group, Faecalibacterium prausnitzii, and clostridial cluster IX (P < 0.05). Compared to the other substrates, AcGGM promoted butyrogenic fermentation whereas AcAGX was more propiogenic. Although further in vivo confirmation is necessary, these results demonstrate that both AcGGM and AcAGX from lignocellulosic feedstocks can be used to direct the promotion of beneficial bacteria, thus exhibiting a promising prebiotic ability to improve or restore gut health.

A method for the high-throughput analysis of the relative lignin contents of Cryptomeria japonica samples over a wide concentration range (3-73%), independent of the type of chemical pretreatment, was developed by using Fourier transform infrared spectroscopy. First, the assignments of the infrared absorbance related to lignin were reviewed. Then, various chemical treatments, including alkaline, acid, and hydrothermal processes, and a sodium chlorite oxidation treatment, were performed to prepare samples containing a wide range of different lignin contents. Principal component analysis indicated high variability among the chemical treatments in terms of the corresponding lignin contents as well as the resulting changes in the chemical structure of hemicellulose; this conclusion was supported by the loading vectors. The intensity of the key band of lignin at 1508 cm^-1 was calculated using the absorbance at 2900 cm^-1 as a reference; a reliable calibration curve with an R² of 0.968 was obtained independent of the chemical treatment performed. This simple and rapid method for determining the lignin content is expected to be widely applicable for optimizing bioethanol production, as well as monitoring biomass degradation processes.

Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus. Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an α-1,2-linked 4-O-methyl-D-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(−)-MSⁿ) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA²Xyl_n, n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(α/β)₈-catalytic domain, lacking an α6 helix, and a small β-rich domain. This structure and site-directed mutagenesis clarified that Arg⁴⁶, conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage-dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn⁹³ in the β2-α2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo- and exo-xylanase.

Konjac glucomannan (KGM) is a polysaccharide extracted from Amorphophallus konjac, and its degradation product is konjac oligo-glucomannan (KOG). The aim of this study was to produce KOG from KGM and to evaluate its effect on the gut microbiota in fecal batch culture. KOG was produced by enzymatic hydrolysis using β-mannanase. The optimum conditions were as follows: reaction temperature of 48°C, reaction time of 4 hr, pH of 5.5 and E/S of 0.05% followed by purification step using 3,000 NMWC ultrafiltration (UF) membrane pore size. The effect of KOG on changes in human fecal bacterial populations and short-chain fatty acids (SCFAs) production was evaluated. The results showed that low-molecular weight KOG (LKOG) from purification step with concentration of 9.54 mg/ml, and a prebiotic index (PI) of 0.76 was successfully produced. LKOG can enhance the production of butyric acid in the colon with the highest concentration (8.24 mM) found at 72 hr fermentation.

Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLPMO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.

Some β-glucans have attracted attention due to their functionality as an immunostimulant and have been used in processed foods. However, accurately measuring the β-glucan content of processed foods using existing methods is difficult. We demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay, in which β-glucan is extracted using sodium hypochlorite, dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into β-glucan fragments using Westase which is an enzyme having β-1,6- and β-1,3 glucanase activity. The β-glucan fragments are further digested into glucose using exo-1,3-β-D-glucanase and β-glucosidase. We measured β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in various polysaccharide reagents and processed foods using our novel method. The SEED assay was able to quantify β-glucan with good reproducibility, and the recovery rate was >90% for food containing β-glucan. Therefore, the SEED assay is capable of accurately measuring the β-glucan content of processed foods.

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two β-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to β-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp⁷⁹) that stacks against the sugar ring at subsite - 5. The K_m) values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the k_cat) remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.

A gene encoding an endoglucanase belonging to subfamily C of glycoside hydrolase family 45 (GH45) was identified in the brown rot fungus Fomitopsis palustris and functionally expressed in Pichia pastoris. The recombinant protein displayed hydrolytic activities toward various substrates such as carboxymethyl cellulose, phosphoric acid swollen cellulose, glucomannan, lichenan, and β-glucan. In particular, the enzyme had a unique catalytic efficiency on β-1,4-glucans rather than mixed β-1,3/1,4-glucans as compared to other GH45 endoglucanases. The fungal enzyme was relatively thermostable, retaining more than 91.4% activity at 80°C for 1 h. Site-directed mutagenesis studies revealed that the mutants N95D and D117N had significantly reduced enzymatic activities, indicating that both residues are essential for the catalytic reaction. Our study expands knowledge and understanding of the catalytic mechanism of GH45 subfamily C enzymes and also suggests that this thermostable endoglucanase from F. palustris has great potential in industrial applications.

Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.