Search

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation., (© 2024. The Author(s).)

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining., (© 2024. The Author(s).)

Understanding the structure of hardwoods can permit better valorization of lignin by enabling the optimization of green, high-yield extraction protocols that preserve the structure of wood biopolymers. To that end, a mild protocol was applied for the extraction of lignin from ball-milled birch. This made it possible to understand the differences in the extractability of lignin in each extraction step. The fractions were extensively characterized using 1D and 2D nuclear magnetic resonance spectroscopy, size exclusion chromatography, and pyrolysis-gas chromatography-mass spectrometry. This comprehensive characterization highlighted that lignin populations extracted by warm water, alkali, and ionic liquid/ethanol diverged in structural features including subunit composition, interunit linkage content, and the abundance of oxidized moieties. Moreover, ether- and ester-type lignin-carbohydrate complexes were identified in the different extracts. Irrespective of whether natively present in the wood or artificially formed during extraction, these complexes play an important role in the extractability of lignin from ball-milled hardwood. Our results contribute to the further improvement of lignin extraction strategies, for both understanding lignin as present in the lignocellulosic matrix and for dedicated lignin valorization efforts., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10-15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL's outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade. IMPORTANCE We report that the genome of the soil bacterium Chitinophaga pinensis encodes three multi-modular carbohydrate-active enzymes that work together to hydrolyze the major polysaccharide components found in fungal cell walls (FCWs). The enzymes are all encoded by one polysaccharide utilization locus and are co-expressed, potentially induced in the presence of β-1,3-glucans. We present biochemical characterization of each enzyme, including the appended carbohydrate-binding modules that likely tether the enzymes to a FCW substrate. Finally, we propose a model for how this so-called fungal cell wall utilization locus might enable C. pinensis to metabolize both chitin and β-1,3-glucans found in complex biomass in the soil., Competing Interests: The authors declare no conflict of interest.

In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non-native ones. In this study, Norway spruce ( Picea abies ) tissue-cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin-carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbiomes and their enzymes process many of the nutrients accessible in the gastrointestinal tract of bilaterians and play an essential role in host health and nutrition. In this review, we describe recent insights into nutrient processing in microbiomes across three exemplary yet contrasting gastrointestinal ecosystems (humans, ruminants and insects), with focus on bacterial mechanisms for the utilization of common and atypical dietary glycans as well as host-derived mucus glycans. In parallel, we discuss findings from multi-omic studies that have provided new perspectives on understanding glycan-dependent interactions and the complex food-webs of microbial populations in their natural habitat. Using key examples, we emphasize how increasing understanding of glycan processing by gut microbiomes can provide critical insights to assist 'microbiome reprogramming', a growing field that seeks to leverage diet to improve animal growth and host health., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cellulose and lignin are the two main components of secondary plant cell walls with substantial impact on stalk in the field and on straw during industrial processing. The amount of fermentable sugar that can be accessed is another important parameter affecting various industrial applications. In the present study, genetic variability of rice (Oryza sativa L.) genotypes for cellulose, lignin, and fermentable sugars contents was analyzed in rice straw. A genome-wide association study of 33,484 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) >0.05 was performed. The genome-wide association study identified seven, three, and three genomic regions to be significantly associated with cellulose, lignin, and fermentable sugar contents, respectively. Candidate genes in the associated genomic regions were enzymes mainly involved in cell wall metabolism. Novel SNP markers associated with cellulose were tagged to GH16, peroxidase, GT6, GT8, and CSLD2. For lignin content, Villin protein, OsWAK1/50/52/53, and GH16 were identified. For fermentable sugar content, UTP-glucose-1-phosphate uridylyltransferase, BRASSINOSTEROID INSENSITIVE 1, and receptor-like protein kinase 5 were found. The results of this study should improve our understanding of the genetic basis of the factors that might be involved in biosynthesis, turnover, and modification of major cell wall components and saccharides in rice straw., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

The Bacteroidetes phylum is renowned for its ability to degrade a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use polysaccharide utilization loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum-exclusive type IX secretion system (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL-encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non-PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that the literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions that can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems., (© 2021 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The glucan content of rice is a key factor defining its nutritional and economic value. Starch and its derivatives have many industrial applications such as in fuel and material production. Non-starch glucans such as (1,3;1,4)-β-D-glucan (mixed-linkage β-glucan, MLG) have many benefits in human health, including lowering cholesterol, boosting the immune system, and modulating the gut microbiome. In this study, the genetic variability of MLG and starch contents were analyzed in rice ( Oryza sativa L.) whole grain, by performing a new quantitative analysis of the polysaccharide content of rice grains. The 197 rice accessions investigated had an average MLG content of 252 μg/mg, which was negatively correlated with the grain starch content. A new genome-wide association study revealed seven significant quantitative trait loci (QTLs) associated with the MLG content and two QTLs associated with the starch content in rice whole grain. Novel genes associated with the MLG content were a hexose transporter and anthocyanidin 5,3- O -glucosyltransferase. Also, the novel gene associated with the starch content was a nodulin-like domain. The data pave the way for a better understanding of the genes involved in determining both MLG and starch contents in rice grains and should facilitate future plant breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Panahabadi, Ahmadikhah, McKee, Ingvarsson and Farrokhi.)

β-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of β-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here, we show that a dominant butyrate producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various β-mannooligosaccharides (β-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two β-MOS utilization loci (F. prausnitzii β-MOS utilization loci [ Fp MULs]) supported a concerted model whereby the imported β-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degraders Bacteroides ovatus and Roseburia intestinalis on polymeric β-mannan resulted in syntrophic growth, thus confirming the high efficiency of the Fp MULs' uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2,441 public human metagenomes revealed that Fp MULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of β-mannan metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. IMPORTANCE Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic, and detailed biochemical analyses, this work reveals the mechanism enabling F. prausnitzii , as a model Ruminococcaceae within Firmicutes , to cross-feed and access β-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that Fp MULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of β-mannans/β-MOS as a common dietary component. Our findings provide a mechanistic understanding of the β-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, and thus butyrate production, in the gut.

The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment., Competing Interests: Conflict of interest The authors declare no conflicts of interest regarding this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches - the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis , have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto , primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish "natto." Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto , as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material., (Copyright © 2020 Schönbichler, Díaz-Moreno, Srivastava and McKee.)

The secretion of extracellular enzymes by soil microbes is rate-limiting in the recycling of biomass. Fungi and bacteria compete and collaborate for nutrients in the soil, with wide ranging ecological impacts. Within soil microbiota, the Bacteroidetes tend to be a dominant phylum, just like in human and animal intestines. The Bacteroidetes thrive because of their ability to secrete diverse arrays of carbohydrate-active enzymes (CAZymes) that target the highly varied glycans in the soil. Bacteroidetes use an energy-saving system of genomic organization, whereby most of their CAZymes are grouped into Polysaccharide Utilization Loci (PULs). These loci enable high level production of specific CAZymes only when their substrate glycans are abundant in the local environment. This gives the Bacteroidetes a clear advantage over other species in the competitive soil environment, further enhanced by the phylum-specific Type IX Secretion System (T9SS). The T9SS is highly effective at secreting CAZymes and/or tethering them to the cell surface, and is tightly coupled to the ability to rapidly glide over solid surfaces, a connection that promotes an active hunt for nutrition. Although the soil Bacteroidetes are less well studied than human gut symbionts, research is uncovering important biochemical and physiological phenomena. In this review, we summarize the state of the art on research into the CAZymes secreted by soil Bacteroidetes in the contexts of microbial soil ecology and the discovery of novel CAZymes for use in industrial biotechnology. We hope that this review will stimulate further investigations into the somewhat neglected enzymology of non-gut Bacteroidetes., (© 2020 Elsevier Inc. All rights reserved.)

The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of β-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of β-glucans isolated from different sources. Most noncrystalline β-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, β-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of β-glucan metabolism. The significant accumulation of some types of β-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for β-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community. IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis , a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota., (Copyright © 2019 American Society for Microbiology.)

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. However, their commercial exploitation is currently limited by a lack of appropriate and efficient enzymes for converting alginate into metabolizable building blocks, such as 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein, we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-β-D-mannuronate (poly M) alginate, with a k cat of 135.8 s -1 , and a 5-fold lower k cat of 25 s -1 against poly-α-L-guluronate (poly G alginate). We propose that this preference for poly M is due to a structural feature of the protein's active site. The mode of action and specificity of this enzyme has made it possible to design an effective and environmentally friendly process for the production of DEH and low molecular weight guluronate-enriched alginate.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses., (© 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs α-chaconine and α-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes., Significance of This Study: Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The secretion of carbohydrate-degrading enzymes by a bacterium sourced from a softwood forest environment has been investigated by mass spectrometry. The findings are discussed in full in the research article "Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis " in Journal of Proteomics by Larsbrink et al. ([1], doi: 10.1016/j.jprot.2017.01.003). The bacterium was grown on three carbon sources (glucose, glucomannan, and galactomannan) which are likely to be nutrient sources or carbohydrate degradation products found in its natural habitat. The bacterium was grown on solid agarose plates to mimic the natural behaviour of growth on a solid surface. Secreted proteins were collected from the agarose following trypsin-mediated hydrolysis to peptides. The different carbon sources led to the secretion of different numbers and types of proteins. Most carbohydrate-degrading enzymes were found in the glucomannan-induced cultures. Several of these enzymes may have biotechnological potential in plant cell wall deconstruction for biofuel or biomaterial production, and several may have novel activities. A subset of carbohydrate-active enzymes (CAZymes) with predicted activities not obviously related to the growth substrates were also found in samples grown on each of the three carbohydrates. The full dataset is accessible at the PRIDE partner repository (ProteomeXchange Consortium) with the identifier PXD004305, and the full list of proteins detected is given in the supplementary material attached to this report.

The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets., Competing Interests: The authors have declared that no competing interests exist.

Use of the 3,5-dinitrosalicylic acid reagent allows the simple and rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in the characterization of enzyme reactions. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance and sugar concentration.

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica , a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

Unlabelled: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes., Database: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK., (© 2016 Federation of European Biochemical Societies.)

Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses., Results: We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a β-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate., Conclusions: Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.

The genome of the soil bacterium Chitinophaga pinensis encodes a diverse array of carbohydrate active enzymes, including nearly 200 representatives from over 50 glycoside hydrolase (GH) families, the enzymology of which is essentially unexplored. In light of this genetic potential, we reveal that C. pinensis has a broader saprophytic capacity to thrive on plant cell wall polysaccharides than previously reported, and specifically that secretion of β-l-arabinopyranosidase activity is induced during growth on arabinogalactan. We subsequently correlated this activity with the product of the Cpin_5740 gene, which encodes the sole member of glycoside hydrolase family 27 (GH27) in C. pinensis, CpArap27. Historically, GH27 is most commonly associated with α-d-galactopyranosidase and α-d-N-acetylgalactosaminidase activity. A new phylogenetic analysis of GH27 highlighted the likely importance of several conserved secondary structural features in determining substrate specificity and provides a predictive framework for identifying enzymes with the less common β-l-arabinopyranosidase activity.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta., (© 2014 American Society of Plant Biologists. All rights reserved.)

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.

Background: The commercially important glycoside hydrolase family 3 (GH3) beta-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined., Methods: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 beta-glucosidase from A. niger ASKU28., Results: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K(m) values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl beta-glucosides and yielded beta-glucosyl azide as a product., Conclusions and General Significance: These results strongly support the assignment of E490 as the acid/base catalyst in a beta-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.

The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated β-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed β-propeller domain and a C-terminal β-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall.

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.