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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 u, QC 20230630

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., QC 20230619

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., QC 20220615

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., QC 20220321

beta-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 beta-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 beta-mannooligosaccharides (beta-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two beta-MOS utilization loci (F. prausnitzii beta-MOS utilization loci [FpMULs]) supported a concerted model whereby the imported beta-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 beta-mannan resulted in syntrophic growth, thus confirming the high efficiency of the FpMULs' uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2,441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of beta-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, QC 20211015

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., QC 20210914

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 wi, QC 20200624

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., QC 20210215

The production of cellulose nanofibres (CNFs) typically requires harsh chemistry and strong mechanical fibrillation, both of which have negative environmental impacts. A possible solution is offered by lytic polysaccharide monooxygenases (LPMOs), oxidative enzymes that boost cellulose fibrillation. Although the role of LPMOs in oxidative modification of cellulosic substrates is rather well established, their use in the production of cellulose nanomaterials is not fully explored, and the effect of the carbohydrate-binding module (CBM) on nanofibrillation has not yet been reported. Herein, we studied the activity of two LPMOs, one of which was appended to a CBM, on delignified softwood fibres for green and energy-efficient production of CNFs. The CNFs were used to prepare cellulose nanopapers, and the structure and properties of both nanofibres and nanopapers were determined. Both enzymes were able to facilitate nanocellulose fibrillation and increase colloidal stability of the produced CNFs. However, the CBM-lacking LPMO was more efficient in introducing carboxyl groups (0.53 mmol/g) on the cellulose fibre surfaces and releasing CNFs with thinner width (4.3 ± 1.5 nm) from delignified spruce fibres than the modular LPMO (carboxylate content of 0.38 mmol/g and nanofibre width of 6.7± 2.5 nm through LPMO pretreatment followed by mild homogenisation. The prepared nanopapers showed improved mechanical properties (tensile strength of 262 MPa, and modulus of 16.2 GPa) compared to conventional CNFs preparation methods, demonstrating the potential of LPMOs as green alternatives for cellulose nanomaterials preparation., QC 20191009

QC 20190925

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 beta-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of beta-glucans isolated from different sources. Most noncrystalline beta-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, beta-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 beta-glucan metabolism. The significant accumulation of some types of beta-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 beta-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 count, QC 20190130

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. Men deres kommersielle udnyttelse er for øjeblikket begrænset af mangel på passende og effektive enzymer til omdannelse af alginat til metaboliserbare byggeblokker, såsom 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 kcat of 135.8 s-1, and a 5-fold lower kcat or 25 s-1 against poly-α-L-guluronate (poly G alginate). We suggest that this preference for poly M is due to a structural feature of the protein's active site., QC 20180209

The gene CCT67099 from Fusarium fujikuroi was shown to encode a novel enzyme from the Lytic Polysaccharide Monooxygenase (LPMO) Family AA11. The gene was expressed and a truncated version of the enzyme, designated as FfAA11, was purified from the periplasmic space of Escherichia coli cells at high yield. FfAA11 exhibited oxidative activity against α- and β-chitins, as well as lobster shells. Under optimised conditions, FfAA11 introduced 35 nmol of carboxylate (COO−) moieties per milligram of α-chitin. These carboxylate groups were introduced onto the chitin surface under mild enzymatic oxidation conditions in an aqueous solution without changes to the crystallinity of the chitin fibres. FfAA11 was also combined with a simple and environmentally friendly chemical method that transforms recalcitrant chitins into desirable functionalised (nano)materials. The use of ethyl(hydroxyimino)cyanoacetate (Oxyma)-assisted click chemistry allowed the rapid modification of the surface of FfAA11-oxidized chitins, with a fluorescent probe, a peptide, and gold nanoparticles. The chemical steps performed, including the FfAA11 oxidase treatment and surface chemical modification, were achieved without the production of any toxic by-products or waste organic solvents. This approach represents a novel method for the greener production of chitin-based biomaterials., QC 20180521

Several different methods for the extraction, separation, and purification of wood constituents were combined in this work as a unified process with the purpose of achieving a high overall efficiency of material extraction and utilization. This study aimed to present a laboratory-scale demonstrator biorefinery that illustrated how the different wood constituents could be separated from the wood matrix for later use in the production of new bio-based materials and chemicals by combining several approaches. This study builds on several publications and ongoing activities within the Wallenberg Wood Science Center (WWSC) in Sweden on the theme "From wood to material components." Combining the approaches developed in these WWSC projects - including mild steam explosion, membrane and chromatographic separation, enzymatic treatment and leaching, ionic liquid extraction, and fractionation together with Kraft pulping - formed an outline for a complete materials-biorefinery. The process steps involved were tested as integral steps in a linked process. The scale of operations ranged from the kilogram-scale to the gram-scale. The feasibility and efficiency of these process steps in a biorefinery system were assessed, based on the data, beginning with whole wood., QC 20180216

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., QC 20170321

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., Funding details: Knut och Alice Wallenbergs Stiftelse; Funding details: KTH, Kungliga Tekniska Högskolan; Funding details: Svenska Forskningsrådet Formas; Funding text: The author thanks the support of the Knut and Alice Wallenberg Foundation through the Wallenberg Wood Science Centre, Sweden, as well as the KTH Carbohydrate Materials Consortium (CarboMat) supported by the Swedish Research Council FORMAS.QC 20171108

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., QC 20170329

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 a-chaconine and a-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., QC 20170713

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 G- (agb1-2) or G-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. Significance Statement The plant heterotrimeric G protein complex is an essential component of Pathogen Associated Molecular Pattern-triggered immunity (PTI) and of plant disease resistance, QC 20171124