Phlorotannins and polysaccharides are widespread in brown seaweeds. This study shows that ultrasound-assisted extraction combined with natural deep eutectic solvents (NaDES-UAE) enhances the yields of phlorotannins and polysaccharides from the brown seaweed Ecklonia radiata. Among the NaDES solvents tested, choline chloride:lactic acid (molar ratio of 1:3) supplemented with 20 % water was the most effective. The extraction parameters were further optimized using a Box-Behnken design and response surface methodology. The phlorotannin and polysaccharide yields obtained after optimized NaDES-UAE extraction were 12.8 ± 0.3 % and 26.5 ± 0.9 %, respectively-both significantly higher than yields obtained using conventional organic solvent extraction. Twenty-one phlorotannins and isomers were identified using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry, while ten different monosaccharides were detected in the polysaccharide fractions. The relative abundance of specific phlorotannins and monosaccharides varied depending on the extraction method. Compared to conventional aqueous and organic solvent extractions, NaDES-UAE-extracted phlorotannins exhibited higher antioxidant activity, while the polysaccharide fraction, mainly consisting of low-molecular-weight polysaccharides and oligosaccharides, showed enhanced antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. In conclusion, our study reveals that the NaDES-UAE method is an efficient, eco-friendly alternative for extracting bioactive compounds from brown seaweeds, with strong potential for applications in the food, nutraceutical, biopharmaceutical, and other industrial sectors.

Brown algae (BA) represent a great source of sustainable bioproducts such as bioactive substances with beneficial health properties and biopolymers with industrial applications. Increasing demand for these bioproducts necessitates a holistic BA utilisation approach, requiring integrated biorefinery concepts that combine biomass pre-treatment, fractionation and downstream separation and purification technologies. This review discusses holistic BA valorisation focusing on bioactive secondary metabolites and their incorporation as products of BA biorefineries. It explores their structural diversity, identification and characterisation methods, and sustainable processing technologies. Polar phlorotannins, non-polar carotenoids, and sterols predominate bioactive compounds in BA. Phlorotannins in BA are structurally diverse and complex due to the presence of structural isomers. A cascade biorefinery concept can be developed using green technologies, potentially enabling the sequential recovery of up to six high-purity products. Lipophilic and phlorotannin enriched fractions are separately recovered using polarity-based fractionation assisted by cell wall disruption techniques, and separation and purification follow to recover proteins, fucoidan, alginate and algal cellulose-enriched fractions. Encapsulation is necessary to turn BA bioactive compounds into ready-to-use ingredients in functional food and nutraceutical industries.

Apple pomace, the waste product of the apple processing industry, is a significant global waste-management problem and an underutilized source of bioactive molecules, including polyphenols. To help reduce the amount of apple waste sent to landfill, efficient and ecologically sustainable methods to extract bioactive compounds are needed to facilitate the development of value-added products. To this end, we prepared apple pomace extracts with an environmentally friendly deep eutectic solvent (DES) and a commonly used organic solvent. The composition and bioactivity of both extracts are compared. HPLC-DAD MS/MS analysis revealed similar profiles and content of polyphenols in both extracts. Moreover, the extracts have comparable chemical and cellular antioxidant activity. In murine and human models of LPS-induced inflammation, both extracts exhibited potent anti-inflammatory activity as measured by the suppression of pro-inflammatory cytokine gene expression. Mechanistically, the anti-inflammatory effects of the extracts were mediated, at least in part, by inhibiting the pro-inflammatory master transcription factor NF-kappa B and by inhibiting the rate-liming enzyme of prostaglandin biosynthesis, COX-2. Our findings underscore the value of apple pomace as a rich source of bioactive compounds and the utility of DES extraction. Further refinement and upscale of the DES extraction method could facilitate product development in the food and health industries.

Anthocyanins are plant-derived pigments with diverse applications due to their favourable spectral and biological properties, which are often modulated through enzymatic addition of structural modifications. However, evidence indicates that anthocyanin glycosyl groups may be modified by organic acids in solution through an acid-catalysed esterification. Here, the anthocyanin standard cyanidin-3-glucoside is shown to undergo esterification in aqueous conditions with citric acid, acetic acid, and most efficiently, formic acid. Extended to a biological sample, red cabbage anthocyanins incubated with formic acid resulted in 53 unique species from 9 original anthocyanins. From these findings a key structural determinant to predict formylation patterns is identified: the precise glucosyl unit where hydroxycinnamic acid acylation occurs, which dictates the number of free primary alcohol groups in the anthocyanin available for formylation. This study demonstrates the unexpected structural complexity these reactions introduce into samples that necessitate careful consideration in anthocyanin handling. Harnessing these reactions in future may help produce anthocyanins with controlled structures and properties for downstream applications.

ABSTRACT The phylum Oomycota contains economically important pathogens of animals and plants, including Saprolegnia parasitica , the causal agent of the fish disease saprolegniasis. Due to intense fish farming and banning of the most effective control measures, saprolegniasis has re-emerged as a major challenge for the aquaculture industry. Oomycete cells are surrounded by a polysaccharide-rich cell wall matrix that, in addition to being essential for cell growth, also functions as a protective “armor.” Consequently, the enzymes responsible for cell wall synthesis provide potential targets for disease control. Oomycete cell wall biosynthetic enzymes are predicted to be plasma membrane proteins. To identify these proteins, we applied a quantitative (iTRAQ) mass spectrometry-based proteomics approach to the plasma membrane of the hyphal cells of S. parasitica , providing the first complete plasma membrane proteome of an oomycete species. Of significance is the identification of 65 proteins enriched in detergent-resistant microdomains (DRMs). In silico analysis showed that DRM-enriched proteins are mainly involved in molecular transport and β-1,3-glucan synthesis, potentially contributing to pathogenesis. Moreover, biochemical characterization of the glycosyltransferase activity in these microdomains further supported their role in β-1,3-glucan synthesis. Altogether, the knowledge gained in this study provides a basis for developing disease control measures targeting specific plasma membrane proteins in S. parasitica . IMPORTANCE The significance of this research lies in its potential to combat saprolegniasis, a detrimental fish disease, which has resurged due to intensive fish farming and regulatory restrictions. By targeting enzymes responsible for cell wall synthesis in Saprolegnia parasitica , this study uncovers potential avenues for disease control. Particularly noteworthy is the identification of several proteins enriched in membrane microdomains, offering insights into molecular mechanisms potentially involved in pathogenesis. Understanding the role of these proteins provides a foundation for developing targeted disease control measures. Overall, this research holds promise for safeguarding the aquaculture industry against the challenges posed by saprolegniasis.

Linear, unbranched (1,3;1,4)-β-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-β-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-β- and (1,4)-β-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-β-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

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.

ABSTRACT Fungal cell walls are dynamic extracellular matrices that enable efficient adaptation to changing environments. While the cell wall compositions of yeasts, human, and plant pathogenic fungi have been studied to some extent, the cell walls of mycoparasites remain poorly characterized. Trichoderma species comprise a diverse group of soil fungi with different survival strategies and lifestyles. The comparative study of cell wall carbohydrate-active enzymes in 13 Trichoderma spp. revealed that the types of enzymes involved in chitin and chitosan metabolism are phylogenetically distant between mycoparasitic and saprotrophic species. Here, we compare the carbohydrate composition and function of the cell wall of a saprotrophic strain Trichoderma reesei with that of the mycoparasitic, biological control agent Trichoderma atroviride . Monosaccharide and glycosidic linkage analyses as well as dual in situ interaction assays showed that the cell wall polysaccharide composition is conserved between both species, except for the amounts of chitin detected. The results suggest that the observed accumulation of chitosan during mycoparasitism may prevent host recognition. Remarkably, Trichoderma atroviride undergoes dynamic cell wall adaptations during both vegetative development and mycoparasitism, which appears to be confirmed by an evolutionarily expanded group of specialized enzymes. Overall, our analyses support the notion that habitat specialization is reflected in cell wall architecture and that plastic chitin remodeling may confer an advantage to mycoparasites, ultimately enabling the successful invasion and parasitism of plant pathogens. This information may potentially be exploited for the control of crop diseases using biological agents. IMPORTANCE Trichoderma species are emerging model fungi for the development of biocontrol agents and are used in industrial biotechnology as efficient enzyme producers. Fungal cell walls are complex structures that differ in carbohydrate, protein, and enzyme composition across taxa. Here, we present a chemical characterization of the cell walls of two Trichoderma spp., namely the predominantly saprotrophic Trichoderma reesei and the mycoparasite Trichoderma atroviride . Chemical profiling revealed that Trichoderma spp. remodel their cell wall to adapt to particular lifestyles, with dynamic changes during vegetative development. Importantly, we found that chitosan accumulation during mycoparasitism of a fungal host emerged as a sophisticated strategy underpinning an effective attack. These insights shed light on the molecular mechanisms that allow mycoparasites to overcome host defenses and can be exploited to improve the application of T. atroviride in biological pest control. Moreover, our results provide valuable information for targeting the fungal cell wall for therapeutic purposes.

AbstractCarbohydrate-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.

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in β−1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether β−1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to β−1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a β−1,3-glucanase in the female germline transiently perturbed β−1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline β−1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

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.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin–cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

As the most abundant biopolymer in the oceans, the significance of chitin and its biosynthesis is rarely demonstrated in diatoms, which are the main contributors to the primary productivity of the oceans, ascribed to their huge biomass and efficient photosynthesis. We retrieved genes involved in chitin-based metabolism against the Tara Oceans Gene Atlas to expand our knowledge about their diversity and distribution in the marine environment. Potential new producers of chitin were found from the analysis of various algal transcriptome and genome databases.

Cultivated brown algae represent an important potential source of carbohydrate polymers for packaging and other biobased materials. However, their exploitation is currently limited by a short harvest season and a lack of cost-effective and sustainable methods to preserve biopolymer quality. In the present study, cultivated Saccharina latissima (SL) and Alaria esculenta (AE) were preserved with formic acid at 4, 13 and 20 degrees C for up to 16 weeks prior to extraction and characterization of alginate and cellulose. The data show up to 40 % increased yield of alginate from preserved biomass compared with fresh and non-preserved biomass, primarily due to removal of minerals and other soluble compounds during the acid wash. Acid preservation and storage caused a reduction in alginate weight average molecular weight (Mw) that was mainly dependent on storage temperature and to a lesser extent on storage time; storage at 4 degrees C maintained the Mw of alginates at 350-500 kDa. Preservation had no effect on the guluronate block structure of the extracted alginates, but guluronic acid content and block length increased in the non-preserved samples, presumably due to enzymatic degradation of the alginate's M-rich re-gions. Preservation of the seaweed resulted in an increased cellulose yield compared with fresh and non -preserved biomass, again due to the biomass being reduced during acid wash. The molecular weight and crys-tallinity of cellulose were not altered by the process. Altogether our findings demonstrate that acid preservation at low temperatures can effectively stabilize seaweed biomass while preserving alginate and cellulose quality for biomaterials and other applications.

AbstractExtracellular vesicles (EVs) from human fungal pathogens have been implicated in fungal virulence, yet little is known about their role in the host‐pathogen interaction. Progress has been hampered by the lack of a specific marker for fungal EVs that can be used to monitor EV isolation and tracking in biological systems. Here we report the effect of a SUR7 gene knockout on the production, properties, and role of EVs in the virulence of Candida albicans. Sur7 is a component of the membrane compartment of Can1 (MCC) complex and is enriched in the EVs from C. albicans and other fungal species. MCC is a plasma membrane complex which together with the eisosome, a cytoplasmic protein complex, is a key regulator in plasma membrane organisation and plasma membrane associated processes. The SUR7 knockout strain produces smaller EVs than the wild‐type (WT) with different protein and carbohydrate cargos. Furthermore, proteins with known roles in Candida pathogenesis were present in WT EVs and absent or diminished in the sur7Δ EVs. We demonstrate that the reduced virulence of the sur7Δ cells can be partially restored with EVs from a WT strain. These findings demonstrate the importance of Sur7‐like proteins in the biogenesis of EVs in fungi and enhance our understanding of the role of fungal EVs in human pathogenesis.

In the last 50 years, giant strides have been made in our understanding of cell wall biology in the Poaceae, which include our most important cereal crops. These achievements have been made possible by impressive and parallel advances in biotechnologies for defining the structures, biochemistry and cell biology of cell walls. In particular, the advent of DNA cloning technologies enabled us to quickly define the complete amino sequences of enzymes involved in wall synthesis and degradation, and opened the way for solving three-dimensional structures of key enzymes. Functional genomics technologies, supported by spectacular advances in computing power, enabled complete sets of expressed RNA transcripts, proteins and metabolites to be defined in single experiments. Despite these advances, important questions remain about the identities of genes and enzymic mechanisms that mediate (1,3;1,4)-β-glucan and heteroxylan synthesis and the biological roles of xyloglucan transglycosylases. Moreover, there is not yet agreement as to the sites of synthesis of several non-cellulosic wall polysaccharides. Sustained interest in cereals and grasses for feeding the burgeoning global population, as sources of renewable biofuels and in human health, continues to drive the development of exciting new technologies, including crucially important methods for harnessing beneficial natural and induced genetic variation.

Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control.

Pectin methylesterases (PMEs) are enzymes that play a critical role in modifying pectins, a class of complex polysaccharides in plant cell walls. These enzymes catalyze the removal of methyl ester groups from pectins, resulting in a change in the degree of esterification and consequently, the physicochemical properties of the polymers. PMEs are found in various plant tissues and organs, and their activity is tightly regulated in response to developmental and environmental factors. In addition to the biochemical modification of pectins, PMEs have been implicated in various biological processes, including fruit ripening, defense against pathogens, and cell wall remodelling. This review presents updated information on PMEs, including their sources, sequences and structural diversity, biochemical properties and function in plant development. The article also explores the mechanism of PME action and the factors influencing enzyme activity. In addition, the review highlights the potential applications of PMEs in various industrial sectors related to biomass exploitation, food, and textile industries, with a focus on development of bioproducts based on eco-friendly and efficient industrial processes.