Anikó Várnai

Reply to Cosgrove: Catalytic activity in expansin-like proteins does not explain wall creep but raises a flag

Lignocellulose is a major component of plant biomass and is recalcitrant, with efficient degradation typically requiring oxygen-dependent oxidative and carbohydrate-active enzymes (CAZymes). Anaerobic turnover is slower but can be supported by microbes capable of nitrate respiration, including denitrifiers and dissimilatory nitrate reduction to ammonium (DNRA) bacteria, which may use nitrate or nitric oxide as alternative oxidants. Anoxic layers beneath the oxic zones of eutrophic lake sediments, where nitrate penetrates from surface waters, provide a natural habitat for such organisms. To investigate these processes, we established nitrate-amended enrichments from organic-rich sediments of 10 eutrophic lakes and applied gas kinetics alongside metagenomics and metaproteomics to characterize the microbial communities. We identified a set of core microbial metagenome-assembled genomes (MAGs) present in all enrichments, dominated by Pseudomonadota , Bacteroidota , Verrucomicrobiota , and Actinomycetota , which played key roles in denitrification and fermentation. Lignocellulose degradation, however, was largely carried out by species outside the core microbiome—that is, different key degraders between lakes, suggesting lake-specific specialization. Among these, we observed potential respiratory DNRA pathways and a broad repertoire of CAZymes targeting various lignocellulose subfractions. Interestingly, many MAGs also encoded nitric oxide dismutases (NODs), enzymes postulated to convert NO to molecular oxygen and dinitrogen gas. Together, these findings advance our understanding of anaerobic biomass degradation and nitrogen cycling in eutrophic freshwater sediments, while highlighting the unexplored functional diversity of NOD-containing bacteria as an intriguing open question for future research. IMPORTANCE: Lignocellulose, the main structural component of plant biomass, represents a vast reservoir of organic carbon in natural environments. Although lignocellulose breakdown is commonly associated with oxygen-rich conditions, it also occurs in oxygen-depleted habitats such as lake sediments, where the responsible microbes and processes are poorly understood. This study reveals how diverse microbial communities can degrade lignocellulose while respiring nitrate, linking carbon turnover to nitrogen cycling in anoxic environments. By identifying shared and lake-specific microbial strategies, as well as a widespread but poorly characterized class of enzymes associated with nitric oxide metabolism, our work advances our understanding of anaerobic biomass degradation. These insights have implications for ecosystem functioning in nutrient-rich waters and for the development of sustainable, oxygen-free biotechnological processes.

Expansin-related proteins (ERPs) are a broad group of plant cell wall–loosening proteins and are considered noncatalytic, as, to date, no cell wall–derived products have been observed as a result of catalysis, despite the presence of a domain that resembles the catalytic domains of GH45 endoglucanases. Here, we report catalytic activity for a single-domain ERP, Gt EXPN_133317, from the brown-rot fungus Gloeophyllum trabeum , which is highly expressed in the early phase of spruce colonization. We demonstrate enzyme-dependent formation of xylan-derived products, such as glucuronylated xylo-oligosaccharides, using high-performance anion exchange chromatography with pulsed amperometric detection. Structure-based multiple sequence alignment of ERPs with GH45 endoglucanases showed that, next to a single conserved aspartate (Asp87 in Gt EXPN_133317) present in all ERPs and GH45s, fungal ERPs contain a second conserved acidic residue (Asp25 in Gt EXPN_133317). Mutation of these two conserved amino acids, Asp87 and Asp25, led to a nearly complete loss of xylanolytic activity. While these findings do not exclude the possibility of a noncatalytic plant cell wall–loosening mechanism, they show that ERPs likely have other modes of action besides what the current paradigm states.

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.

This study assessed mango seed husk (MSH) fractions for producing glucose and phenolic compounds using commercial enzymes. We focused on cleaving lignin–carbohydrate linkages, specifically feruloyl and glucuronoyl esters, to decrease biomass recalcitrance and enhance product extraction. For saccharification studies, we used Sigma’s C1184 cellulase from Aspergillus niger . Characterisation results of ground MSH using phloroglucinol and scanning electron microscopy revealed that it could be separated into a fine fraction, containing less lignin and cellulose fibres with parallel orientation, and a coarse fraction, with higher lignin content and cellulose fibres at an angled orientation. Activity assays and zymogram analysis of the C1184 preparation prior to saccharification studies revealed diverse CAZyme activities associated with distinct proteins, with xylanolytic activity dominating. Saccharification studies with ground MSH found that the C1184 preparation supplemented with feruloyl or glucuronoyl esterases was suitable for extracting phenolic compounds (0.4–1.7% w/w) from MSH while converting up to 20% of the total biomass as glucose. Interestingly, when replacing 50% (w/w) of the C1184 preparation with glucuronoyl esterase, glucose release nearly doubled from both MSH fractions. Additionally, phenolics attached to carbohydrates may be less condensed in the fine fraction, as all three esterases released three-to-five times more phenolics from the fine fraction compared to the coarse fraction with higher lignin content. Saccharification trials with alkali-pretreated ground MSH showed that the C1184 preparation supplemented with β-glucosidase produced low glucose levels (170–250 mg/g dry biomass) from the substrate after 24 h, even at 50 mg/g biomass protein loading. Overall, this work advances our understanding of the importance of lignin–carbohydrate linkages formed via glucuronoyl esters in biomass recalcitrance. Furthermore, our study corroborates the potential of MSH as a valuable feedstock for producing value-added products in the biorefinery sector. Graphical abstract

Expansin-related proteins (ERPs) are a broad group of plant cell wall-loosening proteins and are considered non-catalytic as, to date, no cell wall-derived products have been observed as a result of catalysis, despite the presence of a domain that resembles the catalytic domains of GH45 endoglucanases. Here, we report catalytic activity for a single-domain ERP, Gt EXPN_133317, from the brown-rot fungus Gloeophyllum trabeum , which is highly expressed in the early phase of spruce colonization. We demonstrate enzyme-dependent formation of xylan-derived products, such as glucuronylated xylo-oligosaccharides, using high-performance anion exchange chromatography with pulsed amperometric detection. Structure-based multiple sequence alignment of ERPs with GH45 endoglucanases showed that, next to a single conserved aspartate (Asp87 in Gt EXPN_133317) present in all ERPs and GH45s, fungal ERPs contain a second conserved acidic residue (Asp25 in Gt EXPN_133317). Mutation of these two conserved amino acids, Asp87 and Asp25, led to a nearly complete loss of xylanolytic activity. While these findings do not exclude the possibility of a non-catalytic plant cell wall-loosening mechanism, they show that ERPs likely have other modes of action besides what the current paradigm states. Significance statement: Protein-mediated plant cell wall-loosening is a natural process that enables plant growth and improves cell wall accessibility for microorganisms. Expansin-related proteins (ERPs) are key to this process, but their mechanism is not fully understood. Traditionally, ERPs are seen as non-catalytic, disrupting non-covalent bonds holding the cellulose network together. We have investigated an expansin-like protein from a fungal saprotroph and demonstrate its catalytic activity by showing product formation from glucuronoxylan and identifying key residues associated with this activity. This activity, although so far only shown for one protein, challenges the current paradigm that ERPs are non-catalytic, shedding new light on plant cell wall architecture and dynamics as well as on the potential roles of ERPs in plant–pathogen interactions.

Cellulose-derived biomaterials offer a sustainable and versatile platform for various applications. Enzymatic engineering of these fibers, particularly using lytic polysaccharide monooxygenases (LPMOs), shows promise due to the ability to introduce functional groups onto cellulose surfaces, potentially enabling further functionalization. However, harnessing LPMOs for fiber engineering remains challenging, partly because controlling the enzymatic reaction is difficult and partly because limited information is available about how LPMOs modify the fibers. In this study, we explored controlling LPMO-mediated fiber oxidation by sequentially adding a reductant (gallic acid, GA) and H₂O₂, using three different carbohydrate-binding module (CBM)-containing LPMOs. An in-depth analysis of the soluble products and the M _n, M _w, and carbonyl content in the fiber fraction indicates that fiber oxidation can indeed be controlled by adjusting the amount of GA and H₂O₂ added to the reaction. In particular, at lower overall dosages of GA and H₂O₂, corresponding to low oxidation levels, fiber oxidation occurs rapidly with almost no release of soluble oxidized products. Conversely, at higher dosages, fiber oxidation levels off, while oxidized oligosaccharides continue to be released and the fibers are eroded. Importantly, next to demonstrating controlled fiber oxidation, this study shows that different cellulose-active LPMOs modify the fibers in different manners.

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors and their active enzymes. Our findings highlight a microbial community enriched for (ligno)cellulose-degrading denitrifiers with key players from the taxa Giesbergeria , Cellulomonas , Azonexus, and UBA5070 ( Fibrobacterota ). A wide substrate specificity is observed among the many expressed carbohydrate-active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. IMPORTANCE: Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria , Cellulomonas , Azonexus , and UBA5070 ( Fibrobacterota ) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. Results: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. Conclusions: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.