Varnai, A. | Search bioRxiv

bioRxiv2025

A single-domain expansin-like protein from Gloeophyllum trabeum able to cleave xylan

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, GtEXPN_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 GtEXPN_133317) present in all ERPs and GH45s, fungal ERPs contain a second conserved acidic residue (Asp25 in GtEXPN_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 statementProtein-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.

biochemistry↗

bioRxiv2024

Microbial consortia driving lignocellulose transformation in agricultural woodchip bioreactors

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 lignocellulose-degrading denitrifiers with key players from 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, even including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. ImportanceFreshwater 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 showcases 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 improving water quality, protecting aquatic ecosystems, and reducing greenhouse gas emissions from WBRs.

microbiology↗