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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Population ecology and biogeochemical implications of ssDNA and dsDNA viruses along a permafrost thaw gradient

Anthropogenic-driven climate change is accelerating permafrost thaw, threatening to release vast carbon stores through increased microbial activity. While microbial roles are increasingly studied, the contributions of viruses remain largely unexplored, in part due to soil-associated technical challenges that have hindered their detection and characterization. Here, we applied an optimized virion enrichment workflow along a permafrost thaw gradient, identifying 9,963 viral populations (vOTUs), including single- and double-stranded DNA viruses, with 99.9% novelty compared to other soils. Hosts were predicted for 38% of vOTUs, spanning nine archaeal, and 36 bacterial phyla, 22% of which were linked to metagenome-assembled genomes, including key carbon-cycling taxa. Genomic analyses revealed 811 putative auxiliary metabolic genes (AMGs) from 658 vOTUs, nearly half involved in carbon processing. These included 59 glycoside hydrolases (GH) across nine GH families, 45 for monosaccharide degradation, and seven involved in short-chain fatty acid and C1 metabolism, linking viruses to both early and late stages of carbon turnover. Additionally, six vOTUs carried racD, which may stabilize microbial necromass and promote long-term carbon storage. Viral and AMG functional diversity increased with thaw stage, indicating that viruses might participate in a broadening range of microbial metabolic processes as permafrost thaws. These findings expand our understanding of virus contributions in microbial carbon processing and suggest their important role in deciphering soil carbon fate under changing climate conditions.

Biological and medical sciences↗
DOE OSTI2022

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome’s functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon “Musculibacterium intestinale”, which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Unlocking saponin biosynthesis in soapwort

Abstract Soapwort ( Saponaria officinalis ) is a flowering plant from the Caryophyllaceae family with a long history of human use as a traditional source of soap. Its detergent properties are because of the production of polar compounds (saponins), of which the oleanane-based triterpenoid saponins, saponariosides A and B, are the major components. Soapwort saponins have anticancer properties and are also of interest as endosomal escape enhancers for targeted tumor therapies. Intriguingly, these saponins share common structural features with the vaccine adjuvant QS-21 and, thus, represent a potential alternative supply of saponin adjuvant precursors. Here, we sequence the S . officinalis genome and, through genome mining and combinatorial expression, identify 14 enzymes that complete the biosynthetic pathway to saponarioside B. These enzymes include a noncanonical cytosolic GH1 (glycoside hydrolase family 1) transglycosidase required for the addition of d- quinovose. Our results open avenues for accessing and engineering natural and new-to-nature pharmaceuticals, drug delivery agents and potential immunostimulants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Stereoselective synthesis of a 4-α-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-α-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-α-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E 2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Self-driving laboratories to autonomously navigate the protein fitness landscape

Abstract Protein engineering has nearly limitless applications across chemistry, energy and medicine, but creating new proteins with improved or novel functions remains slow, labor-intensive and inefficient. Here we present the Self-driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform for fully autonomous protein engineering. SAMPLE is driven by an intelligent agent that learns protein sequence–function relationships, designs new proteins and sends designs to a fully automated robotic system that experimentally tests the designed proteins and provides feedback to improve the agent’s understanding of the system. We deploy four SAMPLE agents with the goal of engineering glycoside hydrolase enzymes with enhanced thermal tolerance. Despite showing individual differences in their search behavior, all four agents quickly converge on thermostable enzymes. Self-driving laboratories automate and accelerate the scientific discovery process and hold great potential for the fields of protein engineering and synthetic biology.

Rapp, Jacob T. (ORCID:0000000226835204)↗
DOE OSTI2025

Genomic and transcriptomic characterization of carbohydrate-active enzymes in the anaerobic fungus Neocallimastix cameroonii var. constans

Anaerobic gut fungi effectively degrade lignocellulose in the guts of large herbivores, but there remain a limited number of isolated, publicly available, and sequenced strains that impede our understanding of the role of anaerobic fungi within microbial communities. We isolated and characterized a new fungal isolate, Neocallimastix cameroonii var. constans, providing a transcriptomic and genomic understanding of its ability to degrade diverse carbohydrates. This anaerobic fungal strain was stably cultivated for multiple years in vitro among members of an initial enrichment microbial community derived from goat feces, and it demonstrated the ability to pair with other microbial members, namely, archaeal methanogens to produce methane from lignocellulose. Genomic analysis revealed a higher number of predicted carbohydrate-active enzymes encoded in the N. cameroonii var. constans genome compared to most other sequenced anaerobic fungi. The carbohydrate-active enzyme profile for this isolate contained 660 glycoside hydrolases, 160 carbohydrate esterases, 194 glycosyltransferases, and 85 polysaccharide lyases. Differential gene expression analysis showed the upregulation of thousands of genes (including predicted carbohydrate-active enzymes) when N. cameroonii var. constans was grown on lignocellulose (reed canary grass) compared to less complex substrates, such as cellulose (filter paper), cellobiose, and glucose. AlphaFold was used to predict functions of transcriptionally active yet poorly annotated genes, revealing feruloyl esterases that likely play an important role in lignocellulose degradation by anaerobic fungi. The combination of this strain's genomic and transcriptomic characterization, omics-informed structural prediction, and robustness in microbial co-culture make it a well-suited platform to conduct future investigations into bioprocessing and enzyme discovery.

CAZymes↗
DOE OSTI2020

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity

Abstract As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates—namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose–methanol–choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases—we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Virus ecology and 7‐year temporal dynamics across a permafrost thaw gradient

Abstract Soil microorganisms are pivotal in the global carbon cycle, but the viruses that affect them and their impact on ecosystems are less understood. In this study, we explored the diversity, dynamics, and ecology of soil viruses through 379 metagenomes collected annually from 2010 to 2017. These samples spanned the seasonally thawed active layer of a permafrost thaw gradient, which included palsa, bog, and fen habitats. We identified 5051 virus operational taxonomic units (vOTUs), doubling the known viruses for this site. These vOTUs were largely ephemeral within habitats, suggesting a turnover at the vOTU level from year to year. While the diversity varied by thaw stage and depth‐related patterns were specific to each habitat, the virus communities did not significantly change over time. The abundance ratios of virus to host at the phylum level did not show consistent trends across the thaw gradient, depth, or time. To assess potential ecosystem impacts, we predicted hosts in silico and found viruses linked to microbial lineages involved in the carbon cycle, such as methanotrophy and methanogenesis. This included the identification of viruses of Candidatus Methanoflorens, a significant global methane contributor. We also detected a variety of potential auxiliary metabolic genes, including 24 carbon‐degrading glycoside hydrolases, six of which are uniquely terrestrial. In conclusion, these long‐term observations enhance our understanding of soil viruses in the context of climate‐relevant processes and provide opportunities to explore their role in terrestrial carbon cycling.

Microbiology↗
DOE OSTI2022

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non‐productive binding: insights from crystal structures and MD simulation

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) ( EC3.2.1.176 ) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p‐ nitrophenyl‐β‐ d ‐cellobioside (pNPC), p‐ nitrophenyl‐β‐ d ‐lactoside (pNPL) and methylumbelliferyl‐β‐ d ‐cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o‐nitrophenyl‐β‐ d ‐cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium . Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non‐productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non‐productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates ( k cat ) than TrCel7A, but higher Michaelis–Menten ( K M ) constants as well. Furthermore, oNPC turned out to be useful as an active‐site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Galactomannan utilization by Cellvibrio japonicus relies on a single essential α‐galactosidase encoded by the aga27A gene

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

09 BIOMASS FUELS↗
DOE OSTI2019

Comparative Genomics of Rumen Butyrivibrio spp. Uncovers a Continuum of Polysaccharide-Degrading Capabilities

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Capacity of Arctic fjord sediments to degrade carbohydrates from permafrost active layer

The degradation of organic matter (OM) by microorganisms in thawing permafrost produces greenhouse gases. Terrestrial OM is transported into fjords through hydrological runoff, but it is unclear whether the microbial mechanisms of OM degradation on land persist after soils enter marine environments, which differ greatly in conditions and microbial communities. This question is particularly relevant for low-OM soils, which dominate Arctic landscapes and are more exposed to oxidants. Here, we compared OM-degrading capacity in permafrost-affected active layer soils and adjacent fjord sediments from Kongsfjorden, Svalbard, focusing on carbohydrate-active enzymes (CAZymes), which target some of the most abundant types of organic matter in soils. Using multi-omics approaches—metagenomics, metagenome-assembled genomes (MAGs), metabolomics, metatranscriptomics, and metaproteomics—we examined CAZyme presence, distribution, and activity. Despite environmental differences, both soils and sediments harbored diverse glycoside hydrolases and polysaccharide lyases, most of which showed evidence of activity. Verrucomicrobia expressed the highest number of CAZyme transcripts, indicating that they dominated active carbohydrate degradation in fjord sediments, while Acidobacteria and Actinobacteria were more active in soils. Notably, CAZymes in fjord sediments targeted primarily soil-derived OM, and the proportions of enzymes degrading terrestrial OM, marine OM, and microbial necromass—remnants of dead microbial cells were similar across both environments. These results suggest that microbial communities in both soils and fjord sediments are equipped to degrade carbohydrates, and that burial of terrestrial-derived OM in fjord sediments may not protect it from microbial breakdown under Arctic warming.

Chukwufumnanya, Abuah [University of Tennessee, Kn↗
DOE OSTI2023

Comparative Genomic Analyses of Cellulolytic Machinery Reveal Two Nutritional Strategies of Marine Labyrinthulomycetes Protists

Labyrinthulomycetes are a group of ubiquitous and diverse unicellular Stramenopiles and have long been known for their vital role in ocean carbon cycling. However, their ecological function from the perspective of organic matter degradation remains poorly understood. This study reports high-quality genomes of two newly isolated Labyrinthulomycetes strains, namely, Botryochytrium sp. strain S-28 and Oblongichytrium sp. strain S-429, and provides molecular analysis of their ecological functions using comparative genomics and a biochemical assay. Our results suggest that Labyrinthulomycetes may occupy multiple ecological niches in marine ecosystems because of the significant differences in gene function among different genera. Certain strains could degrade wheat bran independently by secreting cellulase. The key glycoside hydrolase families (GH1, GH5, and GH9) related to cellulase and the functional domains of carbohydrate-active enzymes (CAZymes) were more enriched in their genomes. This group can actively participate in marine biochemical cycles as decomposers. In contrast, other strains that could not produce cellulase may thrive as “leftover scavengers” and act as a source of nutrients to the higher-trophic-level plankton. In addition, our findings emphasize the dual roles of endoglucanase, acting as both exo- and endoglucanases, in the process of cellulose degradation. Using genomic, biochemical, and phylogenetic analyses, our study provides a broader insight into the nutritional patterns and ecological functions of Labyrinthulomycetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Identification and structural analysis of a thermophilic β-1,3-glucanase from compost

β-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic β-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like β-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Multiplexed Probing for Functional Capacity Measurements in Complex and Limited-Size Samples

A new strategy using glass as a solid support for functionalization with chemical probes has been recently developed with successful results. The next step in glass functionalization is to pair chemical probes with fluorescent glass microspheres. This approach gives us a way to directly quantify probe-bound protein using Fluorescence-Activated Cell Sorting (FACS). FACS has already shown to be amendable to glass microspheres, demonstrating changes in probe-bound protein concentration. Suitable probing conditions for gram-positive and negative microbes, complex microbial communities from myriad ecosystems, and eukaryotic cells/tissues have traditionally suffered from set-backs, such as limited protein per sample and conditions atypical for probing. To remedy this, we propose to: (1) Demonstrate probe functionalization specific to glass microspheres by paired fluorophore and use those activity probes successfully with proteomics. Probes that are currently available (CYP5, GSH/GST, Glycoside Hydrolase) can be easily prepared onto glass surfaces. Probe-bound microspheres can be tested in ratios, first with known amounts of purified protein and then complex microbiome lysates. (2) Our validated microspheres are then used to determine quantitative amounts of protein through FACS sorting. Complimenting these results by proteomics, prepared microspheres are then optimized and condensed into a user-friendly kit. Once successfully commercialized, this multiplexed assay can be used with little to no technical expertise, making this available to various industries users.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Functional Genomics, Transcriptomics, and Proteomics Reveal Distinct Combat Strategies Between Lineages of Wood-Degrading Fungi With Redundant Wood Decay Mechanisms

Wood-degrading fungi vary in their strategies for deconstructing wood, and their competitive successes shape the rate and fate of carbon released from wood, Earth’s largest pool of aboveground terrestrial carbon. In this study, one-on-one interspecific interactions between two model brown rot (carbohydrate-selective) fungi, Gloeophyllum trabeum and Rhodonia (Postia) placenta, were studied on wood wafers where a clearly resolved interaction zone (IZ) could be generated, reproducibly. Comparative RNAseq and proteomics between the IZ and non-interacting hyphae of each species identified combative strategies for each fungus. Glycoside hydrolases were a relatively smaller portion of the interaction secretome compared to non-interacting hyphae. The interaction zone showed higher pectinase specific activity than all other sampling locations, and higher laminarinase specific activity (branched ß-glucan proxy) was seen in the IZ secretome relative to equivalent hyphae in single-species cultures. Our efforts also identified two distinct competitive strategies in these two fungi with a shared nutritional mode (brown rot) but polyphyletic ancestral lineages. Gloeophyllum trabeum (Gloeophyllum clade) employed secondary metabolite (SM) synthesis in response to a competitor, as shown by the upregulation of several SM-synthesizing genes in the interaction. R. placenta (Antrodia clade) instead upregulated a larger variety of uncharacterized oxidoreductases in interacting hyphae, suggesting that an oxidative burst may be a response to competitors in this fungus. Both species produced several hypothetical proteins exclusively in the interaction zone, leaving abundant unknowns on the battlefield. This work supports the existence of multiple interaction strategies among brown rot fungi and highlights the functional diversity among wood decay fungi.

59 BASIC BIOLOGICAL SCIENCES↗