Biology subjects

Westereng, B.

Publications and source records attributed to Westereng, B..

13 recordsLinked to original sources

bioRxiv2026

Mild extraction preserves labile structural features of Gram-positive bacterial cell wall glycopolymers

Cell wall glycopolymers in Gram-positive bacteria are crucial for structural integrity, cell division, and stress resistance. Elucidating the structure of these glycopolymers is not only important for understanding fundamental bacterial physiology, but may facilitate vaccine development, phage therapy, antibiotic design and improvements in food fermentation safety. However, extraction protocols have so far relied on harsh chemicals that can damage these polymers, compromise their structural integrity, obscure their biological functions, and result in low yields. We present a straightforward method for isolating cell wall glycopolymers from Gram-positive bacteria that preserves essential structural features including attachment to peptidoglycan. The method was broadly applicable across phylogenetically diverse species and was not restricted to glycopolymer types. In total, we isolated 17 distinct glycopolymers from 11 species, including six with previously unreported structures, with a median yield of 49 mg per litre of culture. The simplicity and mild nature of the method, combined with the exceptional purity of the resulting products, has the potential to transform research on Gram-positive cell wall glycopolymers

microbiology↗

bioRxiv2026

From FODMAPs to prebiotic candidates: enzymatic transglycosylation of raffinose oligosaccharides towards new mixed-linkage oligosaccharides

Raffinose family oligosaccharides (RFOs) are abundant in side streams from food and feed production from legumes, and the transition to plant-based diets increases the volume of such side streams. RFOs in the diet tend to have negative impacts on the consumers gut (e.g., nausea, bloating, diarrhoea), and in many ways, RFOs are comparable to lactose as a side stream from the dairy industry and symptoms associated with lactose intolerance. On the contrary, galactooligosaccharides (GOS) are recognized as prebiotics, and in this study we used a {beta}-galactosidase from Niallia circulans to produce potential prebiotics from RFOs (acceptors) and lactose (donor), which we hypothesized to have a lower fermentability than unmodified RFOs. The transglycosylation reactions resulted in RFO-based -{beta}-GOS, with NMR characterization showing ({beta}1-4) galactosylations on the non-reducing galactose end of RFOs as the major product. In reactions with RFOs, the characteristics were comparable to reactions with lactose alone and the new -{beta}-GOS products made up the largest fraction (by weight). A screening of 11 relevant gut and food microbe strains revealed that the gut commensal Bacteroides ovatus metabolised these modified oligosaccharides for growth whereas other strains grew only after adaption and others did not use them at all. This implies that mixed-linkage -{beta}-GOS are less fermentable by some microbes compared to raffinose, while other (beneficial) bacteria can still ferment them. The enzymatic synthesis established here is an interesting approach to upgrade abundant food side streams towards new prebiotics in a world where functional foods and food waste reduction receive increasing attention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/731070v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@18e0e62org.highwire.dtl.DTLVardef@1525b4borg.highwire.dtl.DTLVardef@1e7be88org.highwire.dtl.DTLVardef@18df278_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

High molecular-weight polysaccharide contamination from yeast extract in semi-defined bacteriological media: Effects on exopolysaccharide production and purity

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.

microbiology↗

bioRxiv2026

In vitro model reveals structural and metabolic insights to the porcine caecal microbiota in response to β-mannan exposure

The gastrointestinal microbiota plays a pivotal role in shaping host physiology and health. By selectively promoting bacteria associated with improved host health, microbiota-directed fibres offer a strategy to enhance the beneficial functions of the microbiota. In this work, we developed a pH-controlled in vitro fermentation system (InVitSim) as a model to evaluate the effects of such a fibre - acetylated galactoglucomannan from Norway spruce - on the composition and functionality of porcine caecal microbial communities. We validated the experimental outcomes by comparing the response of the in vitro model to a previous in vivo feeding trial utilising the same {beta}-mannan fibres. Long-read sequencing with Oxford Nanopore, metatranscriptomics, and short-chain fatty acid measurements were undertaken to survey microbial community dynamics and functionality. Microbial communities in pigs and InVitSim responded similarly to {beta}-mannan supplementation, with taxa like Prevotella, Catenibacterium, and Faecalibacterium increasing in abundance. Intriguingly, some taxa were observed to be more affected by {beta}-mannan supplementation in InVitSim than in vivo. These taxa included several bacterial species that were not previously known to utilise {beta}-mannan, yet exhibited upregulated genes encoding carbohydrate-active enzymes involved in the degradation of this substrate. ImportanceIn this study, we establish a fermenter system able to preserve more than 70% of over 300 distinct microbial taxa identified in the porcine caecal gut. The in vitro model and the functional omic data generated from it enabled us to identify relevant microbial populations that responded to the presence of AcGGM by upregulating {beta}-mannan-specific polysaccharide utilisation loci. Our results highlight the value of in vitro approaches as a complementary tool to in vivo trials for learning about the gastrointestinal microbiomes response to dietary interventions on the host level. Description of supplementary filesO_LIIn-depth analyses for in vitro model validation and investigations. C_LIO_LICommon taxa between in vivo and in vitro systems exposed to {beta}-mannans. C_LIO_LIDifferential abundance analysis result visualisations. C_LIO_LIShort-chain fatty acid concentrations and correlation with microbial abundances. C_LI

microbiology↗

bioRxiv2025

Deacetylation of β-Mannans by Two Complementary Carbohydrate Esterases from the Human Gut Microbe Bacteroides cellulosilyticus

{beta}-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several classes of {beta}-mannans are decorated with acetylations, which are key for their physicochemical properties and protection against enzymatic degradation. While the process for depolymerization of acetylated {beta}-mannans has been described in depth for members of the phylum Bacillota, there is limited mechanistic knowledge on how Bacteroidota utilize these glycans. Here, we combined proteomics and biochemical analyses to functionally characterize a pair of carbohydrate esterases (CEs) from Bacteroides cellulosilyticus that, together, deacetylate complex {beta}-mannans. We demonstrate that the newly identified BcCExxx enzyme, representing a novel carbohydrate esterase (CE) family, exhibits high specificity by selectively removing axially oriented 2-O-acetyl groups from mannose residues. In contrast, BcCE7 functions as a broad-spectrum esterase, capable of deacetylating oligosaccharides derived from structurally diverse substrates, including {beta}-mannans, xylans, and acetylated cellulose. In transesterification reactions, BcCExxx showed activity on both mannooligosaccharides and polymeric glucomannan. Overall, our findings provide new insight into the strategies that beneficial Bacteroides have evolved to deacetylate complex {beta}-mannans in the human gut. Significance Statement{beta}-Mannans, commonly found in various plant-based foods and used as food additives, can be metabolized by gut microbes, potentially impacting host health. Acetylation of {beta}-mannans enhances their resistance to enzymatic breakdown, making carbohydrate esterases crucial for gut bacteria to utilize these carbohydrates. This study explores the roles of two carbohydrate esterases (CEs) from a human commensal Bacteroides species in deacetylating complex {beta}-mannans. The two esterases together remove acetylations at different positions on mannose units. BcCE7 is a versatile esterase that deacetylates a variety of oligosaccharides, while the newly discovered BcCExxx is highly active towards 2-O-acetylations on mannose units in both oligosaccharides and polymeric {beta}-mannan. This research enhances our understanding of how Bacteroidota species deacetylate {beta}-mannans.

biochemistry↗

bioRxiv2025

Integrative holo-omic data analysis predicts interactions across the host-microbiome axis

Understanding the interplay between host organisms and their microbiomes is central to the development of sustainable food systems. However, high dimensionality and spurious associations remain major obstacles to extracting meaningful biological insight from multi-omic host-associated microbiome data; a challenge further exacerbated when "holo-omic" analyses across the host-microbiome boundary is considered. Here, we show that a computational method designed for multi-omic analysis in eukaryotes can be leveraged to integrate and analyse five layers of holo-omic data from porcine hosts and their gut microbiomes. We collected caecal tissue and digesta samples during a feeding trial that tested the impact of microbiota-directed fibres (acetylated galactoglucomannan) at critical developmental stages. From 800,000 features including microbial and host genes, metagenome-assembled genomes, and metabolites from caecal tissue and digesta, we used multiset correlation and factor analysis to select the most relevant features for capturing coordinated patterns across omic layers. From these features, we predicted over 2,000 putative host-microbiome interactions based on co-occurrence. Some of them reflected previously known relationships between animal and microbiome features, such as microbial genes for carbohydrate metabolism being linked to glycoside abundances in host tissue. Other predicted co-occurrences included features that were not detected in single-omic analysis and offer new hypotheses of host-microbiome interactions that warrant future investigation. Hence, we showcase an application of holo-omic analysis that avoids common pitfalls in high-dimensional data analysis; identifies known interactions as a form of validation; and most importantly, predicts new leads for understanding host-microbiome symbiosis. ImportanceWhile study systems involving mammalian hosts and their microbiomes are inherently complex, multi- and holo-omic analyses promise to provide interpretable results with translational value for the animal production industry. Unfortunately, computational methods capable of this kind of integration are currently scarce, as most existing multi-omics approaches have been developed for analysis of data layers within a single multicellular organism. We propose to adapt existing multi-omic methods for holo-omics by combining feature selection and interaction inference. This two-step analysis approach addresses common challenges in data-driven studies and can be implemented with a variety of tools for feature selection and interaction modelling. Through this holistic approach, we show that both known and novel relationships across the holobiont axis can be identified in a data-driven manner, offering new targets for the continued study of host-microbiome interactions and the effect of dietary interventions on production animals.

bioinformatics↗

bioRxiv2024

A Pilot-Scale Process for the Extraction of Raffinose-Oligosaccharides from Pulse Protein Concentrates

Plant protein concentrates have gained popularity in recent years as part of a shift towards healthier and sustainable food production. Their production via dry fractionation is well established and leads to a protein ingredient that is also rich in raffinose family oligosaccharides (RFOs). Although these can cause gut discomfort, they might also serve as a valuable substrate fowr growth of probiotic bacteria or as substrate in other biotechnological applications. By using a process including mildly acidic extraction, separation and sequential filtration, a carbohydrate fraction enriched in RFOs was isolated from peas (48 %) and faba beans (26 %) on a kilogram scale. Simultaneously a fraction with elevated protein concentration (5-6 % increase compared to the starting material) and only minor changes in amino acid composition is produced. This process could serve ingredient manufacturers that seek to reduce RFOs in protein concentrates or to produce RFOs for biotechnological or chemical conversion towards new products. It also has potential to reduce the amount of acid and base compared to protein isolate production through wet fractionation. Furthermore, the whole process was performed with industrially relevant and scalable equipment. Graphic for table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/590199v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@cc3837org.highwire.dtl.DTLVardef@17eb7cdorg.highwire.dtl.DTLVardef@553105org.highwire.dtl.DTLVardef@1054fd8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The need for high-resolution gut microbiome characterization to design efficient strategies for sustainable aquaculture production

Microbiome-directed dietary interventions such as microbiota-directed fibers (MDFs) have a proven track record in eliciting responses in beneficial gut microbes and are increasingly being promoted as an effective strategy to improve animal production systems. Here we used initial metataxonomic data on fish gut microbiomes as well as a wealth of a priori mammalian microbiome knowledge on -MOS and {beta}-mannan-derived MDFs to study effects of such feed supplements in Atlantic salmon (Salmo salar) and their hitherto poorly characterized gut microbiomes. Our multi-omic analysis revealed that the investigated MDFs (two -mannans and an acetylated {beta}-galactoglucomannan), at a dose of 0.2%, had negligible effects on both host gene expression, and gut microbiome structure and function under studied conditions. While a subsequent trial using a higher (4%) dietary inclusion of {beta}-mannan significantly shifted the gut microbiome composition, there were still no biologically relevant effects on salmon metabolism and physiology. Only a single Burkholderia-Caballeronia-Paraburkholderia (BCP) population demonstrated consistent and significant abundance shifts across both feeding trials, although with no evidence of {beta}-mannan utilization capabilities or changes in gene transcripts for producing metabolites beneficial to the host. In light of these findings, we revisited our omics data to predict and outline novel and potentially beneficial endogenous lactic acid bacteria that should be targeted with future, conceivably more suitable, MDF strategies for salmon. IMPORTANCEThis study focuses on the potential of MDFs to improve aquaculture production. Despite preliminary 16S rRNA amplicon data suggested that populations in the salmon gut microbiome could utilize structurally complex mannans, our findings indicates that endogenous microbes could not metabolize it, nor the host responds to its dietary inclusion, at least not under the trial conditions investigated in this study. We highlight that high-resolution and host-specific microbiome characterization can greatly improve trial design and selection of candidate MDFs for future nutritional interventions. Understanding the intricate interplay between host and its gut microbiome is paramount in studies seeking to leverage endogenous microbial communities to benefit the host. While each new condition, whether it is a disease onset or a nutritional stressor, has the potential to profoundly reshape the microbial diversity, composition and outputs, the functional microbiome information gained under healthy conditions represent a pivotal step towards designing more effective trials involving microbiome-reprogramming feed additives. Overall, we envisage that these results will lead to improved focus on coupling fundamental microbiome characterization to the design of next-generation feeds for salmon aquaculture.

microbiology↗

bioRxiv2021

A technical pipeline for screening microbial communities as a function of substrate specificity through single cell fluorescent imaging

The study of specific glycan uptake and metabolism has been shown to be an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards target. In this study, we successfully used the fluorescent label 2-aminobenzamide (2-AB), most commonly employed for enhancing the detection of protein anchored glycans, to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, were able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it was possible to comprehensively track the metabolism of the labelled glycans in both supernatants and at a single cell level. We envisage this combination of complimentary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities. IMPORTANCEInformation on how bacterial consortia utilize polysaccharides at strain level, whilst progressing rapidly in recent years still lacks a suitable way to study the vast range of ornamentations and structural motifs found in the natural glycans we consume in everyday life. As multi-omic approaches commonly require complex and costly analysis, a screening platform, as described in our work, could be seen as both a complementary and essential new tool in the understanding of microbial polysaccharide metabolism. Our study demonstrates a fast and efficient glycan labelling technique composed of several integrated procedures and advanced analytical methodologies. Chromatography and mass spectrometry are applied in the tracking of metabolized labelled glycans whilst microscopy and flow cytometry are used in the visualization of labelled bacteria at a single cell level.

biochemistry↗

bioRxiv2020

Human gut Faecalibacterium prausnitzii deploy a highly efficient conserved system to cross-feed on β-mannan-derived oligosaccharides

{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}-mannooligosaccharides ({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 degrader Bacteroides ovatus on polymeric {beta}-mannan resulted in syntrophic growth and production of butyrate, thus confirming the high efficiency of the FpMULs uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of {beta}-mannans metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. ImportanceCommensal 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 biochemical analyses this work reveals the mechanism enabling F. prausnitzii, as a model clostridial cluster IV Firmicute, to cross-feed and access {beta}-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of {beta}-mannans/{beta}-MOS as a common dietary component. Our findings provide a mechanistic understanding of the {beta}-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, thus butyrate production, in the gut.

microbiology↗

bioRxiv2020

Synthesis of Glycoconjugates Utilizing the Regioselectivity of a Lytic Polysaccharide Monooxygenase

Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

biochemistry↗

bioRxiv2019

Engineered fibre enables targeted activation of butyrate producing microbiota in the distal gut

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. The accuracy of AcGGM was tested within the complex endogenous gut microbiome of pigs, wherein we resolved 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially expressed AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifested a "butterfly effect", whereby numerous metabolic changes and interdependent cross-feeding pathways were detected in neighboring non-mannolytic populations that produce short-chain fatty acids. Our findings show that intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large.

microbiology↗

bioRxiv2019

A pair of esterases from a commensal gut bacterium remove acetylations from all positions on complex β-mannans

{beta}-Mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. {beta}-Mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs), however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCEX, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The 3D-structure of RiCEX with a mannopentaose in the active site shows that the CBM35 domain of RiCEX forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points towards the Ser41 of the catalytic triad. Cavities on the RiCEX surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In depth characterization of the two enzymes using time-resolved NMR, HPLC and mass spectrometry demonstrates that they work in a complementary manner. RiCEX exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O- and 6-O-acetylations. Activity of RiCE2 is dependent on RiCEX removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O specific RiCEX provided new insight to how temperature and pH affects acetyl migration on mannooligosaccharides.\n\nSignificance statementAcetylations are an important feature of hemicellulose, altering the physical properties of the plant cell wall, and limiting enzyme accessibility. Removal of acetyl groups from beta-mannan is a key step towards efficient utilization of mannans as a carbon source for gut microbiota and in biorefineries. We present detailed insight into mannan deacetylation by two highly substrate-specific acetyl-mannan esterases (AcMEs) from a prevalent gut commensal Firmicute, which cooperatively deacetylate complex galactoglucomannan. The 3D structure of RiCEX with mannopentaose in the active site has a unique two-domain architecture including a CBM35 and an SGNH superfamily hydrolytic domain. Discovery of mannan specific esterases improves the understanding of an important step in dietary fiber utilization by gut commensal Firmicutes.

biochemistry↗