Biology subjects

Vaaje-Kolstad, G.

Publications and source records attributed to Vaaje-Kolstad, G..

16 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

Biodegradation of components from an oxidized polyethylene by a Rhodococcus strain isolated from the gut of Atlantic Salmon

Polyethylene (PE) is the most produced synthetic polymer and as a result, a major source of microplastic waste accumulating globally. Exposure to photo- and thermo-oxidative conditions in the environment can cause PE to degrade into carbonyl-containing compounds, hydrocarbons, and low molecular weight PE (LMWPE). In both marine and freshwater ecosystems, fish, including Atlantic salmon, can ingest PE and its derivatives, creating opportunities for interactions with their gut microbes. Here, we investigated the ability of a bacterial isolate from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, to grow on an LMWPE model substrate for partially depolymerized and oxidized PE. Comparative genomic analyses showed that ASF-10 has a smaller genome than other Rhodococcus species yet retaining conserved functions including those related to utilization of medium- and long-chain hydrocarbons. In-depth characterization of the substrate following growth with ASF-10 confirmed depletion of alkanes and 2-ketones deriving from LMWPE, while the polymeric component remained unchanged. Proteomic analysis identified multiple enzymes that were likely to be involved in the degradation of LMWPE-derivatives, including an alkane 1-monooxygenase, cytochrome P450 hydroxylases and Baeyer-Villiger monooxygenases, as well as proteins for production of biofilm and a surfactant that may enhance accessibility to the substrate. Collectively, our findings advance the understanding of the ecology and enzymatic mechanisms underlying utilization of medium- to long-chain alkanes and oxidized variants thereof, that resemble molecules that can occur from abiotic PE degradation, by a fish gut-associated microbe. This metabolic capacity could be harnessed to develop sustainable strategies for bioremediation of oxidized, LMWPE-derivatives. ImportanceThe widespread presence of plastics in marine and freshwater environments has raised concerns due to their toxicity when ingested by fish. Microbial mechanisms driving breakdown of microplastic components, such as LMWPE and derivatives, in gut systems remain poorly understood. This study reveals how a bacterium isolated from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, metabolizes alkanes and oxidized variants thereof, that can result from abiotic PE decomposition. We identified key enzymes that are potentially involved in this process as well as in the production of biofilm and surfactants that may facilitate access to the substrate. Besides extending the knowledge of the enzymatic basis for degradation of PE-derivatives in gut-associated microbes from aquatic organisms, our results provide a framework that couples advanced compositional characterization of the substrate with omics techniques, offering valuable insight to support future studies aimed at unequivocally identifying microbes and their enzymes implicated in transformation of PE-derivatives.

microbiology↗

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↗

bioRxiv2025

Discovery and characterization of bacterial unspecific peroxygenase-like heme-thiolate enzymes

Unspecific peroxygenases (UPOs, EC. 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds due to their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have ever been reported in prokaryotic organisms. Here, we describe the discovery of a novel family of diverse bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial UPO-like proteins (BUPOs) are structurally analogous to family I ("short") fungal UPOs, despite having sequence similarity below 20%. Expression of one of these proteins (HydBUPO) in its native host (Hydrogenophaga sp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, and two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol and 16-hydroxypalmitic acid, using hydrogen peroxide as co-substrate. These enzymes thus represent a new family of bacterial heme-thiolate peroxygenases that share structural and functional features with eukaryotic UPOs, offering new potential candidates for developing industrially relevant biocatalysts.

biochemistry↗

bioRxiv2025

Microbial degradation of a widely used model polyethylene is restricted to medium- and long-chain alkanes and their oxidized derivatives

Plastics are widely used materials, yet their chemical stability hinders biodegradation, exacerbating pollution on a global scale. Soils contaminated with plastic may foster microbes adapted to degrade plastics or plastic derivatives, and these organisms and their enzymes offer promising avenues for the development of biotechnological recycling strategies. Here, two microbial communities originating from soil collected at a plastic-contaminated landfill in Norway were enriched to select for bacteria involved in the decomposition of a commonly used, model polyethylene (PE; weight average molecular weight (Mw) [~]4000 g/mol). We leveraged genome-resolved metatranscriptomics to identify active population affiliated with Acinetobacter guillouiae and Pseudomonas sp., showing a suite of upregulated genes (including those encoding alkane 1-monooxygenases, flavin-containing monooxygenases FMOs, cytochrome P450 monooxygenases) with functions compatible with degradation of oxidized products as well as medium- and long-chain hydrocarbons. Strikingly, spectroscopic, spectrometric and chromatographic analyses revealed the unexpected presence of medium- and long-chain alkanes and 2-ketones in the model PE substrate, preventing the erroneous conclusion that the community was interacting with the polymeric component. Consistently, only alkanes and 2-ketones with chain length of 10-35 were selectively degraded by an A. guillouiae isolate, as confirmed by proteomics analyses and substrate characterization following bacterial growth. Besides extending the knowledge on the enzymatic basis for degradation of PE-derivatives in soil-associated microbial systems, our results provide an advanced compositional characterization of a widely used model "PE" material, while offering valuable insight to support future studies aimed at unequivocally identifying organisms and their enzymes implicated in PE transformation.

microbiology↗

bioRxiv2025

Production and evaluation of fluorophore-doped polymer substrates to screen for plastic-degrading enzymes

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. While liquid and gas chromatography with mass spectrometry detection offer great power, the cost of the equipment is high, and the methods may not be straightforward when studying crude environmental samples. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent probe, rhodamine 6G, and we evaluate this screening method using poly(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 engineered PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

biochemistry↗

bioRxiv2025

Functional characterization of multi-domain LPMOs from marine Vibrio species reveals modulation of enzyme activity by domain-domain interactions

Several bacterial pathogens secrete multi-domain enzymes known as lytic polysaccharide monooxygenases (LPMOs) that are important for virulence. One example is the Vibrio cholerae virulence factor GbpA (VcGbpA), in which an N-terminal LPMO domain is followed by two domains of unknown function called GbpA2 and GbpA3, and a C-terminal chitin-binding domain, called CBM73. In-depth functional characterization of full-length and truncated variants of VcGbpA and a homologue from V. campbellii (previously V. harveyi; VhGbpA) showed that the catalytic LPMO domains of these proteins exhibit properties similar to natural single-domain LPMOs with established roles in chitin degradation. Interestingly, binding to chitin and efficient degradation of this substrate were affected by the presence of the GbpA2 and GbpA3 domains. Combined with structural predictions and analyses of sequence conservation, our data suggests that GbpA3 has evolved to interact with the reduced copper site to prevent off-pathway reactions in the absence of substrate. Substrate binding by CBM73 weakens this interaction, enabling the activation of the LPMO only when substrate is present. These observations shed new light into the functionality of these multi-domain LPMOs and uncover a novel mechanism for regulating LPMO activity.

biochemistry↗

bioRxiv2025

Whole genome assembly and annotation of the Bumblebee Wax Moth, Aphomia sociella

The bumble bee wax moth, Apshomia sociella, is an important lepidopteran pest impacting bee colonies essential for pollination and apiculture. Despite its relevance, sequence efforts aimed at understanding the genetic makeup of this species have not yet been undertaken. In this work, we successfully achieved a high-quality de novo genome assembly of A. sociella and comprehensive gene annotations generated from long-read DNA and RNA sequencing with Oxford Nanopore technology. The haploid assembly includes 347 contigs, with an N50 of 4.96 Mb, and contains 27 242 protein-coding genes. Benchmarking Universal Single Copy Orthologs (BUSCO) analyses indicates that the assembly has a high level of completeness (98.3%) and low level of fragmentation (0.6%) and duplication (3.9%). Phylogenomic analyses with other members of the Lepidoptera order placed A. sociella in the same clade as Corcyra cephalonica and indicates close evolutionary relationships with the other two species in the subfamily Galleriinae, namely Achroia grisella and Galleria mellonella. This new high-quality genome assembly represents a valuable resource for investigating the genomic basis of ecological specialization of this species and offers critical support for research aimed at developing sustainable and effective pest management strategies.

genomics↗

bioRxiv2024

Revisiting the activity of two poly(vinyl chloride)- and polyethylene-degrading enzymes

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. We attempted to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments with the recombinant proteins, we did not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted, leading to the erroneous claim that enzymatic deconstruction of polyethylene and poly(vinyl chloride) had occurred. Our results should lead to caution when interpreting the growing amount of literature claiming enzymatic degradation of non-hydrolyzable plastics.

biochemistry↗

bioRxiv2023

Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environment survival and infection

Despite major efforts towards its eradication, cholera remains a major health and economic burden in many developing countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, e.g., on zooplankton. N-acetyl glucosamine binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca2+, Mg2+ or K+ to bind close to its carbohydrate-binding surface. In addition to the X-ray crystal structures, we explored how the presence of ions affects the stability of the protein, compared the new GbpA LPMO structures to those of other LPMOs, and discussed the relevance of our discovery for bacterial survival. Calcium ions, abundant in natural sources of chitin, have been found to have the strongest effect on GbpA stability. Our findings suggest a V. cholerae-specific cation-binding site in GbpA that may fine-tune activity and binding to the different substrates during environmental survival and host infection.

biochemistry↗

bioRxiv2023

Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that bind to and oxidize insoluble carbohydrate substrates, such as chitin or cellulose. This class of enzymes has attracted considerable attention due to their ability to convert biomaterials of high abundance into oligosaccharides that can be useful for producing biofuels and bioplastics. However, processes at the interface between solution and insoluble substrates represent a major challenge to biochemical and structural characterization. Here, we investigated the four-domain LPMO from Vibrio cholerae, N-acetyl glucosamine binding protein A (GbpA), to elucidate how it docks onto its insoluble substrate with its two terminal domains. First, we developed a protocol that allowed GbpA and chitin to form a stable complex in suspension, overcoming incompatibilities of the two binding partners with respect to pH. Using contrast variation small-angle neutron scattering (SANS), after determining the neutron scattering contrast match point for chitin (47% D2O), we characterized the structure of GbpA in complex with chitin by SANS, and by electron microscopy. We found that GbpA binds rapidly to chitin, where it spreads out on the chitin fibers, and smoothens their surface. In some locations, GbpA binding induces the formation of protein-chitin clumps containing hundreds of GbpA molecules. Together, this suggests how the secretion of GbpA efficiently prepares the ground for microcolony formation by the bacteria.

biochemistry↗

bioRxiv2023

Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara

Cellulases play a key role in enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. The discovery of novel cellulases from exotic biological niches is of interest as they may present properties that are valuable in biorefining of lignocellulose. We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multidomain enzyme from the unusual bacterial endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The cellulase enzyme, TwCel5, was produced with and without a native C-terminal family 10 carbohydrate-binding module belongs to GH5, subfamily 2. Both variants showed hydrolytic endo-activity on soluble substrates such as, {beta}-glucan, carboxymethylcellulose and konjac glucomannan. However, low activity was observed towards a crystalline form of cellulose. Interestingly, when co-incubated with a cellulose active LPMO, a clear synergy was observed that boosted hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 [A] resolution and revealed a substrate binding cleft containing a putative +3 subsite, which is uncommon in this enzyme family. The enzyme TwCel5 was active in a wide range of pH and temperatures and showed high tolerance for NaCl. This study provides an important advance on discovery new enzymes from shipworm and shed new light on biochemical and structural characterization of cellulolytic cellulase and showed boost in hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose active LPMO. These findings will be relevant for the development of future enzyme cocktail that may be useful for the biotechnological conversion of lignocellulose.

biochemistry↗

bioRxiv2022

The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases

The Atlantic salmon (Salmo salar) genome contains 10 chitinase encoding genes, but little is known about the function of these chitinases. We show that the protein products of three genes, the family 18 glycoside hydrolase (GH18) chitinases Chia.3, Chia.4, and Chia.7 are secreted in the stomach mucosa and are amongst the most abundant proteins in this matrix. Chia.3 and Chia.4, sharing 95% sequence identity, were not possible to separate by standard chromatographic methods and were thus purified as a chitinase pair. Biochemical analysis revealed chitinolytic activity towards {beta}-chitin for up to 24 hours at pH 2-6. Further in vitro analysis showed that this chitinase pair efficiently degraded various chitin-containing substrates to chitobiose (GlcNAc2) suggesting that Atlantic salmon has the potential to utilize novel chitin-containing feed sources.

biochemistry↗

bioRxiv2022

Gene family expansion and functional diversification of chitinase and chitin synthase genes in Atlantic salmon (Salmo salar)

BackgroundChitin is one of the most abundant polysaccharides in nature, forming important structures in insects, crustaceans, and fungal cell walls. Vertebrates on the other hand are generally considered "non-chitinous" organisms, despite having highly conserved chitin metabolism associated genes. Recent work has revealed that the largest group of vertebrates, the teleosts, have the potential to both synthesize and degrade endogenous chitin. Yet little is still known about the genes and proteins responsible for these dynamic processes. Here we used comparative genomics, transcriptomics, and chromatin accessibility data to characterize the repertoire, evolution, and regulation of genes involved in chitin-metabolism in teleosts, with a particular focus on Atlantic salmon. ResultsReconstruction of gene family phylogenies provide evidence for an expansion of teleost and salmonid chitinase and chitin synthase genes after multiple whole-genome duplications. Analyses of multi-tissue gene expression data demonstrated a strong bias of gastrointestinal tract expression for chitin metabolism genes, but with different spatial and temporal tissue specificities. Finally, we integrated transcriptomes from a developmental time series of the gastrointestinal tract with chromatin accessibility data to identify putative transcription factors responsible for regulating chitin-metabolism gene expression (CDX1 and CDX2) as well as tissue-specific divergence in the regulation of gene duplicates (FOXJ2). These transcription factors are also potential regulators of multiple glycosyltransferases being co-expressed with the chitin remodeling genes. ConclusionThe findings presented here add support to the hypothesis that chitin metabolism genes in teleosts play a role in developing and maintaining a chitin-based barrier in the teleost gut and provide a basis for further investigations into the molecular basis of this barrier.

genetics↗

bioRxiv2021

The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc)2), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both - and {beta}-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on {beta}-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on {beta}-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCEThe ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study we demonstrate that Al. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

biochemistry↗

bioRxiv2016

Fenton-type chemistry by a copper enzyme: molecular mechanism of polysaccharide oxidative cleavage

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been instrumental for the development of economically sustainable lignocellulose biorefineries. Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of action remains enigmatic and their activity and stability under process conditions are hard to control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show that H2O2, and not O2 as previously thought, is the co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics and high enzymatic rates are achieved, the LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of reductants is alleviated. These results offer completely new perspectives regarding the mode of action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, and industrial biorefining.

biochemistry↗