Biology subjects

van Munster, J. M.

Publications and source records attributed to van Munster, J. M..

9 recordsLinked to original sources

bioRxiv2026

Caledoniamyces theodoroui, gen. nov., sp. nov., a bulbous anaerobic gut fungus from the bovine rumen

Anaerobic gut fungi (AGF) are members of the microbiome of herbivore digestive tracts, where they are important degraders of plant material. Culture-independent diversity studies have indicated that a broad diversity of these fungi still remain uncultured so far. This limits understanding of their ecology, physiology and potential for biotechnological exploitation. Here we describe the isolation and characterisation of the first cultured representatives of the Neocallimastigomycota clade NY9 / YL1, previously proposed as genus based on culture-independent sequence diversity studies. Two fungal strains, RF6 and RF9, were isolated from Jersey cow rumen fluid, alongside representatives of other genera including Orpinomyces and Neocallimastix. Phylogenetic analysis of the RF6 and RF9 rDNA ITS1 region and D1/D2 domain of 28S LSU rDNA gene revealed formation of a well-supported genus-level cluster. This cluster also contained sequences deposited for NY9 and YL1 and was distinct from other described genera. Isolates RF6 and RF9 displayed small 1-2 mm colonies with a compact structure on solid medium. Microscopy showed bulbous rhizoidal cells encased in amorphous material, possibly extracellular polymeric substances, indicating that the genus represents the third known genus of AGF with bulbous morphology. Both endogenous and exogenous sporangia were observed, which produced mono-flagellated zoospores. Isolate RF6 was able to grow robustly using cellulose and wheat straw as sole carbon sources. For this new genus, for which the type strain is RF6, we propose the name Caledoniamyces, referring to the origin in Scotland, and the species name C. theodoroui.

microbiology↗

bioRxiv2026

Comparative sugar utilisation and metabolism of mannose as co-substrate indicate flexibility in carbon metabolism in anaerobic gut fungi

Anaerobic gut fungi (AGF) are key degraders of plant biomass in ruminants, yet there is limited knowledge of how AGF respond to mixtures of plant-derived sugars. Here, we assessed monosaccharide and disaccharide utilisation by Neocallimastix frontalis CoB3, Caecomyces communis SHB, and Piromyces edwardsiae SHC, which are abundant in the rumen microbiome. While all AGF isolates shared a core set of sugars that supported growth, they had different hierarchies of uptake. Co-substrate experiments using glucose and lignocellulose-derived sugars revealed species-specific responses, with N. frontalis displaying a novel concentration-dependent co-utilisation of glucose and mannose, whereas growth of P. edwardsiae was inhibited under the same conditions, and C. communis exhibited growth inhibition in glucose and xylose co-substrate cultures. Together, these findings demonstrate functional diversity in monosaccharide and disaccharide metabolism amongst the AGF investigated here. Understanding such sugar utilisation phenotypes provides a foundation for evaluating AGF isolate suitability for lignocellulosic biomass valorisation.

microbiology↗

bioRxiv2026

Sugar-mediated inhibition of growth and lignocellulose degradation in anaerobic gut fungi revealed using cellulose filter paper

Anaerobic gut fungi (AGF) are central to the degradation of plant material in the digestive systems of herbivores. However, how their environment influences their colonisation and degradation of complex biomass is unclear. Here, cellulose filter paper was used as a simplified model of the plant cell wall to investigate how the presence of free sugars in the rumen can affect AGF growth and degradative responses of phylogenetically distinct AGF isolates. From this, galactose was revealed to be inhibitory to both Neocallimastix frontalis and Caecomyces communis, and mannose inhibitory to C. communis. Complete inhibition of C. communis growth was conserved when galactose and mannose were added in their polymeric forms, whereas in contrast, N. frontalis growth was unaffected. This indicates, depending on the AGF isolate, the presence of free sugars and their polymeric form may influence AGF growth through regulatory and metabolic interactions - even if the sugar cannot be utilised for growth as the sole substrate. Collectively, this work highlights the functional diversity in AGF carbohydrate responses and the need for greater understanding of their metabolic regulation for applications in lignocellulosic bioconversion and ruminant nutrition.

microbiology↗

bioRxiv2026

Genetic profiling via a novel PCR-RFLP method enabled identification of four genera of anaerobic gut fungi isolated from nyala, giraffe, and zebra hosts

Herbivore gut microbiomes may contain a diversity of anaerobic gut fungi (AGF, phylum Neocallimastigomycota), important for fibre degradation. To perform functional studies and elucidate niches of different AGF species, representative fungal isolates must be obtained into axenic culture, which is a resource-intensive process. Here we leverage the integration of morphological and functional assessments of AGF isolates with a newly developed PCR-RFLP strategy, to distinguish and identify isolates of interest from faecal samples from zoo-housed animals. In silico prediction of PCR-RFLP profiles of cultured genera, followed by experimental validation, confirmed that LSU-based PCR-RFLP with AluI and Hyp188I digestion was effective in identification of fungi of distinct genera. Together our workflow resulted in isolation of a so far uncultured Piromyces (NY08) species and Neocallimastix cameroonii from nyala samples, as well as Feramyces austinii from giraffe and Khoyollomyces ramosus from zebra. Amplicon sequencing confirmed that these species dominated AGF communities in their hosts, likely benefiting isolation success, and we identified enrichment conditions which also affected cultivability. The workflow developed here aids efficient AGF isolations, which are instrumental in expanding opportunities for functional studies that provide insight into the physiology and ecology of these fungi and help realise applications in white and green biotechnology. One sentence summaryA validated PCR-RFLP strategy, developed based on genetic diversity data from Neocallimastigomycota, enables efficient identification of isolates of these anaerobic gut fungi from environmental samples, as demonstrated via targeted enrichment of anaerobic gut fungi common in faeces of giraffe, zebra and nyala, resulting in isolation of species of genera Feramyces, Neocallimastix, Khoyollomyces, and a novel Piromyces/NY08 species.

microbiology↗

bioRxiv2025

Anaerobic gut fungi Caecomyces communis, Neocallimastix frontalis and Piromyces spp. nov., have distinct effects on plant fibres during digestion.

Anaerobic gut fungi are the first colonizers of plant material that enters the digestive system of ruminants. However, it is unclear how different fungal species contribute to the ability of the rumen microbiome to convert feed to nutrients. Here we isolated three species of anaerobic fungi, including a novel Piromyces species. We investigated if these species have distinct roles in the digestion of fibrous feed components, via assessment of changes caused to plant material itself. We found Neocallimastix frontalis isolate CoB3 and Piromyces isolate SHC digested plant materials more effectively than Caecomyces communis isolate SHB. The three fungi had distinct effects on feed composition. N. frontalis CoB3 degraded hemicelluloses and cellulose to a similar extent, Piromyces spp. SHC preferentially degraded hemicellulose, while C. communis SHB preference depended on the substrate. From the panel of monosaccharides that may result from such degradative activity, all fungi consumed only glucose, suggesting involvement of mechanisms more complex than only fungal carbon source usage. Overall, this indicates that each of these fungal species have distinct roles in the degradation of plant material, and different niches in the rumen. Exploring these roles creates functional understanding of the rumen microbiome, critical for developing more sustainable agriculture.

microbiology↗

bioRxiv2024

Determining the culturability of the bovine rumen bacterial and archaeal microbiota

Ruminants play an important part in global food security, but also emit methane which contributes to global warming. Microbes in the rumen strongly influence the energy retention efficiency from the hosts plant-based diet and produce methane as a by-product. While thousands of novel microbial genomes have been assembled from metagenome sequence data, their culturability is ill-defined. Here different media were used to isolate microbes from rumen fluid. 34 genera were grown, and the majority belonged to the phylum Bacillota (75.28% {+/-} 6.34), Bacteroidota (19.99% {+/-} 4.85), Pseudomonadota (2.46% {+/-} 2.01), and Actinomycetota (2.09% {+/-} 1.07). The most abundant genera were Selenomonas (28.08% {+/-} 11.71), Streptococcus (22.67% {+/-} 6.06), Prevotella (18.71% {+/-} 4.02), and unclassified Lachnospiraceae (11.50% {+/-} 2.54). When comparing the mean relative abundance of these genera between media, 31 were significantly enriched on at least one medium. The composition of the source rumen fluid was vastly different to those cultured. Bacteroidota (52.53% {+/-} 5.10) predominated, with by Bacillota (41.00% {+/-} 3.96), the archaeal Euryarchaeota (5.12% {+/-} 1.94), Pseudomonadota (1.22% {+/-} 0.78), and Actinomycetota (0.12% {+/-} 0.08) comprising the rest. The most abundant genera were Prevotella (29.13% {+/-} 4.16), Butyrivibrio (18.21% {+/-} 2.08), Succiniclasticum (15.57% {+/-} 5.03), unclassified Bacteroidetes (13.91% {+/-} 1.67), and unclassified Prevotellaceae (9.50% {+/-} 2.01). These data further emphasise the importance of using defined media to selectively enrich for different microbial taxa. This is essential to understand the complex workings of the rumen microbes to enhance digestion efficiency and reduce the loss of energy as methane.

microbiology↗

bioRxiv2021

Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

{beta}-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative {beta}-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 {degrees}C towards 4-Nitrophenyl-{beta}-galactofuranoside (pNP-{beta}-Galf), with a Km of 17.9 {+/-} 1.9 mM and Vmax of 70.6 {+/-} 5.3 mol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

biochemistry↗

bioRxiv2020

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a promiscuous glycosyltransferase

The {beta}-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of {beta}-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

biochemistry↗

bioRxiv2019

Succession of physiological stages hallmarks the transcriptomic response of fungus Aspergillus niger to lignocellulose

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergilllus niger over its life time to six substrates important for biofuel production.\n\nResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.\n\nConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

microbiology↗