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A holistic understanding of lignocellulose degradation
We aim to create a holistic understanding of how fungi degrade lignocellulose. To achieve this, we combine understanding of fungal responses to lignocellulose with biochemical knowledge of degradative enzymes. We further integrate this with insights in the result of such degradative activity on the composition, structure and architecture of the lignocellulose itself. We are also interested in the interactions that fungi have with other microorganisms during lignocellulose degradation, for example in signaling or in cross-feeding. Experimentally we focus on in vitro microbiology combined with molecular biology and biochemistry. We work with fungal axenic cultures as well as with co-cultures and synthetic communities of microbes.
Our fungi
Our current focus is on the anaerobic fungi (Neocallimastigomycota) that are part of the microbiome in the digestive system of ruminants. These fungi are first colonizers of plant material during its digestion in the rumen, and have a strong fiber degrading activity. The lab also a track record in working with industrially important fungus Aspergillus niger.
The rumen mycobiome - from lab to farm
We create fundamental insights in the roles of anaerobic fungi in the rumen microbiome that we integrate with data from applied in vitro and in vivo ruminant myco- and microbiome studies. Via this route we address the challenge of generating functional understanding of the rumen microbiome. This underpins the formulation of strategies to modulate activity of the rumen microbiome to benefit efficient ruminant digestion while reducing microbiome-mediated production of the potent greenhouse gas methane.
Below selected publications illustrate some of the topics that we have explored in previous projects
Biochemistry of Carbohydrate Active Enzymes
We are interested in uncovering the biochemical activity of fungal CAZymes, with a focus on enzymes active on plant-derived polysaccharides. We have expertise in heterologous enzyme expression, the purification of native and tagged enzymes, in CAZyme activity assays and the detection of enzyme products and substrates. We also have an interest in testing and developing fungal CAZymes for diverse applications such as in food and baking, as feed additives for poultry, or for (selective) digestion of plant biomass for renewables-based biotechnology processes.
Bulmer G, Yuen FW, Begum N, Jones BS, Flitsch SL, van Munster JM. Enzyme Microb Technol (2023) 164:110170
β-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. Analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members via conserved unique peptide patterns and phylogeny, revealed the occurrence of 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 β-D-galactofuranosidase from A. niger which was recombinantly produced in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-nitrophenyl-β-galactofuranoside (pNP-β-Galf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 µM min−1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures.
Garbelotti CV, Bulmer GS, Ward, RJ#, van Munster JM#. Food Chemistry (2023) 45B; 134939
Ferulic acid has antioxidant properties of interest to the food industry and can be released from natural plant fibres using feruloyl esterases. Esterases active at high temperatures are highly desirable but currently underrepresented. Here we report the biochemical characterization of the feruloyl esterase from Thermobacillus xylanilyticus. Specific activity of recombinant Tx-Est1 with ethyl ferulate was 29.2 ± 2.9 U mg−1, with a catalytic efficiency (Kcat/Km) of 393.7 ± 9.8 s-1mM−1. The temperature and pH optima were 60 °C and 7.5, whereby Tx-Est1 retains 70% activity after 25 h at 40 °C. MALDI-TOF MS revealed Tx-ESTI released ferulic acid from xylooligosaccharides with DP4-DP13, and from DP6-8 containing two ferulic acid groups. HPLC demonstrated ferulic acid release from destarched wheat bran was strongly potentiated by co-incubation with xylanase. These properties, especially the high activity at elevated temperatures, suggest Tx-Est1 can be employed for production of high-value compounds from agricultural waste or during plant polysaccharide saccharification.
Recent advances in enzymatic synthesis of beta-glucan and cellulose
Bulmer GS* , de Andrade P*, Field RS, van Munster JM. Carbohydrate Research (2021), 508: 108411
Bottom-up synthesis of β-glucans such as callose, fungal β-(1,3)(1,6)-glucan and cellulose, can create the defined compounds that are needed to perform fundamental studies on glucan properties and develop applications. With the importance of β-glucans and cellulose in high-profile fields such as nutrition, renewables-based biotechnology and materials science, the enzymatic synthesis of such relevant carbohydrates and their derivatives has attracted much attention. Here we review recent developments in enzymatic synthesis of β-glucans and cellulose, with a focus on progress made over the last five years. We cover the different types of biocatalysts employed, their incorporation in cascades, the exploitation of enzyme promiscuity and their engineering, and reaction conditions affecting the production as well as in situ self-assembly of (non)functionalised glucans. The recent achievements in the application of glycosyl transferases and β-1,4- and β-1,3-glucan phosphorylases demonstrate the high potential and versatility of these biocatalysts in glucan synthesis in both industrial and academic contexts.
Bulmer GS, Mattey AP, Parmeggiani F, Williams R, Marchesi A, Seibt LS, Both P, Huang K, Galan MC, Flitsch SL, Green AP, van Munster JM. Organic and Biomolecular Chemistry (2021) 19 (25) 5529-5533
Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise cello-oligosaccharides with tailored functionalities on a scale suitable for employment in mass spectrometry-based assays. The resulting glycan structures enabled reporting of the activity and selectivity of celluloltic enzymes.
Fungal biology and genetics of lignocellulose degradation
We are interested in understanding how fungi respond to the presence of lignocellulose or other plant-derived complex carbohydrates. This includes the sensing of carbohydrates derived from plant material, the activation of genes that encode degradative enzymes, and also the changes in fungal physiology in response to exposure to plant material. We have expertise in aerobic and anaerobic fungal cultivations and a range of modern 'omics tools. In this topic we have a strong track record of working with Aspergillus niger, a biotechnology workhorse important for the industrial-scale production of organic acids, enzymes and other chemicals. We have several ongoing projects that focus on anaerobic rumen fungi.
van Munster JM*, Daly P*, Blythe MJ, Ibbett R, Kokolski M, Gaddipati S, Lindquist E, Singan VR, Barry KW, Lipzen AM, Ngan CY, Petzold CJ, Chan, LJG, Arvas M, Raulo R, Pullan ST, Delmas S, Grigoriev IV, Tucker GA, Simmons BA, Archer DB. Biotech for Biofuels (2020) 13:69
Understanding 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 Aspergillus niger over its life time to six substrates important for biofuel production.
We 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.
In 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.
Growing a circular economy with fungal biotechnology: a white paper
Meyer V, Basenko EY, Benz JP, Braus GH, Caddick MX, Csukai M, de Vries RP, Endy D, Frisvad JC, Gunde-Cimerman N, Haarmann T, Hadar Y, Hansen K, Johnson RI, Keller NP, Kraševec N, Mortensen UH, Perez R, Ram AFJ, Record E, Ross P, Shapaval V, Steiniger C, van den Brink H, van Munster JM, Yarden O, Wösten HAB. Fungal Biol Biotechnology (2020) 7:5
Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation’s sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.
Daly P*, van Munster JM*, Blythe MJ, Ibbett R, Kokolski M, Gaddipati S, Lindquist E, Singan VR, Barry KW, Lipzen A, Ngan CY, Petzold CJ, Chan, LJG, Pullan ST, Delmas S, Waldron PR, Grigoriev IV, Tucker GA, Simmons BA, Archer DB. Biotech for Biofuels (2017) 10:35.
The capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails.
We investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitated identification of temporally limited gene induction patterns.
The presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.
van Munster JM*, Daly P*, Delmas S, Pullan ST, Blythe MJ, Malla S, Kokolski M, Notlorp EC, Wennberg K, Fetherston R, Beniston R, Yu X, Dupree P, Archer DB. Fungal Genet Biol. (2014) 72, 34-47.
Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6 h of exposure to wheat straw was very different from the response at 24 h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24 h of exposure to wheat straw, were also induced after 6 h exposure. Importantly, over a third of the genes induced after 6 h of exposure to wheat straw were also induced during 6 h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes.
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