Carsten Pohl

Background: Fungal-based materials are currently studied as renewable and sustainable alternatives to fossil-based materials used in construction, packaging, textile, and fashion industries. The polypore fungus Fomes fomentarius produces a range of lightweight, durable, and flexible materials from diverse plant residual streams. Genetic engineering of this fungus is an important technological objective, both from fundamental perspectives and to optimize performance characteristics of F. fomentarius materials. Results: We provide proof of principle that F. fomentarius can be genetically transformed. Hyphae produced high amounts of protoplasts using standard protocols. These protoplasts were transformed using nourseothricin selection and a nourseothricin N-acetyl transferase resistance marker ( natR ). Expression of natR was successful using a F. fomentarius translation elongation factor promoter. Stable, multicopy, and non-targeted integration of exogenous cassettes with recipient genome was confirmed by diagnostic PCR and Southern blotting. Preliminary attempts at genome editing based disruption of a putative ku70 encoding gene were, however, unsuccessful. We validated expression of two fluorescent proteins using a Schizophyllum commune H2B promoter using qRT-PCR and fluorescent microscopy. These data confirm functionality of mCherry and eGFP in F. fomentarius and suggest these proteins can be used for future protein localisation studies. Finally, we report DNase activity in F. fomentarius which we hypothesize may be a significant technical challenge for genetic transformation of this fungus. Conclusion: F. fomentarius can be genetically transformed using the promoters, marker, and nourseothricin selection system developed in this study. Expression of both eGFP and mCherry is a significant step towards cell biological and gene functional analyses using fluorescent microscopy. We have laid the foundations for overexpression studies, and, additionally, identified several challenges which need to be overcome for targeted genetic engineering in this important material producing fungus.

Fungal mycelium‐based materials are highly valued for their versatility and for harnessing renewable plant biomass through sustainable biotechnological production. Heat pressing is often used to produce fungal particle boards and leather substitutes. In this study, the impact of heat pressing on fungal mycelium from the basidiomycete Fomes fomentarius under lignocellulose‐based solid‐state and glucose‐based liquid cultivation conditions is investigated, focusing on the changes in cell wall gene expression, morphology, chemical composition, hydrophobicity, and mechanical characteristics. With varying effects between two cultivations, heat pressing at 100 MPa changes hydrophobicity, increases tensile strength and stiffness, in line with hyphal compaction and bonding. Compared to mycelium grown on glucose‐based medium, lignocellulosic substrate leads to mycelium with higher β ‐glucan content, less densely packed mycelium structure and thus lower mechanical strength, probably due to the different expression of genes involved in glucan and chitin biosynthesis. This study highlights the impact of heat pressing on fungal mycelium obtained from different cultivation conditions, offering insights to tailor their performance.

Background: Fungal-based composites have emerged as renewable, high-performance biomaterials that are produced on lignocellulosic residual streams from forestry and agriculture. Production at an industrial scale promises to revolutionize the world humans inhabit by generating sustainable, low emission, non-toxic and biodegradable construction, packaging, textile, and other materials. The polypore Fomes fomentarius is one of the basidiomycete species used for biomaterial production, yet nothing is known about the transcriptional basis of substrate decomposition, nutrient uptake, or fungal growth during composite formation. Co-expression network analysis based on RNA-Seq profiling has enabled remarkable insights into a range of fungi, and we thus aimed to develop such resources for F. fomentarius. Results: We analysed gene expression from a wide range of laboratory cultures (n = 9) or biomaterial formation (n = 18) to determine the transcriptional landscape of F. fomentarius during substrate decomposition and to identify genes important for (i) the enzymatic degradation of lignocellulose and other plant-based substrates, (ii) the uptake of their carbon monomers, and (iii) genes guiding mycelium formation through hyphal growth and cell wall biosynthesis. Simple scripts for co-expression network construction were generated and tested, and harnessed to identify a fungal-specific transcription factor named CacA strongly co-expressed with multiple chitin and glucan biosynthetic genes or Rho GTPase encoding genes, suggesting this protein is a high-priority target for engineering adhesion and branching during composite growth. We then updated carbohydrate activated enzymes (CAZymes) encoding gene annotation, used phylogenetics to assign putative uptake systems, and applied network analysis to predict repressing/activating transcription factors for lignocellulose degradation. Finally, we identified entirely new types of co-expressed contiguous clusters not previously described in fungi, including genes predicted to encode CAZymes, hydrophobins, kinases, lipases, F-box domains, chitin synthases, amongst others. Conclusion: The systems biology data generated in this study will enable us to understand the genetic basis of F. fomentarius biomaterial formation in unprecedented detail. We provided proof-of-principle for accurate network-derived predictions of gene function in F. fomentarius and generated the necessary data and scripts for analysis by any end user. Entirely new classes of contiguous co-expressed gene clusters were discovered, and multiple transcription factor encoding genes which are high-priority targets for genetic engineering were identified.

Background: To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO₂ emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material. Results: In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K)⁻¹ which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites. Conclusion: This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards.

Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (Pyo_Fun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The Pyo_Fun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of Pyo_Fun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (Pyo_Syn). The results confirmed successful production of Pyo_Fun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of Pyo_Fun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, Pyo_Fun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by Pyo_Syn. The findings of this study suggest that Pyo_Fun has significant potential as a biological shield against harmful radiation. Notably, Pyo_Fun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes Pyo_Fun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation.

Background: Filamentous fungi of the phylum Basidiomycota are considered as an attractive source for the biotechnological production of composite materials. The ability of many basidiomycetes to accept residual lignocellulosic plant biomass from agriculture and forestry such as straw, shives and sawdust as substrates and to bind and glue together these otherwise loose but reinforcing substrate particles into their mycelial network, makes them ideal candidates to produce biological composites to replace petroleum-based synthetic plastics and foams in the near future. Results: Here, we describe for the first time the application potential of the tinder fungus Fomes fomentarius for lab-scale production of mycelium composites. We used fine, medium and coarse particle fractions of hemp shives and rapeseed straw to produce a set of diverse composite materials and show that the mechanical materials properties are dependent on the nature and particle size of the substrates. Compression tests and scanning electron microscopy were used to characterize composite material properties and to model their compression behaviour by numerical simulations. Their properties were compared amongst each other and with the benchmark expanded polystyrene (EPS), a petroleum-based foam used for thermal isolation in the construction industry. Our analyses uncovered that EPS shows an elastic modulus of 2.37 ± 0.17 MPa which is 4-times higher compared to the F. fomentarius composite materials whereas the compressive strength of 0.09 ± 0.003 MPa is in the range of the fungal composite material. However, when comparing the ability to take up compressive forces at higher strain values, the fungal composites performed better than EPS. Hemp-shive based composites were able to resist a compressive force of 0.2 MPa at 50% compression, rapeseed composites 0.3 MPa but EPS only 0.15 MPa. Conclusion: The data obtained in this study suggest that F. fomentarius constitutes a promising cell factory for the future production of fungal composite materials with similar mechanical behaviour as synthetic foams such as EPS. Future work will focus on designing materials characteristics through optimizing substrate properties, cultivation conditions and by modulating growth and cell wall composition of F. fomentarius, i.e. factors that contribute on the meso- and microscale level to the composite behaviour.

Background: Recent efforts in fungal biotechnology aim to develop new concepts and technologies that convert renewable plant biomass into innovative biomaterials. Hereby, plant substrates become metabolized by filamentous fungi to transform them into new fungal-based materials. Current research is thus focused on both understanding and optimizing the biology and genetics underlying filamentous fungal growth and on the development of new technologies to produce customized fungal-based materials. Results: This manuscript reports the production of stable pastes, composed of Fomes fomentarius mycelium, alginate and water with 71 wt.% mycelium in the solid content, for additive manufacturing of fungal-based composite materials. After printing complex shapes, such as hollow stars with up to 39 mm in height, a combination of freeze-drying and calcium-crosslinking processes allowed the printed shapes to remain stable even in the presence of water. The printed objects show low bulk densities of 0.12 ± 0.01 g/cm³ with interconnected macropores. Conclusions: This work reports for the first time the application of mycelium obtained from the tinder fungus F. fomentarius for an extrusion-based additive manufacturing approach to fabricate customized light-weight 3D objects. The process holds great promise for developing light-weight, stable, and porous fungal-based materials that could replace expanded polystyrene produced from fossil resources.

There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities.

Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent in typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression datasets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene co-expression networks based on Spearman’s correlation coefficients (SCC) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCC). By mining these networks, we predicted six transcription factors named MjkA – MjkF to concomitantly regulate secondary metabolism in A. niger . Over-expression of each transcription factor using the Tet-on cassette modulated production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of global (SCC) and pathway-specific (MR-PCC) transcription factors, respectively. These results highlight the great potential of co-expression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that novel drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. Importance: There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates.