Hierarchical structure and chemical composition of complementary segments of the fruiting bodies of Fomes fomentarius fungi fine-tune the compressive properties.

Humanity is often fascinated by structures and materials developed by Nature. While structural materials such as wood have been widely studied, the structural and mechanical properties of fungi are still largely unknown. One of the structurally interesting fungi is the polypore Fomes fomentarius. The present study deals with the investigation of the light but robust fruiting body of F. fomentarius. The four segments of the fruiting body (crust, trama, hymenium, and mycelial core) were examined. The comprehensive analysis included structural, chemical, and mechanical characterization with particular attention to cell wall composition, such as chitin/chitosan and glucan content, degree of deacetylation, and distribution of trace elements. The hymenium exhibited the best mechanical properties even though having the highest porosity. Our results suggest that this outstanding strength is due to the high proportion of skeletal hyphae and the highest chitin/chitosan content in the cell wall, next to its honeycomb structure. In addition, an increased calcium content was found in the hymenium and crust, and the presence of calcium oxalate crystals was confirmed by SEM-EDX. Interestingly, layers with different densities as well as layers of varying calcium and potassium depletion were found in the crust. Our results show the importance of considering the different structural and compositional characteristics of the segments when developing fungal-inspired materials and products. Moreover, the porous yet robust structure of hymenium is a promising blueprint for the development of advanced smart materials.

Mechanical, physical and thermal properties of composite materials produced with the basidiomycete Fomes fomentarius.

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.

Aspergillus niger as a cell factory for the production of pyomelanin, a molecule with UV-C radiation shielding activity.

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 (PyoFun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The PyoFun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of PyoFun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (PyoSyn). The results confirmed successful production of PyoFun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of PyoFun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, PyoFun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by PyoSyn. The findings of this study suggest that PyoFun has significant potential as a biological shield against harmful radiation. Notably, PyoFun 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 PyoFun 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.

Establishment of the basidiomycete Fomes fomentarius for the production of composite materials.

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.

Effect of Fomes fomentarius Cultivation Conditions on Its Adsorption Performance for Anionic and Cationic Dyes.

Lab-cultivated mycelia of Fomes fomentarius (FF), grown on a solid lignocellulose medium (FF-SM) and a liquid glucose medium (FF-LM), and naturally grown fruiting bodies (FF-FB) were studied as biosorbents for the removal of organic dyes methylene blue and Congo red (CR). Both the chemical and microstructural differences were revealed using X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, zeta potential analysis, and scanning electron microscopy, illuminating the superiority of FF-LM and FF-SM over FF-FB in dye adsorption. The adsorption process of CR on FF-LM and FF-SM is best described by the Redlich-Peterson model with ß constants close to 1, that is, approaching the monolayer Langmuir model, which reach maximum adsorption capacities of 48.8 and 13.4 mg g-1, respectively, in neutral solutions. Adsorption kinetics follow the pseudo-second-order model where chemisorption is the rate-controlling step. While the desorption efficiencies were low, adsorption performances were preserved and even enhanced under simulated dye effluent conditions. The results suggest that F. fomentarius can be considered an attractive biosorbent in industrial wastewater treatment and that its cultivation conditions can be specifically tailored to tune its cell wall composition and adsorption performance.

Extrusion-based additive manufacturing of fungal-based composite materials using the tinder fungus Fomes fomentarius.

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.

S2k guideline diagnosis and treatment of carbon monoxide poisoning.

Carbon monoxide (CO) can occur in numerous situations and ambient conditions, such as fire smoke, indoor fireplaces, silos containing large quantities of wood pellets, engine exhaust fumes, and when using hookahs. Symptoms of CO poisoning are nonspecific and can range from dizziness, headache, and angina pectoris to unconsciousness and death. This guideline presents the current state of knowledge and national recommendations on the diagnosis and treatment of patients with CO poisoning. The diagnosis of CO poisoning is based on clinical symptoms and proven or probable exposure to CO. Negative carboxyhemoglobin (COHb) levels should not rule out CO poisoning if the history and symptoms are consistent with this phenomenon. Reduced oxygen-carrying capacity, impairment of the cellular respiratory chain, and immunomodulatory processes may result in myocardial and central nervous tissue damage even after a reduction in COHb. If CO poisoning is suspected, 100% oxygen breathing should be immediately initiated in the prehospital setting. Clinical symptoms do not correlate with COHb elimination from the blood; therefore, COHb monitoring alone is unsuitable for treatment management. Especially in the absence of improvement despite treatment, a reevaluation for other possible differential diagnoses ought to be performed. Evidence regarding the benefit of hyperbaric oxygen therapy (HBOT) is scant and the subject of controversy due to the heterogeneity of studies. If required, HBOT should be initiated within 6 h. All patients with CO poisoning should be informed about the risk of delayed neurological sequelae (DNS).

Modular Synthetic Biology Toolkit for Filamentous Fungi.

Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.

Beyond the Biosynthetic Gene Cluster Paradigm: Genome-Wide Coexpression Networks Connect Clustered and Unclustered Transcription Factors to Secondary Metabolic Pathways.

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 under 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 data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman's correlation coefficients (SCCs) and on mutual rank-transformed Pearson's correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger. Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that 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 the 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 putative 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.

A Penicillium rubens platform strain for secondary metabolite production.

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.

Fungi as source for new bio-based materials: a patent review.

BACKGROUND: The circular economy closes loops in industrial manufacturing processes and minimizes waste. A bio-based economy aims to replace fossil-based resources and processes by sustainable alternatives which exploits renewable biomass for the generation of products used in our daily live. A current trend in fungal biotechnology-the production of fungal-based biomaterials-will contribute to both. RESULTS: This study gives an overview of various trends and development applications in which fungal mycelium is used as new and sustainable biomaterial. A patent survey covering the last decade (2009-2018) yielded 47 patents and patent applications claiming fungal biomass or fungal composite materials for new applications in the packaging, textile, leather and automotive industries. Furthermore, fungal-based materials are envisaged for thermal insulation and as fire protection materials. Most patents and patent applications describe the use of different lignin- and cellulose-containing waste biomass as substrate for fungal cultivations, covering 27 different fungal species in total. Our search uncovered that most patent activities are on-going in the United States and in China. CONCLUSION: Current patent developments in the field suggest that fungal bio-based materials will considerable shape the future of material sciences and material applications. Fungal materials can be considered as an excellent renewable and degradable material alternative with a high innovation potential and have the potential to replace current petroleum-based materials.

Bacterial MbtH-like Proteins Stimulate Nonribosomal Peptide Synthetase-Derived Secondary Metabolism in Filamentous Fungi.

Filamentous fungi are known producers of bioactive natural products, low molecular weight molecules that arise from secondary metabolism. MbtH-like proteins (MLPs) are small (∼10 kDa) proteins, which associate noncovalently with adenylation domains of some bacterial nonribosomal peptide synthetases (NRPS). MLPs promote the folding, stability, and activity of NRPS enzymes. MLPs are highly conserved among a wide range of bacteria; however, they are absent from all fungal species sequenced to date. We analyzed the interaction potential of bacterial MLPs with eukaryotic NRPS enzymes first using crystal structures, with results suggesting a conservation of the interaction surface. Subsequently, we transformed five MLPs into Penicillium chrysogenum strains and analyzed changes in NRPS-derived metabolite profiles. Three of the five transformed MLPs increased the rate of nonribosomal peptide formation and elevated the concentrations of intermediate and final products of the penicillin, roquefortine, chrysogine, and fungisporin biosynthetic pathways. Our results suggest that even though MLPs are not found in the fungal domain of life, they can be used in fungal hosts as a tool for natural product discovery and biotechnological production.

Genome Editing in Penicillium chrysogenum Using Cas9 Ribonucleoprotein Particles.

Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences . This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.

Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin.

BACKGROUND: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. RESULTS: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. CONCLUSION: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.

Instructed illiteracy reveals expertise-effects on unconscious processing.

We used a new methodological approach to investigate whether top-down influences like expertise determine the extent of unconscious processing. This approach does not rely on preexisting differences between experts and novices, but instructs essentially the same task in a way that either addresses a domain of expertise or not. Participants either were instructed to perform a lexical decision task (expert task) or to respond to a combination of single features of word and non-word stimuli (novel task). The stimuli and importantly also the mapping of responses to those stimuli, however, were exactly the same in both groups. We analyzed congruency effects of masked primes depending on the instructed task. Participants performing the expert task responded faster and less error prone when the prime was response congruent rather than incongruent. This effect was significantly reduced in the novel task, and even reversed when excluding identical prime-target pairs. This indicates that the primes in the novel task had an effect on a perceptual level, but were not able to impact on response activation. Overall, these results demonstrate an expertise-based top-down modulation of unconscious processing that cannot be explained by confounds that are otherwise inherent in comparisons between novices and experts.

Gaming to see: action video gaming is associated with enhanced processing of masked stimuli.

Recent research revealed that action video game players outperform non-players in a wide range of attentional, perceptual and cognitive tasks. Here we tested if expertise in action video games is related to differences regarding the potential of shortly presented stimuli to bias behavior. In a response priming paradigm, participants classified four animal pictures functioning as targets as being smaller or larger than a reference frame. Before each target, one of the same four animal pictures was presented as a masked prime to influence participants' responses in a congruent or incongruent way. Masked primes induced congruence effects, that is, faster responses for congruent compared to incongruent conditions, indicating processing of hardly visible primes. Results also suggested that action video game players showed a larger congruence effect than non-players for 20 ms primes, whereas there was no group difference for 60 ms primes. In addition, there was a tendency for action video game players to detect masked primes for some prime durations better than non-players. Thus, action video game expertise may be accompanied by faster and more efficient processing of shortly presented visual stimuli.

Your unconscious knows your name.

One's own name constitutes a unique part of conscious awareness - but does this also hold true for unconscious processing? The present study shows that the own name has the power to bias a person's actions unconsciously even in conditions that render any other name ineffective. Participants judged whether a letter string on the screen was a name or a non-word while this target stimulus was preceded by a masked prime stimulus. Crucially, the participant's own name was among these prime stimuli and facilitated reactions to following name targets whereas the name of another, yoked participant did not. Signal detection results confirmed that participants were not aware of any of the prime stimuli, including their own name. These results extend traditional findings on "breakthrough" phenomena of personally relevant stimuli to the domain of unconscious processing. Thus, the brain seems to possess adroit mechanisms to identify and process such stimuli even in the absence of conscious awareness.

Development and characterization of a disposable plastic microarray printhead.

During the last decade microarrays have become a powerful analytical tool. Commonly microarrays are produced in a non-contact manner using silicone printheads. However, silicone printheads are expensive and not able to be used as a disposable. Here, we show the development and functional characterization of 8-channel plastic microarray printheads that overcome both disadvantages of their conventional silicone counterparts. A combination of injection-molding and laser processing allows us to produce a high quantity of cheap, customizable and disposable microarray printheads. The use of plastics (e.g., polystyrene) minimizes the need for surface modifications required previously for proper printing results. Time-consuming regeneration processes, cleaning procedures and contaminations caused by residual samples are avoided. The utilization of plastic printheads for viscous liquids, such as cell suspensions or whole blood, is possible. Furthermore, functional parts within the plastic printhead (e.g., particle filters) can be included. Our printhead is compatible with commercially available TopSpot devices but provides additional economic and technical benefits as compared to conventional TopSpot printheads, while fulfilling all requirements demanded on the latter. All in all, this work describes how the field of traditional microarray spotting can be extended significantly by low cost plastic printheads.

It takes two-skilled recognition of objects engages lateral areas in both hemispheres.

Our object recognition abilities, a direct product of our experience with objects, are fine-tuned to perfection. Left temporal and lateral areas along the dorsal, action related stream, as well as left infero-temporal areas along the ventral, object related stream are engaged in object recognition. Here we show that expertise modulates the activity of dorsal areas in the recognition of man-made objects with clearly specified functions. Expert chess players were faster than chess novices in identifying chess objects and their functional relations. Experts' advantage was domain-specific as there were no differences between groups in a control task featuring geometrical shapes. The pattern of eye movements supported the notion that experts' extensive knowledge about domain objects and their functions enabled superior recognition even when experts were not directly fixating the objects of interest. Functional magnetic resonance imaging (fMRI) related exclusively the areas along the dorsal stream to chess specific object recognition. Besides the commonly involved left temporal and parietal lateral brain areas, we found that only in experts homologous areas on the right hemisphere were also engaged in chess specific object recognition. Based on these results, we discuss whether skilled object recognition does not only involve a more efficient version of the processes found in non-skilled recognition, but also qualitatively different cognitive processes which engage additional brain areas.