The colors of life: an interdisciplinary artist-in-residence project to research fungal pigments as a gateway to empathy and understanding of microbial life

Background Biological pigmentation is one of the most intriguing traits of many fungi. It holds significance to scientists, as a sign of biochemical metabolism and organism-environment interaction, and to artists, as the source of natural colors that capture the beauty of the microbial world. Furthermore, the functional roles and aesthetic appeal of biological pigmentation may be a path to inspiring human empathy for microorganisms, which is key to understanding and preserving microbial biodiversity. A project focused on cross-species empathy was initiated and conducted as part of an artist-in-residence program in 2021. The aim of this residency is to bridge the current divide between science and art through interdisciplinary practice focused on fungi. Results The residency resulted in multiple products that are designed for artistic and scientific audiences with the central theme of biological pigmentation in fungi and other microorganisms. The first product is a video artwork that focuses on Aspergillus niger as a model organism that produces melanin pigment in a biosynthetic process similar to that of humans. The growth and morphology of this commonplace organism are displayed through video, photo, animation, and time-lapse footage, inviting the viewer to examine the likenesses and overlaps between humans and fungi. The second product is The Living Color Database, an online compendium of biological colors for scientists, artists, and designers. It links organisms across the tree of life, focusing on fungi, bacteria, and archaea, and the colors they express through biological pigmentation. Each pigment is represented in terms of its chemistry, its related biosynthesis, and its color expressions according to different indices: HEX, RGB, and Pantone. It is available at color.bio. Conclusions As fungal biotechnology continues to mature into new application areas, it is as important as ever that there is human empathy for these organisms to promote the preservation and appreciation of fungal biodiversity. The products presented here provide paths for artists, scientists, and designers to understand microorganisms through the lens of color, promoting interspecies empathy through research, teaching, and practice.

DC-SIGN targets amphotericin B-loaded liposomes to diverse pathogenic fungi

Background Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans. Results We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs. Conclusions DC-SIGN’s CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.

Deciphering the ChitoCode: fungal chitins and chitosans as functional biopolymers

Chitins and chitosans are among the most widespread and versatile functional biopolymers, with interesting biological activities and superior material properties. While chitins are evolutionary ancient and present in many eukaryotes except for higher plants and mammals, the natural distribution of chitosans, i.e. extensively deacetylated derivatives of chitin, is more limited. Unequivocal evidence for its presence is only available for fungi where chitosans are produced from chitin by the action of chitin deacetylases. However, neither the structural details such as fraction and pattern of acetylation nor the physiological roles of natural chitosans are known at present. We hypothesise that the chitin deacetylases are generating chitins and chitosans with specific acetylation patterns and that these provide information for the interaction with specific chitin- and chitosan-binding proteins. These may be structural proteins involved in the assembly of the complex chitin- and chitosan-containing matrices such as fungal cell walls and insect cuticles, chitin- and chitosan-modifying and -degrading enzymes such as chitin deacetylases, chitinases, and chitosanases, but also chitin- and chitosan-recognising receptors of the innate immune systems of plants, animals, and humans. The acetylation pattern, thus, may constitute a kind of ‘ChitoCode’, and we are convinced that new in silico, in vitro, and in situ analytical tools as well as new synthetic methods of enzyme biotechnology and organic synthesis are currently offering an unprecedented opportunity to decipher this code. We anticipate a deeper understanding of the biology of chitin- and chitosan-containing matrices, including their synthesis, assembly, mineralisation, degradation, and perception. This in turn will improve chitin and chitosan biotechnology and the development of reliable chitin- and chitosan-based products and applications, e.g. in medicine and agriculture, food and feed sciences, as well as cosmetics and material sciences.

Mechanical characteristics of bacterial cellulose-reinforced mycelium composite materials

Background While mycelium is considered a promising alternative for fossil-based resins in lignocellulosic materials, the mechanical properties of mycelium composite materials remain suboptimal, among other reasons due to the weak internal bonds between the hyphae and the natural fibres. A solution could be provided by the hybridisation of mycelium materials with organic additives. More specifically, bacterial cellulose seems to be a promising additive that could result in reinforcing mycelium composites; however, this strategy is underreported in scientific literature. Results In this study, we set out to investigate the mechanical properties of mycelium composites, produced with the white-rot fungus Trametes versicolor, and supplemented with bacterial cellulose as an organic additive. A methodological framework is developed for the facile production of bacterial cellulose and subsequent fabrication of mycelium composite particle boards based on a hybrid substrate consisting of bacterial cellulose and hemp in combination with a heat-pressing approach. We found that, upon adding bacterial cellulose, the internal bond of the composite particle boards significantly improved. Conclusions The addition of bacterial cellulose to mycelium composite materials not only results in a strengthening of internal bonding of mycelium material, but also renders tuneable mechanical properties to the material. As such, this study contributes to the ongoing development of fully biological hybrid materials with performant mechanical characteristics.

Current state and future prospects of pure mycelium materials

In the context of the ongoing transition from a linear to a circular economy, ecologically friendly renewable solutions are put in place. Filamentous fungi can be grown on various organic feedstocks and functionalized into a range of diverse material types which are biobased and thus more sustainable in terms of their production, use and recycling. Pure mycelium materials, consisting only of mycelial biomass, can adopt versatile properties and appear promising as a substitute for current petrochemically produced polymeric materials or, in the case of myco-leather, as a substitute for animal-based leather. In recent years, a handful of private companies have been innovating to bring products based on pure mycelium materials to the market while scientific interest in these promising biomaterials is now starting to gain momentum. In this primer, we introduce pure mycelium materials, frame different production methods, review existing and potential future applications, thereby offering a vision on future advances for this emerging fungi-based technology.

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.

Filamentous fungal applications in biotechnology: a combined bibliometric and patentometric assessment

Background Processes and products employing filamentous fungi are increasing contributors to biotechnology. These organisms are used as cell factories for the synthesis of platform chemicals, enzymes, acids, foodstuffs and therapeutics. More recent applications include processing biomass into construction or textile materials. These exciting advances raise several interrelated questions regarding the contributions of filamentous fungi to biotechnology. For example, are advances in this discipline a major contributor compared to other organisms, e.g. plants or bacteria? From a geographical perspective, where is this work conducted? Which species are predominantly used? How do biotech companies actually use these organisms? Results To glean a snapshot of the state of the discipline, literature (bibliometry) and patent (patentometry) outputs of filamentous fungal applications and the related fields were quantitatively surveyed. How these outputs vary across fungal species, industrial application(s), geographical locations and biotechnological companies were analysed. Results identified (i) fungi as crucial drivers for publications and patents in biotechnology, (ii) enzyme and organic acid production as the main applications, (iii) Aspergillus as the most commonly used genus by biotechnologists, (iv) China, the United States, Brazil, and Europe as the leaders in filamentous fungal science, and (v) the key players in industrial biotechnology. Conclusions This study generated a summary of the status of filamentous fungal applications in biotechnology. Both bibliometric and patentometric data have identified several key trends, breakthroughs and challenges faced by the fungal research community. The analysis suggests that the future is bright for filamentous fungal research worldwide.

A review on architecture with fungal biomaterials: the desired and the feasible

Fungal biomaterials are becoming increasingly popular in the fields of architecture and design, with a significant bloom of projects having taken place during the last 10 years. Using mycelium as a stabilizing compound for fibers from agricultural waste, new building elements can be manufactured according to the circular economy model and be used for architectural construction to transform the building industry towards an increased environmental and economic sustainability. Simultaneously, research on those materials and especially fungal biocomposites is producing knowledge that allows for the materials themselves to inspire and transform the architectural design. Novel research on those materials is not only allowing for their use as construction materials, but it inspires and affects the architectural design process through the discovery and variation of the materials’ properties. Today, many interdisciplinary teams are working on this emerging field to integrate fungal biocomposites in the construction industry and to merge science, art, and architecture responsibly.This study provides an overview of the progress that has been made in this field during the last 10 years, focusing on six works that are presented in more detail. Those six works are spaces at an architectural scale which showcase unique elements and innovative aspects for the use of fungal biomaterials in architecture. Each work has followed different design strategies, different fabrication methods, or different post-processing methods. All of them together have produced significant technical knowledge as well as a cultural impact for the field of architecture but also for the field of fungal biotechnology.

A review on the potential of filamentous fungi for microbial self-healing of concrete

Concrete is the most used construction material worldwide due to its abundant availability and inherent ease of manufacturing and application. However, the material bears several drawbacks such as the high susceptibility for crack formation, leading to reinforcement corrosion and structural degradation. Extensive research has therefore been performed on the use of microorganisms for biologically mediated self-healing of concrete by means of CaCO3 precipitation. Recently, filamentous fungi have been recognized as high-potential microorganisms for this application as their hyphae grow in an interwoven three-dimensional network which serves as nucleation site for CaCO3 precipitation to heal the crack. This potential is corroborated by the current state of the art on fungi-mediated self-healing concrete, which is not yet extensive but valuable to direct further research. In this review, we aim to broaden the perspectives on the use of fungi for concrete self-healing applications by first summarizing the major progress made in the field of microbial self-healing of concrete and then discussing pioneering work that has been done with fungi. Starting from insights and hypotheses on the types and principles of biomineralization that occur during microbial self-healing, novel potentially promising candidate species are proposed based on their abilities to promote CaCO3 formation or to survive in extreme conditions that are relevant for concrete. Additionally, an overview will be provided on the challenges, knowledge gaps and future perspectives in the field of fungi-mediated self-healing concrete.

CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.