Fungal Biology and Biotechnology最新文献

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.

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.

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.

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.

Background: Fungi are prolific producers of secondary metabolites (SMs), which are bioactive small molecules with important applications in medicine, agriculture and other industries. The backbones of a large proportion of fungal SMs are generated through the action of large, multi-domain megasynth(et)ases such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The structure of these backbones is determined by the domain architecture of the corresponding megasynth(et)ase, and thus accurate annotation and classification of these architectures is an important step in linking SMs to their biosynthetic origins in the genome.

Results: Here we report synthaser, a Python package leveraging the NCBI's conserved domain search tool for remote prediction and classification of fungal megasynth(et)ase domain architectures. Synthaser is capable of batch sequence analysis, and produces rich textual output and interactive visualisations which allow for quick assessment of the megasynth(et)ase diversity of a fungal genome. Synthaser uses a hierarchical rule-based classification system, which can be extensively customised by the user through a web application ( http://gamcil.github.io/synthaser ). We show that synthaser provides more accurate domain architecture predictions than comparable tools which rely on curated profile hidden Markov model (pHMM)-based approaches; the utilisation of the NCBI conserved domain database also allows for significantly greater flexibility compared to pHMM approaches. In addition, we demonstrate how synthaser can be applied to large scale genome mining pipelines through the construction of an Aspergillus PKS similarity network.

Conclusions: Synthaser is an easy to use tool that represents a significant upgrade to previous domain architecture analysis tools. It is freely available under a MIT license from PyPI ( https://pypi.org/project/synthaser ) and GitHub ( https://github.com/gamcil/synthaser ).

Material development based on fungal mycelium is a fast-rising field of study as researchers, industry, and society actively search for new sustainable materials to address contemporary material challenges. The compelling potential of fungal mycelium materials is currently being explored in relation to various applications, including construction, packaging, "meatless" meat, and leather-like textiles. Here, we highlight the discussions and outcomes from a recent 1-day conference on the topic of fungal mycelium materials ("Fungal Mycelium Materials Mini Meeting"), where a group of researchers from diverse academic disciplines met to discuss the current state of the art, their visions for the future of the material, and thoughts on the challenges surrounding widescale implementation.

Fungi of the genus Trichoderma are routinely used as biocontrol agents and for the production of industrial enzymes. Trichoderma spp. are interesting hosts for heterologous gene expression because their saprotrophic and mycoparasitic lifestyles enable them to thrive on a large number of nutrient sources and some members of this genus are generally recognized as safe (GRAS status). In this review, we summarize and discuss several aspects involved in heterologous gene expression in Trichoderma, including transformation methods, genome editing strategies, native and synthetic expression systems and implications of protein secretion. This review focuses on the industrial workhorse Trichoderma reesei because this fungus is the best-studied member of this genus for protein expression and secretion. However, the discussed strategies and tools can be expected to be transferable to other Trichoderma species.

Background: In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD.

Results: Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9.

Conclusions: Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure-function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

Background: The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.

Results: In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.

Conclusion: With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.