Fungal Biology and Biotechnology最新文献

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.

Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.

A fungal skin is a thin flexible sheet of a living homogeneous mycelium made by a filamentous fungus. The skin could be used in future living architectures of adaptive buildings and as a sensing living skin for soft self-growing/adaptive robots. In experimental laboratory studies we demonstrate that the fungal skin is capable for recognising mechanical and optical stimulation. The skin reacts differently to loading of a weight, removal of the weight, and switching illumination on and off. These are the first experimental evidences that fungal materials can be used not only as mechanical 'skeletons' in architecture and robotics but also as intelligent skins capable for recognition of external stimuli and sensorial fusion.

Background: Chitin and its derivative chitosan are readily exploited, especially in food, cosmetic, pharmaceutical, biomedical, chemical, and textile industries. The biopolymers are currently recovered from the crustacean shells after purification from the large amount of proteins and minerals. The key problems are centered around a lot of chemical waste and allergenic potential of the heat-stable remaining proteins. Fungi can be considered as an alternative eco-friendlier source of the chitin and chitosan due to the lower level of inorganic materials and absence of the allergenic proteins.

Results: The work presents a new chemical assay to change the composition of the milled Fomes fomentarius fruiting bodies. A gradual 13-fold increase of the chitin amount accompanied by 14-fold decrease of the glucan content was obtained after repetitive alkali-acidic treatment. Raw material contained mainly chitin with 30% degree of deacetylation. After the first and second alkali treatment, the polymer was defined as chitosan with comparable amounts of N-acetyl-D-glucosamine and D-glucosamine units. The last treated samples showed an increase of the chitin amount to 80%, along with typical for the natural tinder fibers degree of deacetylation and three-dimensional fibrous hollow structure.

Conclusions: A new approach allowed a gradual enrichment of the pulverized Fomes fomentarius fruiting bodies with chitin or chitosan, depending on the extraction conditions. High stability and fibrous structure of the fungal cell walls with a drastically increased chitin ratio let us suggest a possibility of the targeted production of the chitin-enriched fungal material biotechnologically under eco-friendly conditions.