Fungal Biology and Biotechnology最新文献

Laccases are multi-copper oxidases that are usually composed of three Cu-oxidase domains. Domains one and three house the copper binding sites, and the second domain is involved in forming a substrate-binding cleft. However, Streptomyces species are found to have small laccases (SLAC) that lack one of the three Cu-oxidase domains. This type of SLAC with interesting lignocellulose bioconversion activities has not been reported in Aspergillus niger. In our research, we explored the expression and engineering of the SLAC from Streptomyces leeuwenhoekii C34 in A. niger. Genes encoding two versions of the SLAC were expressed. One encoding the SLAC in its native form and a second encoding the SLAC fused to two N-terminal CBM1 domains. The latter is a configuration also known for specific yeast laccases. Both SLAC variants were functionally expressed in A. niger as shown by in vitro activity assays and proteome analysis. Laccase activity was also analyzed toward bioconversion of lignocellulosic rice straw. From this analysis it was clear that the SLAC activity improved the efficiency of saccharification of lignocellulosic biomass by cellulase enzyme cocktails.

From 30 September 2023 to 7 January 2024, the Nobel Prize Museum in Stockholm presented the show Fungi-In Art and Science. For the exhibition, an alliance of scientists, artists, and designers was brought together that overcame all the alleged borders between the disciplines, between the scientific and the creative world. This special exhibition is the starting point to take on a tour where it is about crossing borders and growing connections when working with fungi. My interview partners represent perfectly the different angles from which you can take a look onto the kingdom of fungi. There is the person without previous knowledge but with a profound artistic understanding who got mesmerized by the subject-matter, which he didn't realize it existed before-Karl-Johan Cottman. There is the scientist, being knee-deep in fungi matter who discovered the arts for an extension of her scientific understanding-Vera Meyer. And last but not least there is the person living passionately for the arts who found fungi mesmerizing for both art creation and progressive/sustainable production-Phil Ross. So, there are three threads weaving one fungal fabric. Have fun reading it!

Background: Recent advancements in the collaboration between two scientific disciplines-fungal biotechnology and materials sciences-underscore the potential of fungal mycelium as renewable resource for sustainable biomaterials that can be harnessed in different industries. As fungal mycelium can be biotechnologically obtained from different filamentous fungi and is as a material very versatile, respective research and commercial application should be thriving. However, some granted patents in the field of fungal mycelium-based materials have caused uncertainty in the community as to which subject matter is patent-protected and for how long the protection is expected to last.

Results: This opinion paper therefore maps the patent landscape of fungal mycelium-based materials with a specific focus on technical applications including building construction, insulation, packaging, and the like. We provide an overview of granted patents (73) and pending applications (34) related to granted patents, the dominant patent portfolios (five, with the number of patents and/or applications per owner between six and 44), the patent owners, and highlight the key claims formulated to protect the inventions. Additionally, we outline various options towards an increased activity in the field.

Conclusion: Patent developments in the field leave the impression that fungal materials, despite their high potential as renewable and biodegradable materials, have been held back due to patent over-protection. Considering the need for replacing current petroleum-based materials with renewable biomaterials, coordinated efforts may be called for to intensify efforts in the field.

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3' end and by HDR at the 5' end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.

Background: Renewable materials made using environmentally friendly processes are in high demand as a solution to reduce the pollution created by the fashion industry. In recent years, there has been a growing trend in research on renewable materials focused on bio-based materials derived from fungi.

Results: Recently, fungal cell wall material of a chitosan producing fungus has been wet spun to monofilaments. This paper presents a modification for the fungal monofilament spinning process, by the development of a benign method, dry gel spinning, to produce continuous monofilaments and twisted multifilament yarns, from fungal cell wall, that can be used in textile applications. The fungal biomass of Rhizopus delemar, grown using bread waste as a substrate, was subjected to alkali treatment with a dilute sodium hydroxide solution to isolate alkali-insoluble material (AIM), which mainly consists of the fungal cell wall. The treatment of AIM with dilute lactic acid resulted in hydrogel formation. The morphology of the hydrogels was pH dependent, and they exhibited shear thinning viscoelastic behavior. Dry gel spinning of the fungal hydrogels was first conducted using a simple lab-scale syringe pump to inject the hydrogels through a needle to form a monofilament, which was directly placed on a rotating receiver and left to dry at room temperature. The resulting monofilament was used to make twisted multifilament yarns. The process was then improved by incorporating a heated chamber for the quicker drying of the monofilaments (at 30⁰C). Finally, the spinning process was scaled up using a twin-screw microcompounder instead of the syringe pump. The monofilaments were several meters long and reached a tensile strength of 63 MPa with a % elongation at break of 14. When spinning was performed in the heated chamber, the tensile strength increased to 80 MPa and further increased to 103 MPa when a micro-compounder was used for spinning.

Conclusion: The developed dry gel spinning method shows promising results in scalability and demonstrates the potential for renewable material production using fungi. This novel approach produces materials with mechanical properties comparable to those of conventional textile fibers.

Background: Penicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use.

Results: Increasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54-83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol.

Conclusion: Genome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.

Medicinal mushrooms, such as Taiwanofungus camphoratus, Inonotus obliquus, and Tropicoporus linteus, have been used in traditional medicine for therapeutic purposes and promotion of overall health in China and many East Asian countries for centuries. Modern pharmacological studies have demonstrated the large amounts of bioactive constituents (such as polysaccharides, triterpenoids, and phenolic compounds) available in these medicinal mushrooms and their potential therapeutic properties. Due to the rising demand for the health-promoting medicinal mushrooms, various cultivation methods have been explored to combat over-harvesting of the fungi. Evidence of the robust pharmacological properties, including their anticancer, hypoglycemic, hypolipidemic, antioxidant, and antiviral activities, have been provided in various studies, where the health-benefiting properties of the medicinal fungi have been further proven through numerous clinical trials. In this review, the cultivation methods, available bioactive constituents, therapeutic properties, and potential uses of T. camphoratus, I. obliquus and T. linteus are explored.

Background: Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.

Results: The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.

Conclusion: The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Background: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking.

Results: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and β-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports.

Conclusions: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.