Fungal Biology and Biotechnology最新文献

Background: Fomitopsis pinicola is one of the most common fungi found in temperate zone of Europe, widely distributed spread in Asia and North America. Fungus has a wide range of therapeutic activity: antitumor, antimicrobial, anti-inflammatory, antidiabetic, antifungal, hepatoprotective, hemostatic action. A number of studies have confirmed the significant antioxidant activity of F. pinicola fruiting bodies. However, the controlled cultivation conditions that influence fungal growth and metabolite production of F. pinicola, particularly the mycelial growth and biosynthesis of metabolites in its culture broth, as well as the antioxidant activity of its mycelium, remain poorly understood.

Results: This study investigated the impact of cultivation conditions on F. pinicola mycelium growth, phenols synthesis and antioxidant activity. Difference in the biosynthetic activity of F. pinicola under tested cultivation conditions was established. A highest value of 2,2-diphenyl-1-picryl-hydrazyl (DPPH•) inhibition (78.2 ± 0.9%) was found for a mycelium cultivated at 30 ºC, while cultivation at a lower temperature (20 ºC) was suitable for biomass growth (8.5 ± 0.3 g/L) and total phenolic content (TPC) 11.0 ± 0.6 mg GAE/g. Carbon and nitrogen sources in a cultivation broth significantly influenced the studied characteristics. Xylose supported the highest DPPH• inhibition (89.91 ± 0.5%) and TPC (16.55 ± 0.4 mg GAE/g), while galactose yielded the best biomass (4.0 ± 0.3 g/L). Peptone was the most effective nitrogen source for obtaining the mycelium with high potential of DPPH• radical inactivation (90.42 ± 0.5%) and TPC (17.41 ± 0.5 mg GAE/g), while the maximum biomass yield (7.8 ± 0.6 g/L) was found with yeast extract in cultivation medium. F. pinicola demonstrated the ability to grow and produce bioactive metabolites across a wide pH range from 2.5 to 7.5. Shaking cultivation resulted in the highest TPC (21.44 ± 0.10 mg GAE/g), though the same level of antioxidant activity (93%) was achieved under both shaking and static cultivation on the 7th and 28th days, respectively.

Conclusion: Controlling cultivation parameters makes it possible to regulate the metabolic and biochemical processes of F. pinicola, facilitating the balance needed to obtain optimal biomass, phenols and antioxidant activity. The findings show the potential to increase phenol production by 2.25 and 2.23 times under shaking and static conditions, respectively, while maintaining a high level of activity.

Background: Spores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores.

Results: In this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers.

Conclusions: This study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Background: Ashbya gossypii is a filamentous fungus widely utilized for industrial riboflavin production and has a great potential as a microbial chassis for synthesizing other valuable metabolites such as folates, biolipids, and limonene. Engineered strains of A. gossypii can effectively use various waste streams, including xylose-rich feedstocks. Notably, A. gossypii has been identified as a proficient biocatalyst for producing limonene from xylose-rich sources. This study aims to investigate the capability of engineered A. gossypii strains to produce various plant monoterpenes using agro-industrial waste as carbon sources.

Results: We overexpressed heterologous terpene synthases to produce acyclic, monocyclic, and bicyclic monoterpenes in two genetic backgrounds of A. gossypii. These backgrounds included an NPP synthase orthogonal pathway and a mutant erg20^F95W allele with reduced FPP synthase activity. Our findings demonstrate that A. gossypii can synthesize linalool, limonene, pinene, and sabinene, with terpene synthases showing differential substrate selectivity for NPP or GPP precursors. Additionally, co-overexpression of endogenous HMG1 and ERG12 with heterologous NPP synthase and terpene synthases significantly increased sabinene yields from xylose-containing media. Using mixed formulations of corn-cob lignocellulosic hydrolysates and either sugarcane or beet molasses, we achieved limonene and sabinene productions of 383 mg/L and 684.5 mg/L, respectively, the latter representing a significant improvement compared to other organisms in flask culture mode.

Conclusions: Engineered A. gossypii strains serve as a suitable platform for assessing plant terpene synthase functionality and substrate selectivity in vivo, which are crucial to understand monoterpene bioproduction. The NPP synthase pathway markedly enhances limonene and sabinene production in A. gossypii, achieving levels comparable to those of other industrial microbial producers. Furthermore, these engineered strains offer a novel approach for producing monoterpenes through the valorization of agro-industrial wastes.

Background: Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.

Results: Here we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13-20% of the transformants while 80-87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35-61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85-90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.

Conclusion: The awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Background: The application of plant-beneficial microorganisms as bio-fertilizer and biocontrol agents has gained traction in recent years, as both agriculture and forestry are facing the challenges of poor soils and climate change. Trichoderma spp. are gaining popularity in agriculture and forestry due to their multifaceted roles in promoting plant growth through e.g. nutrient translocation, hormone production, induction of plant systemic resistance, but also direct antagonism of other fungi. However, the mycotrophic nature of the genus bears the risk of possible interference with other native plant-beneficial fungi, such as ectomycorrhiza, in the rhizosphere. Such interference could yield unpredictable consequences for the host plants of these ecosystems. So far, it remains unclear, whether Trichoderma is able to differentiate between plant-beneficial and plant-pathogenic fungi during the process of plant colonization.

Results: We investigated whether Trichoderma spp. can differentiate between beneficial ectomycorrhizal fungi (represented by Laccaria bicolor and Hebeloma cylindrosporum) and pathogenic fungi (represented by Fusarium graminearum and Alternaria alternata) in different confrontation scenarios, including a newly developed olfactometer "race tube"-like system. Using two independent species, T. harzianum and T. atrobrunneum, with plant-growth-promoting and immune-stimulating properties towards Populus x canescens, our study revealed robustly accelerated growth towards phytopathogens, while showing a contrary response to ectomycorrhizal fungi. Transcriptomic analyses identified distinct genetic programs during interaction corresponding to the lifestyles, emphasizing the expression of mycoparasitism-related genes only in the presence of phytopathogens.

Conclusion: The findings reveal a critical mode of fungal community interactions belowground and suggest that Trichoderma spp. can distinguish between fungal partners of different lifestyles already at a distance. This sheds light on the entangled interactions of fungi in the rhizosphere and emphasizes the potential benefits of using Trichoderma spp. as a biocontrol agent and bio-fertilizer in tree plantations.

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.