Fungal Biology and Biotechnology最新文献

Background: Topical product derived from the fungus Cordyceps militaris was explored as a feasible method for an industrial practice.

Results: The mycelium residue of C. militaris that was industrial biotechnological produced was extracted with water at different time conditions under ambient temperature, filtered and lyophilized. The extracts were all light to dark brown powder. The 24 h extraction was significantly (p < 0.01) highest in an extractive yield and total polysaccharides content (TPC) (43.33 ± 0.99% and 144.02 ± 2.06 mg glucose/g crude extract). This extract was proved to be stable following an accelerated stability test with the insignificant (p > 0.05) reduction of TPC (4.95 ± 2.23%). Topical product containing the extract were developed. Skin care preparation containing 0.2% extract was exhibited as the appropriated amount giving the stable cream. The developed C. militaris polysaccharide cream was confirmed safe and gained more than 70% of the overall preferences examined in 20 female volunteers.

Conclusions: Cordyceps militaris mycelium residue is a beneficial source for pharmaceutical products. The C. militaris polysaccharides extract was prepared and qualified in terms of active content and stability. The extract was shown to be compatible with the available cosmetic ingredients. The safe and preferred C. militaris polysaccharides skin care cosmetics was developed. Accordingly, C. militaris polysaccharides skin care cosmetics that meets all the quality characters which are stable, safe, usable and efficient.

Background: Oleaginous fungi have versatile metabolism and able to transform a wide range of substrates into lipids, accounting up to 20-70% of their total cell mass. Therefore, oleaginous fungi are considered as an alternative source of lipids. Oleaginous fungi can accumulate mainly acyl glycerides and free fatty acids which are localized in lipid droplets. Some of the oleaginous fungi possessing promising lipid productivity are dimorphic and can exhibit three cell forms, flat hyphae, swollen hyphae and yeast-like cells. To develop sustainable targeted fungal lipid production, deep understanding of lipogenesis and lipid droplet chemistry in these cell forms is needed at multiscale level. In this study, we explored the potential of infrared spectroscopy techniques for examining lipid droplet formation and accumulation in different cell forms of the dimorphic and oleaginous fungus Mucor circinelloides.

Results: Both transmission- and reflectance-based spectroscopy techniques are shown to be well suited for studying bulk fungal biomass. Exploring single cells with infrared microspectroscopy reveals differences in chemical profiles and, consequently, lipogenesis process, for different cell forms. Yeast-like cells of M. circinelloides exhibited the highest absorbance intensities for lipid-associated peaks in comparison to hyphae-like cell forms. Lipid-to-protein ratio, which is commonly used in IR spectroscopy to estimate lipid yield was the lowest in flat hyphae. Swollen hyphae are mainly composed of lipids and characterized by more uniform distribution of lipid-to-protein concentration. Yeast-like cells seem to be comprised mostly of lipids having the largest lipid-to-protein ratio among all studied cell forms. With infrared nanospectroscopy, variations in the ratios between lipid fractions triglycerides and free fatty acids and clear evidence of heterogeneity within and between lipid droplets are illustrated for the first time.

Conclusions: Vibrational spectroscopy techniques can provide comprehensive information on lipogenesis in dimorphic and oleaginous fungi at the levels of the bulk of cells, single cells and single lipid droplets. Unicellular spectra showed that various cell forms of M. circinelloides differs in the total lipid content and profile of the accumulated lipids, where yeast-like cells are the fatty ones and, therefore, could be considered as preferable cell form for producing lipid-rich biomass. Spectra of single lipid droplets showed an indication of possible droplet-to-droplet and within-droplet heterogeneity.

Background: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host-pathogen interaction can be studied in more detail.

Results: Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette.

Conclusions: The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen.

The production of petroleum-based plastics increased dramatically following industrialization. Because of multifaceted properties such as durability, thermostability, water resistance, and many others, these plastics have become an indispensable part of daily life. However, while improving people's quality of life, indiscriminate use of plastics has caused pollution and raised environmental concerns. To address this situation and reduce environmental risks, microbially produced biopolymers such as poly-3-hydroxyalkanoates can be used to make bioplastics that are completely biodegradable under normal environmental conditions. At the moment, the cost of bioplastic production is high when compared to petroleum-based plastics, so alternate strategies for making the bioplastic process economical are urgently needed. Agricultural waste is abundant around the world and can be efficiently used as a low-cost renewable feedstock after pretreatment and enzymatic hydrolysis. Fungi are well known as primary degraders of lignocellulosic waste, and this property was used in the current study to enzymatically hydrolyze the pretreated paddy straw for the production of reducing sugars, which were then used in the microbial fermentation for the production of PHB. In this study, Aspergillus nidulans was used to advance a low-cost and efficient enzyme hydrolysis system for the generation of reducing sugars from lignocellulosic biomass. For the production of the holocellulosic enzyme complex, the fungus was grown on wheat straw with Reese mineral medium as a wetting agent. After 216 h of solid-state fermentation at 30 °C, pH 6.0, the enzyme extract from A. nidulans demonstrated the highest activity, CMCase 68.58 (± 0.55), FPase 12.0 (± 0.06), Xylanase 27.17 (± 0.83), and β-glucosidase 1.89 (± 0.037). The initial pH, incubation temperature, and time all had a significant impact on final enzyme activity. Enzymatic hydrolysis of pretreated paddy straw produced reducing sugars (8.484 to 30.91 gL^-1) that were then used to produce poly(3-hydroxybutyrate) using halophilic bacterial isolates. Burkholderia gladioli 2S4R1 and Bacillus cereus LB7 accumulated 26.80% and 20.47% PHB of the cell dry weight, respectively. This suggests that the holocellulosic enzyme cocktail could play a role in the enzymatic hydrolysis of lignocellulosic materials and the production of PHA from less expensive feedstocks such as agricultural waste.

Extracellular vesicles (EVs) are increasingly recognized as an important mechanism for cell-cell interactions. Their role in fungi is still poorly understood and they have been isolated from only a handful of species. Here, we isolated and characterized EVs from Aureobasidium pullulans, a biotechnologically important black yeast-like fungus that is increasingly used for biocontrol of phytopathogenic fungi and bacteria. After optimization of the isolation protocol, characterization of EVs from A. pullulans by transmission electron microscopy (TEM) revealed a typical cup-shaped morphology and different subpopulations of EVs. These results were confirmed by nanoparticle tracking analysis (NTA), which revealed that A. pullulans produced 6.1 × 10⁸ nanoparticles per milliliter of culture medium. Proteomic analysis of EVs detected 642 proteins. A small fraction of them had signal peptides for secretion and transmembrane domains. Proteins characteristic of different synthesis pathways were found, suggesting that EVs are synthesized by multiple pathways in A. pullulans. Enrichment analysis using Gene Ontology showed that most of the proteins found in the EVs were associated with primary metabolism. When sequencing the small RNA fraction of A. pullulans EVs, we found two hypothetical novel mil-RNAs. Finally, we tested the biocontrol potential of EVs from A. pullulans. The EVs did not inhibit the germination of spores of three important phytopathogenic fungi-Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum. However, exposure of grown cultures of C. acutatum and P. expansum to A. pullulans EVs resulted in visible changes in morphology of colonies. These preliminary results suggest that EVs may be part of the antagonistic activity of A. pullulans, which is so far only partially understood. Thus, the first isolation and characterization of EVs from A. pullulans provides a starting point for further studies of EVs in the biotechnologically important traits of the biocontrol black fungus A. pullulans in particular and in the biological role of fungal EVs in general.

Background: Several metabolites released by fungal species are an essential source of biologically active natural substances. Gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) is one of the techniques used in profiling the metabolites produced by microorganisms, including Talaromyces pinophilus. However, there is limited information regarding differential substrates' impacts on this fungal strain's metabolite profiling. This study examined the metabolite profile of T. pinophilus strain SPJ22 cultured on three different media, including solid czapek yeast extract agar (CYA), malt extract agar (MEA) and potato dextrose agar (PDA) using GC-HRTOF-MS. The mycelia including the media were plugged and dissolved in 5 different organic solvents with varying polarities viz.: acetonitrile, dichloromethane, hexane, 80% methanol and water, and extracts analysed on GC-HRTOF-MS.

Results: The study revealed the presence of different classes of metabolites, such as fatty acids (2.13%), amides (4.26%), alkanes (34.04%), furan (2.13%), ketones (4.26%), alcohols (14.89%), aromatic compounds (6.38%), and other miscellaneous compounds (17.02%). Significant metabolites such as acetic acid, 9-octadecenamide, undecanoic acid methyl ester, hydrazine, hexadecane, nonadecane, eicosane, and other compounds reported in this study have been widely documented to have plant growth promoting, antimicrobial, anti-inflammatory, antioxidant, and biofuel properties. Furthermore, T. pinophilus grown on PDA and MEA produced more than twice as many compounds as that grown on CYA.

Conclusion: Thus, our result showed that the production of essential metabolites from T. pinophilus is substrate dependent, with many of these metabolites known to have beneficial characteristics, and as such, this organism can be utilised as a sustainable and natural source for these useful organic molecules.

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

Since the initial detection, in 2007, of fungal ribosomally synthesised and post-translationally modified peptides (RiPPs), this group of natural products has undergone rapid expansion, with four separate classes now recognised: amatoxins/phallotoxins, borosins, dikaritins, and epichloëcyclins. Largely due to their historically anthropocentric employment in medicine and agriculture, novel fungal proteins and peptides are seldom investigated in relation to the fungus itself. Therefore, although the benefits these compounds confer to humans are often realised, their evolutionary advantage to the fungus, the reason for their continued production, is often obscure or ignored. This review sets out to summarise current knowledge on how these small peptide-derived products influence their producing species and surrounding biotic environment.

Fungal specialized metabolites play an important role in the environment and have impacted human health and survival significantly. These specialized metabolites are often the end product of a series of sequential and collaborating biosynthetic enzymes that reside within different subcellular compartments. A wide variety of methods have been developed to understand fungal specialized metabolite biosynthesis in terms of the chemical conversions and the biosynthetic enzymes required, however there are far fewer studies elucidating the compartmentalization of the same enzymes. This review illustrates the biosynthesis of specialized metabolites where the localization of all, or some, of the biosynthetic enzymes have been determined and describes the methods used to identify the sub-cellular localization.