There are two types of autophagy, non-selective (bulk) autophagy, in which substrates are randomly incorporated into autophagosomes, and selective autophagy, in which substrates are specifically ...targeted. In filamentous fungi, the molecular mechanism underlying selective autophagy remains largely unknown. Recently we identified a novel protein, AoAtg8-interacting protein A (AeiA), in the filamentous fungus Aspergillus oryzae. AeiA was localized to peroxisomes and autophagosomal intermediates, such as phagophore assembly site (PAS) and the phagophore. Moreover, pexophagy flux was reduced in AeiA deletants. Taken together, AeiA is a novel selective autophagy-related protein that contributes to pexophagy in A. oryzae. Our findings provide insight into the molecular mechanisms of selective autophagy including pexophagy in filamentous fungi. Abbreviations: AIM, Atg8-family interacting motifs; Atg8, autophagy-related 8; EGFP, enhanced green fluorescent protein; GABARAP, Gamma aminobutyric acid A receptor associated protein; LC3, Microtubule-associated protein light chain 3; MTS, microbody targeting signal; PD, potato dextrose.
Filamentous fungi possess the capacity to produce a wide array of secondary metabolites with diverse biological activities and structures, such as lovastatin and swainsonine. With the advent of the ...post-genomic era, increasing amounts of cryptic or uncharacterized secondary metabolite biosynthetic gene clusters are continually being discovered. However, owing to the longstanding lack of versatile, comparatively simple, and highly efficient genetic manipulation techniques, the broader exploration of industrially important secondary metabolites has been hampered thus far. With the emergence of CRISPR/Cas9-based genome editing technology, this dilemma may be alleviated, as this advanced technique has revolutionized genetic research and enabled the exploitation and discovery of new bioactive compounds from filamentous fungi. In this review, we introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CRISPR/Cas9-mediated genome editing in filamentous fungi. We also briefly introduce the specific applications of the CRISPR/Cas9 system and CRISPRa in the improvement of secondary metabolite contents and discovery of novel biologically active compounds in filamentous fungi, with specific examples noted. Additionally, we highlight and discuss some of the challenges and deficiencies of using the CRISPR/Cas9-based genome editing technology in research on the biosynthesis of secondary metabolites as well as future application of CRISPR/Cas9 strategy in filamentous fungi are highlighted and discussed.
Fungi as a group are some of the most resilient spoilage microorganisms and are capable of overcoming the control strategies utilized by the food industry. Various fungal propagules are rapidly ...dispersed by water and air, survive under extreme conditions, and sustainably increase in biomass. The aim of this study were isolation and identification of microscopic filamentous fungi from traditional cow cheese. Altogether, 40 samples of Slovak traditional cheese “Parenica“ were examined. The samples included non-smoked and smoked cheese samples. Microscopic fungi were cultivated on Malt extract agar. Microscopic fungi count was from 1.89 log cfu.g-1 to 2.65 log cfu.g-1. A total of 129 isolates of microscopic fungi were identified in our study. Alternaria and Penicillium (50%) were the most abundant microscopic fungi. Also Aspergillus (45%) and Cladosporium (45%) were frequently identified. Alternaria sp., Aspergillus niger, Aspergillus sp., Cladosporium sp., Mucor sp., Penicillium sp. and Rhizopus sp. were isolated from non-smoked and smoked cheese.
Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation. Agricultural side products, wheat straw ...and corn stover and forestry energy plant willow chips were selected as plant biomass sources. The substrates were confronted by pure cultures of Penicillium aurantiogriseum (new isolate from rumen), Trichoderma reesei (DSM768), Gilbertella persicaria (SZMC11086) and Rhizomucor miehei (SZMC11005). In addition to total cellulolytic filter paper degradation activity, the production of endoglucanase, cellobiohydrolase, β-glucosidase enzymes were followed during the pretreatment period, which lasted for 10 days at 37 °C. The products of pretreatments were subsequently tested for mesophilic biogas production in batch reactors. All 4 strains effectively pretreated the lignocellulosic substrates albeit in varying degrees, which was related to the level of the tested hydrolytic enzyme activities. Penicillium aurantiogriseum showed outstanding hydrolytic enzyme production and highest biogas yield from the partially degraded substrates. Corn stover was the best substrate for biomass decomposition and biogas production. Scanning electron microscopy confirmed the deep penetration of fungal hyphae into the lignocellulosic substrate in all cases.
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•Lignocellulosic substrates were subjected to pretreatment by hydrolytic enzymes.•Wheat straw, corn stover and woody willow chips were the tested substrates.•Filamentous fungi produced the enzymes in situ during the10-day pretreatment at 37 °C.•Penicillium, Trichoderma, Gilbertella and Rhizomucor strains were assessed.•Substantially increased biogas production was obtained from the pretreated substrates.•Penicillium aurantiogriseum was the most efficient strain for pretreatment and biogas.
Currently, Pb pollution has become a severe environmental problem and filamentous fungi hold a promising potential for the treatment of Pb-containing wastewater. The present study showed that the ...strain Pleurotus ostreatus ISS-1 had a strong ability to tolerate Pb at high concentration and reached a removal rate of 53.7% in liquid media. Pb was removed by extracellular biosorption, intracellular bioaccumulation by mycelia, or precipitation with extracellular oxalic acids. On the cellular level, Pb was mainly distributed in the cell wall, followed by vacuoles and organelles. Fourier transform infrared spectroscopy (FTIR) analysis indicated that hydroxyl, amides, carboxyl, and sulfhydryl groups provided binding sites for Pb. Furthermore, Pb was found on the cell surface in the form of PbS and PbCO3 through X-ray diffraction (XRD). Intracellular chelates such as thiol compounds and oxalic acid, as well as extracellular oxalic acid, might play an important role in the tolerance of Pb. In addition, isobaric tags for relative and absolute quantitation (iTRAQ) analysis showed that ATP-binding cassette (ABC) transporter, cytochrome P450, peroxisome, and the calcium signaling pathway might participate in both accumulation and detoxification of Pb. These results have successfully provided a basis for further developing Pb polluted water treatment technology by fungi.
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•Pleurotus ostreatus ISS-1 had a strong ability to tolerate and remove Pb.•Fungus could remove Pb through biosorption, bioaccumulation and precipitation.•Pb was mainly distributed in the cell wall, followed by vacuoles and organelles.•Cell wall could bind Pb by some special chemical groups in the form of PbS and PbCO3.•Intracellular and extracellular chelating agents participated in the tolerance of Pb.
•The CRISPR/Cas9 system was used for on-site point mutations in Aspergillus niger.•60-mer single stranded oligonucleotides were successfully used as repair templates.•Mutations resulted in increased ...production of carbohydrate active enzymes.•Constitutively active XlnR and GaaR improved saccharification efficiency in A. niger.
The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.
This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δsup.1-progesterone) (2) through microbiological transformation. For the role of catalysts, we used six strains of ...entomopathogenic filamentous fungi (Beauveria bassiana KCh J1.5, Beauveria caledonica KCh J3.3, Isaria fumosorosea KCh J2, Isaria farinosa KCh KW1.1, Isaria tenuipes MU35, and Metarhizium robertsii MU4). The substrate (2) was obtained by carrying out an enzymatic 1,2-dehydrogenation on an increased scale (3.5 g/L) using a recombinant cholest-4-en-3-one Δsup.1-dehydrogenase (AcmB) from Sterolibacterium denitrificans. All selected strains were characterized by the high biotransformation capacity for the used substrate. As a result of the biotransformation, six steroid derivatives were obtained: 11α-hydroxypregn-1,4-diene-3,20-dione (3), 6β,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6β-hydroxypregn-1,4-diene-3,11,20-trione (5), 6β,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6β,17β-dihydroxyandrost-1,4-diene-3-one (7), and 12β,17α-dihydroxypregn-1,4-diene-3,20-dione (8). The results show evident variability of the biotransformation process between strains of the tested biocatalysts from different species described as entomopathogenic filamentous fungi. The obtained products were tested in silico using cheminformatics tools for their pharmacokinetic and pharmacodynamic properties, proving their potentially high biological activities. This study showed that the obtained compounds may have applications as effective inhibitors of testosterone 17β-dehydrogenase. Most of the obtained products should, also with a high probability, find potential uses as androgen antagonists, a prostate as well as menopausal disorders treatment. They should also demonstrate immunosuppressive, erythropoiesis-stimulating, and anti-inflammatory properties.
•Mixtures of agro-industrial wastes improved SSF performing.•Solid state fermentation increased the antioxidant activity 2.3-fold.•Maximum antioxidant activity was achieved in 2 days of ...fermentation.•Brewery spent grain was the best substrate for lignocellulolytic enzymes production.
Agro-food industries face the challenge of re-using their wastes following the circular economy concept. The co-bioprocessing of wastes produced by different agro-food industries located in the same region can be a suitable strategy for the valorization of these wastes. This work evaluated the use of mixtures of wastes from wineries, olive mill and breweries to produce lignocellulolytic enzymes and antioxidant compounds by Aspergillus niger under solid-state fermentation (SSF). The SSF of a mixture of brewery spent grain with crude and exhausted olive pomace led to the highest values of enzymatic activity (xylanase, 837 U/g, cellulase, 87 U/g; β-glucosidase, 254 U/g), being higher than enzymes activities per mass of total substrates. The highest antioxidant activity was achieved after 1 day of fermentation, being 2.3-fold higher in comparison to the unfermented wastes. SSF allowed the co-management of agro-industrial wastes from different industries.
Progressive lung injury in Cystic Fibrosis (CF) patients can lead to chronic colonization with bacteria and fungi. Fungal colonization is obtained from the environment which necessitates locally ...performed epidemiology studies. We prospectively analyzed respiratory samples of CF patients during a 3-year period, using a uniform fungal culture protocol, focusing on filamentous fungi and azole resistance in Aspergillus fumigatus.
Over a 3-year period, all respiratory specimens collected from CF patients in 5 Dutch CF centers, were analyzed. Samples were inoculated onto the fungal culture media Sabouraud dextrose agar (SDA) and Medium B+. All fungal isolates were collected and identified in one centre, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting, rDNA PCR and ITS, calmodulin and β-tubulin sequencing. Azole resistance was assessed for all A. fumigatus using a qPCR assay followed by phenotypic confirmation.
Filamentous fungi were recovered from 699 patients from at least one respiratory sample, corresponding with 3787 cultured fungal species. A. fumigatus was cultured most often with a mean prevalence of 31.7%, followed by Penicillium species (12.6%), non-fumigatus Aspergillus species (5.6%), Scedosporium species (4.5%) and Exophiala dermatitidis and Cladosporium species (1.1% each). In total 107 different fungal species were identified, with 39 Penicillium species and 15 Aspergillus species. Azole resistance frequency in A. fumigatus was 7.1%, with TR34/L98H being the dominant resistance mechanism.
A vast diversity of filamentous fungi was demonstrated, dominated by Aspergillus and Penicillium species. We observed a mean azole resistance prevalence of 7.1% of A. fumigatus culture positive patients.
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