Search Results (14)

Phosphatidylethanolamine-binding proteins (PEBPs) play a crucial role in the growth and development of various organisms. Due to the low sequence similarity compared to plants, humans, and animals, the study of pebp genes in fungi has not received significant attention. The redifferentiation of fruiting bodies is exceedingly rare in fungal development. Hitherto, only a few studies have identified the Capebp2 gene as being associated with this phenomenon in Cyclocybe aegerita. Thus, exploring the role of pebp genes in fruiting body development is imperative. In the present study, four Capebp genes (Capebp1, Capebp3, Capebp4, and Capebp5) were cloned from the AC0007 strain of C. aegerita based on genome sequencing and gene prediction. The findings indicate that the pebp family, in C. aegerita, comprises a total of five genes. Moreover, the sequence similarity was low across the five CAPEBP protein sequences in C. aegerita, and only a few conserved sequences, such as HRY and RHF, were identical. Expression analyses revealed that, similarly to Capebp2, the four Capebp genes exhibit significantly higher expression levels in the fruiting bodies than in the mycelium. Furthermore, overexpressed and RNA interference Capebp1 or Capebp5 transformants were analyzed. The results demonstrate that overexpression of Capebp1 or Capebp5 could induce the regeneration of the lamella or fruiting body, whereas the knockdown of Capebp1 or Capebp5 could lead to the accelerated aging of fruiting bodies. These findings highlight a significant role of Capebp genes in the generation of C. aegerita fruiting bodies and provide a foundation for further exploration into their involvement in basidiomycete growth and development. Full article

The sclerotia of Wolfiporia hoelen (Fr.) Y.C. Dai & V. Papp is an important traditional Chinese medicine with diverse pharmacological properties. This study utilized a combination of PacBio Long-Read Sequencing, Illumina Short-Read Sequencing, and Hi-C Sequencing to generate a high-quality chromosome-level genome assembly of a W. hoelen strain Minling A5. There were 112 contigs in the genome, with 62.95 Mb in total length and 4.21 Mb in length for the contig N50. The average GC content was 51.89%. Based on Hi-C data, we corrected the CCS data and scaffolded them into 14 pseudo-chromosomes. The genome contained 44.37% repetitive sequences and 12,670 protein-coding genes, 86.53% (10,963) of which could be functionally annotated in at least one of the KOG, GO, Pfam, Swissprot, TrEMBL, NR, and KEGG databases. In addition, 240 transfer RNAs, 97 ribosomal RNAs, and 103 other non-coding RNAs were identified in the W. hoelen genome. A total of 755 pseudogenes were also identified, with an average length of 2665.51 bp. Further, there were 398, 100, 2837, 519, and 2068 genes annotated by CAZymes, TCDB, PHI, P450, and DFVF databases, respectively. One notable attribute of W. hoelen is its capacity to thrive in a substrate of fresh pine sawdust. Through an analysis of the growth on various pure wood sawdust culture media, we found that the growth of W. hoelen and Sparassis latifolia on pine sawdust was similar to that on broad-leaved wood sawdust, while the growth of Pleurotus ostreatus, P. eryngii, and Cyclocybe aegerita was slower than that on broad-leaved wood sawdust. By the functional annotation analysis of orthogroups in these five mushroom-forming fungi, it was determined that 645 orthogroups were specifically common in W. hoelen and S. latifolia. The genes in these specific orthogroups were significantly enriched in 12 pathways, including steroid biosynthesis, biosynthesis of antibiotics, and tyrosine metabolism. The high-quality genome and comparative genome analysis results significantly contribute to advancing our foundational knowledge of W. hoelen biology, while also offering valuable insights for the development of innovative biotechnological approaches aimed at enhancing the efficient and sustainable utilization of Pinus. Full article

Ageritin from poplar mushrooms is a specific endonuclease that hydrolyzes a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA, thereby blocking protein synthesis. Considering the possible biotechnological use of this enzyme, here we report its antifungal activity against virulent fungi affecting crops of economic interest. Our results show that ageritin (200 µg/plug; ~13.5 nmole) inhibits the growth of Botrytis cinerea (57%), Colletotrichum truncatum (42%), and Alternaria alternata (57%), when tested on potato dextrose agar plates. At the same time, no effect was observed against Trichoderma harzianum (a fungus promoting beneficial effects in plants). To verify whether the antifungal action of ageritin against B. cinerea and T. harzianum was due to ribosome damage, we tested ageritin in vitro on partially isolated B. cinerea and T. harzianum ribosomes. Interestingly, ageritin was able to release the Endo’s fragment from both tested fungal ribosomes. We therefore decided to test the antifungal effect of ageritin on B. cinerea and T. harzianum using a different growth condition (liquid medium). Differently from the result in solid medium, ageritin can inhibit both B. cinerea and T. harzianum fungal growth in liquid medium in a concentration-dependent manner up to 35.7% and 38.7%, respectively, at the highest concentration tested (~200 µg/mL; 12 µM), and the analysis of RNA isolated from ageritin-treated cells revealed the presence of Endo’s fragment, highlighting its ability to cross the fungal cell wall and reach the ribosomes. Overall, these data highlight that the efficacy of antifungal treatment to prevent or treat a potential fungal disease may depend not only on the fungal species but also on the conditions of toxin application. Full article

Phosphatidylethanolamine-binding protein (PEBP) is widely involved in various physiological behaviors, such as the transition from vegetative growth to reproductive growth in plants, tumorigenesis in the human, etc. However, few functional studies have examined pebp genes affecting the development of fungi. In this study, Capebp2 was cloned from Cyclocybe aegerita AC0007 strains based on the genome sequence and gene prediction, and the sequence alignment of CaPEBP2 with other PEBP proteins from other biological sources including plant, animal, fungi, and bacteria indicated that PEBP had low sequence similarity in fungi, whereas all protein sequences had some conserved motifs such as DPDAP and HRY. Expression analysis showed the transcription level of Capebp2 increased approximately 20-fold in fruiting bodies compared with mycelia. To uncover the function of Capebp2 in C. aegetita development, Capebp2 was cloned into a pATH vector driven by the actin promoter for obtaining overexpression transformant lines. Fruiting experiments showed the transformed strains overexpressing Capebp2 exhibited redifferentiation of the cap on their surface, including intact fruiting bodies or partial lamella during fruiting development stage, and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies. In summary, the sequence characterization of Capebp2, expression level during different development stages, and function on fruiting body development were documented in this study, and these findings provided a reference to study the role of pebp in the development process of basidiomycetes. Importantly, gene mining of pebp, function characterization, and the regulating pathways involved need to be uncovered in further studies. Full article

White button mushroom (Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes. In addition, bisporitin shows magnesium-dependent endonuclease activity and displays a similar far-UV CD spectrum as ageritin, the prototype of RL-Ps, isolated from Cyclocybe aegerita fruiting bodies. Interestingly, bisporitin is the first member of RL-Ps to have noticeably lower thermal stability (T_m = 48.59 ± 0.98 °C) compared to RL-Ps isolated in other mushrooms (T_m > 70 °C). Finally, this protein is only partially hydrolyzed in an in vitro digestive system and does not produce adverse growing effects on eukaryotic cell lines. This evidence paves the way for future investigations on possible bioactivities of this RL-P in the digestive system. Full article

The Black Poplar Mushroom Cyclocybe aegerita (syn. Agrocybe aegerita) is a white-rot fungus that naturally fruits from woody substrates, including buried wood. It is known for its substrate versatility and is equipped with a respective carbohydrate-active enzyme repertoire being intermediate between typical white-rot fungi and plant litter decomposers. Given relative nitrogen scarcity in wood, mobilization of nitrogen from surrounding litter is known as a way to meet nitrogen requirements for cellular homeostasis and reproduction of wood decay fungi. However, the effect of added nitrogen on vegetative and reproductive biomass has not yet been studied in a uniform minimalistic laboratory setup. For C. aegerita, such a growth and fruiting setup has been developed. In the present study, this white-rot fungus has been grown with and without additional β-adenosine, an organic nitrogen source present in plant litter. Elevated β-adenosine levels increased aerial mycelium weight by 30% (1 × β-adenosine) and 55% (10 × β-adenosine), reproductive biomass by 75% (1 × β-adenosine) and by 100% (10 × β-adenosine), number of primordia by 127% (10 × β-adenosine) and accelerated primordium formation by 1.6 days (10 × β-adenosine), compared to the control treatment. These findings imply that C. aegerita invests additional organic nitrogen resources into direct vegetative and reproductive biomass build-up at the same time. Colonization of niches with accessory nitrogen sources, like buried wood, which is near the plant litter layer, may thus provide an evolutionary fitness advantage. Globally anthropogenically altered nitrogen dynamics may affect hyphal-driven processes as well as fruit body-driven food webs. Full article

Unspecific peroxygenases (UPOs), the extracellular enzymes capable of oxygenating a potpourri of aliphatic and aromatic substrates with a peroxide as co-substrate, come out with a new reaction: carbon-chain shortening during the conversion of fatty acids with the well-known UPOs from Coprinopsis cinerea (rCciUPO) and Cyclocybe (Agrocybe) aegerita (AaeUPO). Although a pathway (Cα-oxidation) for shortening the hydrocarbon chain of saturated fatty acids has already been reported for the UPO from Marasmius rotula (MroUPO), it turned out that rCciUPO and AaeUPO shorten the chain length of both saturated and unsaturated fatty acids in a different way. Thus, the reaction sequence does not necessarily start at the Cα-carbon (adjacent to the carboxyl group), as in the case of MroUPO, but proceeds through the subterminal (ω-1 and ω-2) carbons of the chain via several oxygenations. This new type of shortening leads to the formation of a dicarboxylic fatty acid reduced in size by two carbon atoms in the first step, which can subsequently be further shortened, carbon by carbon, by the UPO Cα-oxidation mechanism. Full article

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine. Full article

Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, flavoring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H₂O₂ as cosubstrate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C_12:1 to C_20:1) after an initial optimization of several reaction parameters (cosolvent, cosubstrate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, notable differences were observed between them, with rCciUPO being responsible for the highest substrate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies. Full article

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L⁻¹ within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts. Full article

Cyclocybe parasitica is a wood-destroying parasitic edible mushroom growing on diverse broad-leafed trees in New Zealand and other Pacific areas. Recent molecular systematics of European Cyclocybe aegerita, a newly delimited Asian phylum and of related species, corroborated the distinction of the chiefly saprobic cultivated edible mushroom C. aegerita from C. parasitica. Here, we show that C. parasitica exhibits a morpho-physiological trait characteristic to its European cousin, i.e., monokaryotic fruiting sensu stricto (basidiome formation without mating). Monokaryotic fruiting structures formed by C. parasitica ICMP 11668-derived monokaryons were categorized into four phenotypes. One of them displays ulcer-like structures previously reported from bracket fungi. Histology of dikaryotic and monokaryotic C. parasitica fruiting structures revealed anatomical commonalities and differences between them, and towards monokaryotic fruiting structures of C. aegerita. Mating experiments with C. parasitica strains representative of each fruiting phenotype identified compatible sibling monokaryons. Given reports on hypothetically monokaryotic basidiome field populations of ‘C. aegerita sensu lato’, it seems worthwhile to prospectively investigate whether monokaryotic fruiting s.str. occurs in nature. Sampling from such populations including karyotyping, comparative -omics, and competition assays may help to answer this question and provide evidence whether this trait may confer competitive advantages to a species capable of it. Full article

Ageritin is a specific ribonuclease, extracted from the edible mushroom Cyclocybe aegerita (synonym Agrocybe aegerita), which cleaves a single phosphodiester bond located within the universally conserved alpha-sarcin loop (SRL) of 23–28S rRNAs. This cleavage leads to the inhibition of protein biosynthesis, followed by cellular death through apoptosis. The structural and enzymatic properties show that Ageritin is the prototype of a novel specific ribonucleases family named ‘ribotoxin-like proteins’, recently found in fruiting bodies of other edible basidiomycetes mushrooms (e.g., Ostreatin from Pleurotus ostreatus, Edulitins from Boletus edulis, and Gambositin from Calocybe gambosa). Although the putative role of this toxin, present in high amount in fruiting body (>2.5 mg per 100 g) of C. aegerita, is unknown, its antifungal and insecticidal actions strongly support a role in defense mechanisms. Thus, in this review, we focus on structural, biological, antipathogenic, and enzymatic characteristics of this ribotoxin-like protein. We also highlight its biological relevance and potential biotechnological applications in agriculture as a bio-pesticide and in biomedicine as a therapeutic and diagnostic agent. Full article

Fungi can run feedstock pretreatment to improve the hydrolysis and utilization of recalcitrant lignocellulose-rich biomass during anaerobic digestion (AD). In this study, three fungal strains (Coprinopsis cinerea MUT 6385, Cyclocybe aegerita MUT 5639, Cephalotrichum stemonitis MUT 6326) were inoculated in the non-sterile solid fraction of digestate, with the aim to further (re)use it as a feedstock for AD. The application of fungal pretreatments induced changes in the plant cell wall polymers, and different profiles were observed among strains. Significant increases (p < 0.05) in the cumulative biogas and methane yields with respect to the untreated control were observed. The most effective pretreatment was carried out for 20 days with C. stemonitis, causing the highest hemicellulose, lignin, and cellulose reduction (59.3%, 9.6%, and 8.2%, respectively); the cumulative biogas and methane production showed a 182% and 214% increase, respectively, compared to the untreated control. The increase in AD yields was ascribable both to the addition of fungal biomass, which acted as an organic feedstock, and to the lignocellulose transformation due to fungal activity during pretreatments. The developed technologies have the potential to enhance the anaerobic degradability of solid digestate and untap its biogas potential for a further digestion step, thus allowing an improvement in the environmental and economic sustainability of the AD process and the better management of its by-products. Full article

The current management practice of digestate from biogas plants involves its use for land application as a fertilizer. Nevertheless, the inadequate handling of digestate may cause environmental risks due to losses of ammonia, methane and nitrous oxide. Therefore, the key goals of digestate management are to maximize its value by developing new digestate products, reducing its dependency on soil application and the consequent air pollution. The high nitrogen and lignin content in solid digestate make it a suitable substrate for edible and medicinal mushroom cultivation. To this aim, the mycelial growth rate and degradation capacity of the lignocellulosic component from corn silage digestate, undigested wheat straw and their mixture were investigated on Cyclocybe aegerita, Coprinus comatus, Morchella importuna, Pleurotus cornucopiae and Pleurotus ostreatus. The structural modification of the substrates was performed by using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Preliminary in vitro results demonstrated the ability of P. ostreatus, P. cornucopiae and M. importuna to grow and decay hemicellulose and lignin of digestate. Cultivation trials were carried out on C. aegerita, P. cornucopiae and P. ostreatus. Pleurotus ostreatus showed the highest biological efficiency and fruiting body production in the presence of the digestate; moreover, P. ostreatus and P. cornucopiae were able to degrade the lignin. These results provide attractive perspectives both for more sustainable digestate management and for the improvement of mushroom cultivation efficiency. Full article