Nutritional profiling and in silico analysis of pharmacological activities from local rice Pulu Mandoti fermented with Pleurotus spp.

Pulu Mandoti, a local red rice (Oryza sativa L.) variety popular among Sulawesi residents, has gained recognition for its perceived health benefits, especially as a preferred dietary option for individuals with diabetes or those seeking to prevent obesity. Given the increasing consumption of mushrooms, particularly Pleurotus species, renowned for their nutritional and medicinal attributes, this study delves into the transformative effects of Pleurotus spp. fermentation on Pulu Mandoti, the indigenous rice variety. Proximate analysis disclosed elevated dry matter (91.99 ± 0.61%), crude protein (8.55 ± 0.15%), and crude fat (1.34 ± 0.05%) in Pleurotus cystidiosus fermentation compared to Pleurotus ostreatus and Pleurotus djamor. Concurrently, antioxidant and antidiabetic activities were notably improved in all Pleurotus fermentations. Pulu Mandoti fermented with P. cystidiosus outperformed other treatments, aligning with molecular docking results pinpointing 11-Eicosenoic acid, methyl ester, and butylated hydroxytoluene as optimal interactors with antioxidant receptors 5O0x and 2CKJ. Butylated hydroxytoluene demonstrated interactions with the antidiabetic receptor 2QV4, along with 9-Octadecenoic acid, methyl ester. These compounds, previously unreported in Pleurotus, displayed promising attributes as antioxidants and antidiabetic agents. Furthermore, the investigation delved into the fatty acid profiles, emphasizing the diverse range of potential bioactive compounds in fermented Pulu Mandoti. The findings of this research present a potential functional food rich in natural antioxidants and antidiabetic compounds, highlighting the yet undiscovered capabilities of Pleurotus spp. fermentation in augmenting the nutritional composition and bioactivity of indigenous rice varieties, specifically Pulu Mandoti.

Persistent polycyclic aromatic hydrocarbons removal from sewage sludge-amended soil through phytoremediation combined with solid-state ligninolytic fungal cultures.

Polycyclic aromatic hydrocarbons (PAHs) are widely present in the environment, causing increasing concern because of their impact on soil health, food safety and potential health risks. Four bioremediation strategies were examined to assess the dissipation of PAHs in agricultural soil amended with sewage sludge over a period of 120 days: soil-sludge natural attenuation (SS); phytoremediation using maize (Zea mays L.) (PSS); mycoremediation (MR) separately using three white-rot fungi (Pleurotus ostreatus, Phanerochaete chrysosporium and Irpex lacteus); and plant-assisted mycoremediation (PMR) using a combination of maize and fungi. In the time frame of the experiment, mycoremediation using P. chrysosporium (MR-PH) exhibited a significantly higher (P < 0.05) degradation of total PAHs compared to the SS and PSS treatments, achieving a degradation rate of 52 %. Both the SS and PSS treatments demonstrated a lower degradation rate of total PAHs, with removal rates of 18 % and 32 %, respectively. The PMR treatments showed the highest removal rates of total PAHs at the end of the study, with degradation rates of 48-60 %. In the shoots of maize, only low- and medium-molecular-weight PAHs were found in both the PSS and PMR treatments. The calculated translocation and bioconversion factors always showed values < 1. The analysed enzymatic activities were higher in the PMR treatments compared to other treatments, which can be positively related to the higher degradation of PAHs in the soil.

Degradation of edible mushroom waste by Hermetia illucens L. and consequent adaptation of its gut microbiota.

The edible fungus industry is one of the pillar industries in the Yunnan-Guizhou Plateau, China. The expansion of the planting scale has led to the release of various mushroom residues, such as mushroom feet, and other wastes, which are not treated adequately, resulting in environmental pollution. This study investigated the ability of black soldier fly (Hermetia illucens L.) larvae (BSFL) to degrade mushroom waste. Moreover, this study analyzed changes in the intestinal bacterial community and gene expression of BSFL after feeding on mushroom waste. Under identical feeding conditions, the remaining amount of mushroom waste in Pleurotus ostreatus treatment group was reduced by 18.66%, whereas that in Flammulina velutipes treatment group was increased by 31.08%. Regarding gut microbial diversity, compared with wheat bran-treated control group, Dysgonomonas, Providencia, Enterococcus, Pseudochrobactrum, Actinomyces, Morganella, Ochrobactrum, Raoultella, and Ignatzschineria were the most abundant bacteria in the midgut of BSFL in F. velutipes treatment group. Furthermore, Dysgonomonas, Campylobacter, Providencia, Ignatzschineria, Actinomyces, Enterococcus, Morganella, Raoultella, and Pseudochrobactrum were the most abundant bacteria in the midgut of BSFL in P. ostreatus treatment group. Compared with wheat bran-treated control group, 501 upregulated and 285 downregulated genes were identified in F. velutipes treatment group, whereas 211 upregulated and 43 downregulated genes were identified in P. ostreatus treatment group. Using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses, we identified 14 differentially expressed genes (DEGs) related to amino sugar and nucleotide sugar metabolism in F. velutipes treatment group, followed by 12 DEGs related to protein digestion and absorption. Moreover, in P. ostreatus treatment group, two DEGs were detected for fructose and mannose metabolism, and two were noted for fatty acid metabolism. These results indicate that feeding on edible mushroom waste can alter the intestinal microbial community structure of BSFL; moreover, the larval intestine can generate a corresponding feedback. These changes contribute to the degradation of edible mushroom waste by BSFL and provide a reference for treating edible mushroom waste using BSFL.

A Novel O- and S-Methyltransferase from Pleurotus sapidus Is Involved in Flavor Formation.

Increasing consumer aversion to non-natural flavoring substances is prompting a heightened interest in enzymatic processes for flavor production. This includes methylation reactions, which are often performed by using hazardous chemicals. By correlation of aroma profile data and transcriptomic analysis, a novel O-methyltransferase (OMT) catalyzing a respective reaction within the formation of p-anisaldehyde was identified in the mushroom Pleurotus sapidus. Heterologous expression in E. coli followed by purification allowed for further characterization of the enzyme. Besides p-hydroxybenzaldehyde, the proposed precursor of p-anisaldehyde, the enzyme catalyzed the methylation of further hydroxylated aromatic compounds at the meta- and para-position. The Km values determined for p-hydroxybenzaldehyde and S-adenosyl-l-methionine were 80 and 107 µM, respectively. Surprisingly, the studied enzyme enabled the transmethylation of thiol-nucleophiles, as indicated by the formation of 2-methyl-3-(methylthio)furan from 2-methyl-3-furanthiol. Moreover, the enzyme was crystallized at a resolution of 2.0 Å, representing the first published crystal structure of a basidiomycetous OMT.

Effect of oil contaminants on antioxidant responses and antioxidant properties of Pleurotus florida (P. Kumm).

This research investigated the antioxidant responses of Pleurotus florida at different concentrations of gas oil [0% (control), 2.5%, 5%, and 10% (v:v)] for 30 days. The activities of superoxide dismutase and catalase enzymes decreased in responses to the gas oil presence by an average of 83% and 49%, respectively. In contrast, the activities of the ascorbate peroxidase and glutathione peroxidase enzymes displayed an upward trend in the groups cultured in oil-contaminated media. The gas oil contaminant increased total phenol and flavonoid accumulation, reflecting the variation in secondary metabolism. According to the 1,2-diphenyl-2-picrylhydrazyl radical scavenging, the 2.5% gas oil treatment resulted in the highest antioxidant activity (48 µg mL-1). The highest scavenging activity of nitric oxide radicals (IC50 = 272 µg mL-1) was observed in the treatment with the highest gas oil concentration (10%). Also, this treatment showed an excellent ability to chelate Fe+2 ions (IC50 = 205 µg mL-1). The IC50 values of methanolic extract for nitric oxide scavenging activity and metal chelating ability were significantly reduced by increasing gas oil concentration in the treatments. With increasing the gas oil concentration, malondialdehyde content as a criterion measure of lipid peroxidation level showed significant reduction. These results show that P. florida is resistant to and a compatible mushroom with oil pollutants. Also, the activity of glutathione peroxidase and the ascorbate-glutathione cycle detoxify nitric oxide radicals and products of reactive oxygen species-induced lipid peroxidation in the gas oil treatments.

Biodegradation of malachite green by Pleurotus eryngii: a study on decolorization, mechanism, toxicity, and enzyme.

The purpose of this study was to investigate the biodegradation of malachite green (MG) by Pleurotus eryngii via decolorization. This study also explored the possible mechanisms and toxicity. The results indicated that this fungus exhibited strong decolorizing potential. MG degradation based on UPLC-TOF-Triple-MS analysis revealed the formation of intermediates such as 4-(dimethylamino)benzophenone, 4-(methylamino)benzophenone, and 4-(dimethylamino)phenol. Furthermore, a significant reduction in the toxicity of the degradation products was observed using the zebrafish animal model. A newly discovered dye-decolorizing peroxidase (DyP-PE) from P. eryngii was amplified, cloned, and expressed. The purified 56.4 kDa DyP-PE strongly decolorized MG, suggesting potentially application in the bioremediation of MG pollution. Thus, the DyP-PE derived from P. eryngii may contribute to the degradation of MG.

Pleurotus ostreatus polysaccharide-mediated modulation of skin damage caused by microcystin-LR in tadpoles.

In this study, we aimed to evaluate the effect of dietary supplementation with edible mushroom (Pleurotus ostreatus)-derived polysaccharides on microcystin leucine-arginine (MC-LR)-induced skin damage in Pelophylax nigromaculatus tadpoles. Tadpoles were exposed to 1 µg/L daily MC-LR, with or without 5.0 g/kg of dietary P. ostreatus polysaccharides, for 30 days. P. ostreatus polysaccharide supplementation significantly increased the dermal collagen fibrils, increased tight junction protein gene expression, decreased the amount of MC-LR accumulation in skin tissues, attenuated oxidative stress, downregulated apoptosis-associated gene transcription, decreased eosinophil numbers, and downregulated transcription of inflammation-related genes (e.g. TLR4, NF-κB, and TNF-α). The composition of the skin commensal microbiota of MC-LR-exposed tadpoles supplemented with P. ostreatus polysaccharides was similar to that of the no-treatment control group. Lipopolysaccharide (LPS) content was positively correlated with the abundance of Gram-negative bacteria, including Chryseobacterium and Thauera. Therefore, P. ostreatus polysaccharides may alleviate MC-LR-induced skin barrier damage in tadpoles in two ways: 1) attenuation of oxidative stress-mediated apoptosis mediated by increased glutathione (GSH) content and total superoxide dismutase activity; and 2) alteration of the skin commensal microbiota composition to attenuate the LPS/Toll-like receptor 4 inflammatory pathway response. Furthermore, P. ostreatus polysaccharides may increase skin GSH synthesis by promoting glycine production via the gut microbiota and may restore the MC-LR-damaged skin resistance to pathogenic bacteria by increasing antimicrobial peptide transcripts and lysozyme activity. This study highlights for the first time the potential application of P. ostreatus polysaccharides, an ecologically active substance, in mitigating the skin damage induced by MC-LR exposure, and may provide new insights for its further development in aquaculture.

Copper increases laccase gene transcription and extracellular laccase activity in Pleurotus eryngii KS004.

The white-rot fungus Pleurotus eryngii secretes various laccases involved in the degradation of a wide range of chemical compounds. Since the laccase production is relatively low in fungi, many efforts have been focused on finding ways to increase it, so in this study, we investigated the effect of copper on the transcription of the pel3 laccase gene and extracellular laccase activity. The results indicate that adding 0.5 to 2 mM copper to liquid cultures of P. eryngii KS004 increased both pel3 gene transcription and extracellular laccase activity in a concentration-dependent manner. The most significant increase in enzyme activity occurred at 1 mM Cu2+, where the peak activity was 4.6 times higher than in control flasks. Copper also induced the transcription of the laccase gene pel3. The addition of 1.5 and 2 mM Cu2+ to fungal culture media elevated pel3 transcript levels to more than 13-fold, although the rate of induction slowed down at Cu2+ concentrations higher than 1.5 mM. Our findings suggest that copper acts as an inducer in the regulation of laccase gene expression in P. eryngii KS004. Despite its inhibitory effect on fungal growth, supplementing cultures with copper can lead to an increased extracellular laccase production in P. eryngii.

Oat protein isolate-Pleurotus ostreatus ß-glucan conjugate nanoparticles bound to ß-carotene effectively alleviate immunosuppression by regulating gut microbiota.

Individuals with immune disorders cannot establish an adequate defense to pathogens, leading to gut microbiota dysbiosis. ß-Carotene can regulate immune response, but its bioavailability in vivo is very low. Herein, we developed a glycosylated oat protein-based nanoparticle to improve the application of ß-carotene for mitigating cyclophosphamide-induced immunosuppression and gut microbiota imbalance in mice. The results showed that the nanoparticles facilitated a conversion of ß-carotene to retinol or retinyl palmitate into the systemic circulation, leading to an increased bioavailability of ß-carotene. The encapsulated ß-carotene bolstered humoral immunity by elevating immunoglobulin levels, augmenting splenic T lymphocyte subpopulations, and increasing splenic cytokine concentrations in immunosuppressed mice. This effect was accompanied by the alleviation of pathological features observed in the spleen. In addition, the encapsulated ß-carotene restored the abnormal gut microbiota associated with immunosuppression, including Erysipelotrichaceae, Akkermansia, Bifidobacterium and Roseburia. This study suggested that nanoparticles loaded with ß-carotene have great potential for therapeutic intervention in human immune disorders by specifically targeting the gut microbiota.

Interactions between intestinal microbial fermentation products of Pleurotus eryngii polysaccharide with gut mucus.

Recently, Pleurotus eryngii (P. eryngii) polysaccharide (PEP) has received a lot of attention from many researchers as the primary active substance. The PEP influences the gut microbiota in several ways, including the interaction of fermentation products with the intestinal mucus layer (IML) and intestinal epithelial cells. Herein, we characterized interactions between the IML and PEP after degradation by the gut microbes. Our results showed that fermented P. eryngii polysaccharide (FPEP) can interact with intestinal mucus (IM), and this interaction can reduce the degree of molecular aggregation of polysaccharides. At the same time, the fermentation time of FPEP also affects the interaction between the two. SEM showed that the FPEP solution tended to aggregate into larger particles, while with the addition of IM, the FPEP molecules were dispersed. Particle size measurements unveil substantial differences in the fermented polysaccharides' particle size between the group with supplementary IM (0 hours of fermentation: 485.1 ± 11.3 nm) and the group without IM (0 hours of fermentation: 989.33 ± 21.3 nm). Remarkably, within the group with added IM, the particle size reached its maximum at 24 hours of fermentation (585.87 ± 42.83 nm). Additionally, turbidity assessments demonstrate that, during the 12-hour interaction period, the 24-hour fermented polysaccharides consistently exhibit the highest OD values, ranging between 0.57 and 0.61. This work investigates the interaction between FPEP and IM, predicting the adhesion of polysaccharides to IM. Meanwhile, this provides a theoretical basis for further studies on the absorption and transport pathways of PEP and provides a novel research viewpoint on intestinal digestion and absorption.

Effect of selenium and zinc biofortification on the biochemical parameters of Pleurotus spp. under submerged and solid-state fermentation.

BACKGROUND: Pleurotus has a remarkable nutritional and nutraceutical profile due to mineral mobilization and accumulation abilities from the substrate. The present study aimed to observe the effect of single and dual supplementations Se and Zn on biochemical parameters of P. florida, P. sajor caju and P. djamor. Also, the bioaccumulation of the trace elements in fortified mushrooms was estimated. METHODS: Biomass production and radial growth rate were observed on Se and Zn supplemented broth and agar based medium. Furthermore, the influence of Se and Zn supplementation was recorded on the fruit body yield. The colorimetric assays were employed to estimate total soluble protein, total phenol and total flavonoid contents. The antioxidant activity was assayed as DPPH radical scavenging test. While, ICP-AES was performed to estimate the variation in the Zn and Se content of the fruit bodies. RESULTS: The Se supplementation at low rate resulted in improvement in the radial growth rate and biomass production for P. sajor caju. For solid-state fermentation, a better yield was obtained with inorganic salt supplementation in comparison to organically enriched Se straw. The maximum total soluble protein content and total flavonoid content were observed in fruit bodies of P. sajor caju at 4 mg L -1 of Se and Se-Zn respectively. Pleurotus djamor exhibited the highest total phenolic content on Zn supplementation (10 mg L-1). Improved antioxidant potential was recorded with dual supplementations. Salt supplementations caused shrinkage, distortion of the fungal hyphae, and decreased basidiospores with significant amelioration in elemental composition in fortified mushrooms. CONCLUSION: The inorganic salt supplementation increased the biochemical potential of Pleurotus spp. in comparison to organically enriched substrate which could further be used for the development of dietary supplements.

Copper removal from aqueous solutions by white rot fungus Pleurotus ostreatus GEMB-PO1 and its potential in co-remediation of copper and organic pollutants.

Addressing the environmental contamination from heavy metals and organic pollutants remains a critical challenge. This study explored the resilience and removal potential of Pleurotus ostreatus GEMB-PO1 for copper. P. ostreatus GEMB-PO1 showed significant tolerance, withstanding copper concentrations up to 2 mM. Its copper removal efficiency ranged from 64.56 % at 0.5 mM to 22.90 % at 8 mM. Transcriptomic insights into its response to copper revealed a marked upregulation in xenobiotic degradation-related enzymes, such as laccase and type II peroxidases. Building on these findings, a co-remediation system using P. ostreatus GEMB-PO1 was developed to remove both copper and organic pollutants. While this approach significantly enhanced the degradation efficiency of organic contaminants, it concurrently exhibited a diminished efficacy in copper removal within the composite system. This study underscores the potential of P. ostreatus GEMB-PO1 in environmental remediation. Nevertheless, further investigation is required to optimize the simultaneous removal of organic pollutants and copper.

Thermosensitive polymer-assisted extraction and purification of fungal laccase from citrus pulp wash effluent.

BACKGROUND: This study explores the use of liquid-liquid extraction with thermosensitive polymers for producing laccase (Lac) from Pleurotus sajor-caju. This process leverages liquid waste from the citrus industry, specifically pulp wash. The research delves into extractive fermentation and thermoseparation, both processes being facilitated by a polymer exhibiting a lower critical solution temperature transition. RESULTS: Key factors considered include the choice of polymer, its concentration, pH, separation temperature, and the behavior of the polymer-rich phase post-extractive fermentation concerning the lower critical solution temperature. Notably, under conditions of 45% by weight of Pluronic L-61 and pH 5.0 at 25 °C, the Lac resulted in an enhancement in the purification factor of 28.4-fold, compared with the Lac obtained directly from the fermentation process on the eighth day. There was an 83.6% recovery of the Lac enzyme in the bottom phase of the system. Additionally, the unique properties of Pluronic L-61, which can induce phase separation and also allow for thermoseparation, led to a secondary fraction (aqueous solution) of Lac with purification factor of 2.1 ± 0.1-fold (at 32 ± 0.9 °C and 30 ± 0.3 min without stirring) from the polymeric phase (top phase). Fourier-transform infrared analysis validated the separation data, particularly highlighting the α-helix content in the amide I region (1600-1700 cm-1 ). CONCLUSION: In summary, the insights from this study pave the way for broader industrial applications of these techniques, underscoring benefits like streamlined process integration, heightened selectivity, and superior separation efficacy. © 2023 Society of Chemical Industry.

Novel techniques for the mass production of nutritionally improved, fungus-treated lignocellulosic biomass for ruminant nutrition.

BACKGROUND: Laboratory-scale experiments have shown that treatment with selective lignin-degrading white-rot fungi improves the nutritional value and ruminal degradability of lignocellulosic biomass (LCB). However, the lack of effective field-applicable pasteurization methods has long been recognized as a major obstacle for scaling up the technique for fungal treatment of large quantities of LCB for animal feeding. In this study, wheat straw (an LCB substrate) was subjected to four field-applicable pasteurization methods - hot-water, formaldehyde fumigation, steam, and hydrated lime - and cultured with Pleurotus ostreatus grain spawn for 10, 20, and 30 days under solid-state fermentation. Samples of untreated, pasteurized but non-inoculated and fungus-treated straws were analyzed for chemical composition, aflatoxin B1 (AFB1 ), and in vitro dry matter digestibility (IVDMD), in vitro total gas (IVGP), methane (CH4 ), and volatile fatty acid (VFA) production. RESULTS: During the 30-day fungal treatment, steam and lime pasteurized straws had the greatest loss of lignin, resulting in marked improvements in crude protein (CP), IVDMD, IVGP, and total VFAs. Irrespective of the pasteurization method, the increase in IVDMD during fungal treatment was linearly (R2 = 0.77-0.92) related to lignin-loss in the substrate during fungal treatment. The CH4 production of the fungus-treated straw was not affected by the pasteurization methods. Aflatoxin B1 was within the safe level (<5 µg kg-1 ) in all pasteurized, fungus treated straws. CONCLUSION: Steam and lime were promising field-applicable pasteurization techniques to produce nutritionally improved fungus-treated wheat straw to feed ruminants. Lime pasteurization was more economical and did not require expensive energy inputs. © 2023 Society of Chemical Industry.

Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus.

Pleurotus ostreatus is a white-rot fungus that can degrade lignin in a preferential manner using a variety of extracellular enzymes, including manganese and versatile peroxidases (encoded by the vp1-3 and mnp1-6 genes, respectively). This fungus also secretes a family of structurally related small secreted proteins (SSPs) encoded by the ssp1-6 genes. Using RNA sequencing (RNA-seq), we determined that ssp4 and ssp6 are the predominant members of this gene family that were expressed by P. ostreatus during the first three weeks of growth on wheat straw. Downregulation of ssp4 in a strain harboring an ssp RNAi construct (KDssp1) was then confirmed, which, along with an increase in ssp6 transcript levels, coincided with reduced lignin degradation and the downregulation of vp2 and mnp1. In contrast, we observed an increase in the expression of genes related to pectin and side-chain hemicellulose degradation, which was accompanied by an increase in extracellular pectin-degrading capacity. Genome-wide comparisons between the KDssp1 and the wild-type strains demonstrated that ssp silencing conferred accumulated changes in gene expression at the advanced cultivation stages in an adaptive rather than an inductive mode of transcriptional response. Based on co-expression networking, crucial gene modules were identified and linked to the ssp knockdown genotype at different cultivation times. Based on these data, as well as previous studies, we propose that P. ostreatus SSPs have potential roles in modulating the lignocellulolytic and pectinolytic systems, as well as a variety of fundamental biological processes related to fungal growth and development.

PriA is involved in Pleurotus ostreatus development and defense against Pseudomonas tolaasii.

Pleurotus ostreatus is a crucial commercial mushroom widely cultivated for diverse uses. Scientists have worked on breeding disease-resistant and high-yielding varieties to secure food supply. Studies on the molecular genetic mechanism of growth and development can provide valuable information to facilitate crop breeding programs by genetic engineering. Aegerolysins are pore-forming proteins widely distributed in both prokaryotes and eukaryotes, which are reported to have haemolytic activity and be involved in the early stages of fructification. The present study aimed to explore biological function of a differential expressed aegerolysin gene PriA in P. ostreatus. The expression level of PriA gene was higher in primordium and fruiting body than that in mycelium. The PriA expression in overexpression (OE) and RNAi interference (RNAi) strains was detected by qRT-PCR. The RNAi strains grew at slightly slower rates and advanced producing yellow pigments than the wild type, while OE strains showed no prominent phenotypic characteristics. Furthermore, Pseudomonas tolaasii infection assays showed that the PriA OE strains could enhance mycelia and caps resistance to P. tolaasii. These data demonstrate PriA from P. ostreatus play an essential role in mycelial development and increase antagonism against P. tolaasii. Our study provides some reference information on interactions between edible fungi and pathogenic bacteria and offers a new resistance-conferring gene for breeding.

Visualizing organelles with recombinant fluorescent proteins in the white-rot fungus Pleurotus ostreatus.

White-rot fungi secrete numerous enzymes involved in lignocellulose degradation. However, the secretory mechanisms or pathways, including protein synthesis, folding, modification, and traffic, have not been well studied. In the first place, few experimental tools for molecular cell biological studies have been developed. As the first step toward investigating the mechanisms underlying protein secretion, this study visualized organelles and transport vesicles involved in secretory mechanisms with fluorescent proteins in living cells of the white-rot fungus Pleurotus ostreatus (agaricomycete). To this end, each plasmid containing the expression cassette for fluorescent protein [enhanced green fluorescent protein (EGFP) or mCherry] fused with each protein that may be localized in the endoplasmic reticulum (ER), Golgi, or secretory vesicles (SVs) was introduced into P. ostreatus strain PC9. Fluorescent microscopic analyses of the obtained hygromycin-resistant transformants suggested that Sec13-EGFP and Sec24-EGFP visualize the ER; Sec24-EGFP, mCherry-Sed5, and mCherry-Rer1 visualize the compartment likely corresponding to early Golgi and/or the ER-Golgi intermediate compartment; EGFP/mCherry-pleckstrin homology (PH) visualizes possible late Golgi; and EGFP-Seg1 and mCherry-Rab11 visualize SVs. This study successfully visualized mitochondria and nuclei, thus providing useful tools for future molecular cell biological studies on lignocellulose degradation by P. ostreatus. Furthermore, some differences in the Golgi compartment or apparatus and the ER-Golgi intermediate of P. ostreatus compared to other fungi were also suggested.

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

Evaluation of copper-tolerant fungi isolated from Sarcheshmeh copper mine of Iran.

Mycoremediation, a subset of bioremediation, is considered an advanced method to eliminate environmental contaminations. To identify tolerant fungi to copper contamination and study the related gene expression, sampling was carried out from the soil of "Sarcheshmeh Copper Mine," which is one of the biggest open-cast copper mines in the world. A total of 71 fungal isolates were obtained and purified. Afterward, the inhibitory effect of different concentrations (1000, 1500, 3500, 4000, and 5500 ppm) of copper sulfate on mycelial growth was evaluated. Results indicated that only 5500 ppm of copper sulfate inhibited fungal growth compared to the control. Based on the bioassay experiments, three isolates including S3-1, S3-21, and S1-7, which were able to grow on solid and broth medium containing 5500 ppm of copper sulfate at different pH conditions, were selected and identified using molecular approaches. Also, laccase and metallothionein gene expression has been assessed in these isolates. According to the molecular identification using ITS1-5.8S- ITS2 region, isolates S3-1 and S1-7 were identified as Pleurotus eryngii, and isolate S3-21 belonged to the genus Sarocladium. In addition, P. eryngii showed laccase gene expression reduction after 8 days of exposure to copper sulfate. While in the genus Sarocladium, it increased (almost 2 times) from 6 to 8 days. Besides, metallothionein gene expression has increased from 6 to 8 days of copper sulfate treatment compared to the control which reveals its role in copper tolerance of all studied isolates. In this study, Pleurotus eryngii and Sarocladium sp. are introduced as heavy metal tolerant fungi and the related gene expression to copper tolerance was studied for the first time in Iran.

Use of oyster mushrooms (Pleurotus ostreatus) for increased circularity and valorization of rapeseed residues.

In Europe, rapeseed is a common oilseed crop, resulting in the production of 20 million tons of rapeseed press cake yearly. This press cake can be further upcycled and a protein fraction can be extracted for food purposes, leaving de-proteinized fiber-rich residues. This study examined the use of these residues in the production of oyster mushrooms (Pleurotus ostreatus) and of the spent substrate as feed, since mushroom cultivation may improve the feed properties of substrate. In terms of mushroom production, the addition of rapeseed press residues was beneficial, giving significantly higher biological efficiency (BE = 93.1 ± 11.0%) compared with the control, sugar beet pulp substrate (70.0 ± 6.6%). This increase in productivity can most likely be explained by higher energy content in the substrate supplemented with lipid-rich rapeseed residues. Despite differences in BE between the substrates, high similarity was observed in lipid composition of the fruiting bodies (lipid profile dominated by linoleic acid (18:2), palmitic acid (16:0), and oleic acid (18:1)), and in protein and moisture content. After mushroom harvest, approximately 70% of the initial dry weight of both substrates remained as a possible feed source. Both substrates had significantly lower levels of carbohydrates and unchanged neutral detergent fiber content after mushroom harvest, and both gave lower in vitro digestibility, total gas production, and methane production. However, protein concentration differed between the substrates, with the highest concentration (15.8% of dry weight) found in spent substrate containing rapeseed press residues. The result of the present study suggests that the de-proteinized rapeseed press residue is a resource well-suited for use in the production of mushrooms and feed.