Fungi anaesthesia.

Electrical activity of fungus Pleurotus ostreatus is characterised by slow (h) irregular waves of baseline potential drift and fast (min) action potential likes spikes of the electrical potential. An exposure of the myceliated substrate to a chloroform vapour lead to several fold decrease of the baseline potential waves and increase of their duration. The chloroform vapour also causes either complete cessation of spiking activity or substantial reduction of the spiking frequency. Removal of the chloroform vapour from the growth containers leads to a gradual restoration of the mycelium electrical activity.

Mycoremediation of oil contaminant by Pleurotus florida (P.Kumm) in liquid culture.

This study investigated the potential functions of Pleurotus florida (an edible mushroom) in the biodegradation of gas oil at concentrations of 0 (control), 2.5, 5, and 10% (V: V) for 30 days. The gas oil increased dry weight and protein concentration in all treatments (by an average of 19.5 and 108%, respectively). Moreover, the pH, surface tension (ST), and interfacial tension (IFT) were reduced by the mushroom supplementation. The lowest surface tension (31.9 mN m-1) and the highest biosurfactant production belonged to the 10% gas oil treatment (0.845 ± 0.03 mg mL-1). The results demonstrated that the adsorption isotherm agreed well with the Langmuir isotherm. The maximum Langmuir adsorption capacity was calculated at 0.743 mg g-1 wet biomass of P. florida. The fungal supplementation efficiently remedied the total petroleum hydrocarbons (TPHs) by an average of 55% after 30 days. Gas chromatography (GC) analysis revealed that P. florida effectively detoxified C13-C28 hydrocarbons, Pristane, and Phytane, implying its high mycoremediation function. The toxicity test showed that mycoremediation increased the germination by an average of 35.82% ± 8.89 after 30 days. Laccase activity increased significantly with increasing gas oil concentration in the treatments. The maximum laccase activity was obtained in the 10% gas oil treatment (142.25 ± 0.72 U L-1). The presence of pollutants was also associated with induction in the tyrosinase activity when compared to the control. These results underline the high mycoremediation capacity of P. florida through the involvement of biosurfactants, laccase, and tyrosinase.

Effects of exogenous ascorbic acid on the mycelia growth and primordia formation of Pleurotus ostreatus.

Primordia formation is the first and most critical step in the development of fruiting bodies of edible fungi. In this study, the effects of exogenous ascorbic acid (ASA) on the Pleurotus ostreatus mycelia growth and primordia formation were researched and the results showed that the growth rate of P. ostreatus mycelia was accelerated and the time of primordia formation was advanced. The protein content and ascorbate oxidase (AAO) activity analysis showed that with the increase of ASA concentration, the protein content of mycelia first decreased and then increased, and in a certain concentration range, exogenous ASA could significantly promote the activity of AAO. Further expression analysis of the development regulating genes (Pofst3 and Pofst4) as well as blue light receptor coding genes (PoWC-1 and PoWC-2) showed the expression levels of those four genes all changed after the exogenous ASA addition, which indicated that the expression changes of PoWC-1 and PoWC-2, two key genes in the light morphogenesis, might affect the expression levels of development regulating genes Pofst3 and Pofst4, so as to lead to the formation of primordia in advance.

Lactic acid accumulation under heat stress related to accelerated glycolysis and mitochondrial dysfunction inhibits the mycelial growth of Pleurotus ostreatus.

High temperature is a major threat to Pleurotus ostreatus cultivation. In this study, a potential mechanism by which P. ostreatus mycelia growth is inhibited under heat stress was explored. Lactate, as a microbial fermentation product, was found unexpectedly in the mycelia of P. ostreatus under heat stress, and the time-dependent accumulation and corresponding inhibitory effect of lactate on mycelial growth was further confirmed. The addition of a glycolysis inhibitor, 2-deoxy-D-glucose (2DG), reduced the lactate content in mycelia and slightly restored mycelial growth under high-temperature conditions, which indicated the accumulation of lactate can be inhibited by glycolysis inhibition. Further data revealed mitochondrial dysfunction under high-temperature conditions, with evidence of decreased oxygen consumption and adenosine triphosphate (ATP) synthesis and increased reactive oxygen species (ROS). The removal of ROS with ascorbic acid decreased the lactate content, and mycelial growth recovered to a certain extent, indicating lactate accumulation could be affected by the mitochondrial ROS. Moreover, metabolic data showed that glycolysis and the tricarboxylic acid cycle were enhanced. This study reported the accumulation of lactate in P. ostreatus mycelia under heat stress and the inhibitory effect of lactate on the growth of mycelia, which might provide further insights into the stress response mechanism of edible fungi. Key Points • Lactate can accumulate in Pleurotus ostreatus mycelia under heat stress and inhibit its growth. • The accumulation of lactate may be due to the acceleration of glycolysis and the dysfunction of mitochondria of P. ostreatus mycelia under high-temperature stress. • The glycolysis and tricarboxylic acid cycle of P. ostreatus mycelia were accelerated under high-temperature stress.

Nitric Oxide Improves the Tolerance of Pleurotus ostreatus to Heat Stress by Inhibiting Mitochondrial Aconitase.

Pleurotus ostreatus is widely cultivated in China. However, its cultivation is strongly affected by seasonal temperature changes, especially the high temperatures of summer. Nitric oxide (NO) was previously reported to alleviate oxidative damage to mycelia by regulating trehalose. In this study, we found that NO alleviated oxidative damage to P. ostreatus mycelia by inhibiting the protein and gene expression of aconitase (ACO), and additional studies found that the overexpression and interference of aco could affect the content of citric acid (CA). Furthermore, the addition of exogenous CA can induce alternative oxidase (aox) gene expression under heat stress, reduce the content of H2O2 in mycelium, and consequently protect the mycelia under heat stress. An additional analysis focused on the function of the aox gene in the heat stress response of mycelia. The results show that the colony diameter of the aox overexpression (OE-aox) strains was significantly larger than that of the wild-type (WT) strain under heat stress (32°C). In addition, the mycelia of OE-aox strains showed significantly enhanced tolerance to H2O2 In conclusion, this study demonstrates that NO can affect CA accumulation by regulating aco gene and ACO protein expression and that CA can induce aox gene expression and thereby be a response to heat stress.IMPORTANCE Heat stress is one of the abiotic stresses that affect the growth and development of edible fungi. Our previous study found that exogenous NO had a protective effect on mycelia under heat stress. However, its regulatory mechanism had not been elucidated. In this study, we found that NO altered the respiratory pathway of mycelia under heat stress by regulating aco The results have enhanced our understanding of NO signaling pathways in P. ostreatus.

Comparative Proteomic Analysis of Pleurotus ostreatus Reveals Great Metabolic Differences in the Cap and Stipe Development and the Potential Role of Ca2+ in the Primordium Differentiation.

Pleurotus ostreatus is a widely cultivated edible fungus around the world. At present, studies on the developmental process of the fruiting body are limited. In our study, we compared the differentially expressed proteins (DEPs) in the stipe and cap of the fruiting body by high-throughput proteomics. GO and pathway analysis revealed the great differences in the metabolic levels, including sucrose and starch metabolism, and sphingolipid signaling and metabolism, and the differences of 16 important DEPs were validated further by qPCR analysis in expression level. In order to control the cap and stipe development, several chemical inducers were applied to the primordium of the fruiting body according to the pathway enrichment results. We found that CaCl2 can affect the primordium differentiation through inhibiting the stipe development. EGTA (ethyleneglycol bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) treatment confirmed the inhibitory role of Ca2+ in the stipe development. Our study not only shows great metabolic differences during the cap and stipe development but also reveals the underlying mechanism directing the primordium differentiation in the early development of the fruiting body for the first time. Most importantly, we provide a reliable application strategy for the cultivation and improvement of the Pleurotus ostreatus, which can be an example and reference for a more edible fungus.

Performance of Low-Cost Agar from Gracilaria salicornia on Tissue Culture of Pleurotus HK-37.

Currently, the demand of Pleurotus HK-37 (oyster mushroom) in Tanzania is growing rapidly due to the increasing of awareness on its nutrition, health, and economic benefits. Despite the increasing demand, the availability of strains of Pleurotus HK-37 species is still a challenge due to high cost of tissue culture technology. The high cost of importing agar seems to be among the factors for this failure. This study aimed at investigating the performance of low-cost agar from local Gracilaria salicornia on tissue culture of Pleurotus HK-37. Local extracted agars with different gel strengths ranging between 100, 200, 300, 400, and 500 g/cm2 were used to make PDA media. The average mycelia growth rate (mm/day) ranged between 9.87 ± 1.44 and 14.9 ± 0.85 mm/day. Low-cost agar shows quite similar performance as that of standard agar on active growth of Pleurotus HK-37 mycelia. All PDA plates appeared white and feathery and showed to grow in a circular mode (radial extension). Mycelia growth on standard agar PDA took 5 days while on extracted local agar PDA took 5 to 7 days to fully colonize the plate at 27 ± 2°C. The present study shows that the production cost can be reduced by ∼35-78% by using local agar.

Removal and tolerance mechanism of Pb by a filamentous fungus: A case study.

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.

Mycelial Growth and Antimicrobial Activity of Pleurotus Species (Agaricomycetes).

The objective of this study was to analyze mycelial growth under different culture conditions and antimicrobial activity of Pleurotus ostreatus (Jacq.: Fr.) Kumm. (DPUA 1533) and P. ostreatus (Jacq.: Fr.) Kumm. cv. Florida (DPUA 1534) against fungi and bacteria of medical importance. The growth of Pleurotus species was evaluated in natural and complex media, with and without light, at 25°C and 28°C for 8, 15, and 30 days. Candida albicans (DPUA 1336), Cryptococcus laurentii (DPUA 1501), Aspergillus flavus (DPUA 1836), Escherichia coli (DAUPE 224), and Mycobacterium smegmatis (ATCC 607) were used to test antibiosis. Under all growth conditions in vitro, Pleurotus species evidenced growth and high density of mycelia on potato dextrose agar and Sabouraud agar with yeast extract; mycelial growth but lesser mycelial density was observed on rice bran extract agar. Organic mycelial extracts of Pleurotus species exhibited potential antibacterial and antifungal activity, and were selective for the tested microorganisms.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr = 9.84 mm day-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22 U g-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

Mechanisms of Cd and Cr removal and tolerance by macrofungus Pleurotus ostreatus HAU-2.

Fungi bioaccumulation is a novel and highly promising approach to remediate polluted soil. The present study revealed a high ability to tolerate Cd and Cr in the fungus Pleurotus ostreatus HAU-2. However, high concentrations of Cd and Cr can suppress fungal growth and result in a variation of hypha micromorphology. Batch experiments were performed to investigate Cd and Cr stress effects on the amount of active oxygen in fungi, activity of antioxidant enzyme, as well as the removal efficiency of Cd and Cr. The results revealed that Cd and Cr caused increasing active oxygen and malonaldehyde (MDA) concentrations. Antioxidant enzymes play a central role in removing active oxygen, while glutathione (GSH) aids the Cd detoxification within cells. In fluid culture, fungal removal rates of Cd and Cr ranged from 44.85% to 80.36% and 14.49% to 45.55%, respectively. Intracellular accumulation and extracellular adsorption were the major removal approaches. Bag cultivation testing indicated that the fungus absorbed Cd and Cr contained within soil. In particular, the accumulation ability of Cd (15.6mgkg-1) was higher compared to that of Cr (8.9mgkg-1). These results successfully establish P. ostreatus HAU-2 as promising candidate for the remediation of heavy-metal polluted soils.

A development and an improvement of selectable markers in Pleurotus ostreatus transformation.

Pleurotus ostreatus was transformed using the nourseothricin-resistant gene for the first time. The transformation efficiency was 1.3±0.6transformants/µg plasmid DNA. In addition, the transformation efficiency of the bialaphos-resistant gene was increased to 26.7±11.5transformants/µg plasmid DNA.

Fates of nickel and fluoranthene during the bioremediation by Pleurotus eryngii in three different soils.

This study focused on the bioremediation role of Pleurotus eryngii in different characteristics soils contaminated with nickel (Ni) and fluoranthene. The results of bioremediation experiments showed that fluoranthene had a positive effect on the growth of P. eryngii, whereas Ni exerted a negative influence. The concentration of fluoranthene significantly decreased in inoculated soil accounting for 86.39-91.95% of initial concentration in soils and 71.46-81.76% in non-inoculated soils, which showed that the dissipation of fluoranthene was enhanced by mushroom inoculating. The highest removal rates of fluoranthene in sandy loam, loamy clay, and sandy soils reached to 87.81, 86.39, and 91.95%, respectively, which demonstrated that P. eryngii was more suitable for the bioremediation of sandy soil contaminated with fluoranthene. In addition, the presence of Ni tended to decrease the dissipation of fluoranthene in inoculated soil. Higher ligninolytic enzymes activities were detected in inoculated soils, resulting in the enhanced dissipation of fluoranthene in inoculated soils. Furthermore, P. eryngii had the ability to uptake Ni (4.88-39.53 mg kg-1 ) in co-contamination soil. In conclusion, the inoculating of P. eryngii was effective in remediating of Ni-fluoranthene co-contaminated soils.

Iron translocation in Pleurotus ostreatus basidiocarps: production, bioavailability, and antioxidant activity.

Translocation of minerals from substrate to mushrooms can change the medicinal characteristics, commercial value, and biological efficiency of mushroom. In the present study, we demonstrated that addition of iron to the substrate reduces the yield of Pleurotus ostreatus mushroom. The biological efficiency of the mushroom varied from 36.53% on the unsupplemented substrate to 2.08% for the substrate with 500 mg/kg iron added. The maximum iron concentration obtained for mushroom was 478.66 mg/kg (dry basis) and the maximum solubility in vitro was 293.70 mg/kg (dry basis). Iron translocation increased the ash and protein content, reduced antioxidant activity, and enhanced the aroma and flavor characteristics of the mushroom. However mushroom has higher amounts of iron than vegetables like collard greens, it is not feasible to use mushrooms as the only dietary source of iron. The study also indicated that because of more bioaccumulation of iron in mycelium than in the mushroom, mycelium and not mushroom, could be a better alternative as a non-animal iron source.

Alleviating the toxicity of heavy metals by combined amendments in cultivated bag of Pleurotus cornucopiae.

The substrate of mushroom can be polluted with heavy metals and subsequently contaminate mushroom, which requires alternative solutions to reduce associated environmental and human health risks. The effects of amendment application on alleviating Cu and Cd toxicities to Pleurotus cornucopiae were investigated in a cultivated bag experiment conducted with the naturally contaminated substrate. Addition of combined amendments (sodium bentonite, silicon fertilizer, activated carbon, and potassium dihydrogen phosphate) increased the P. cornucopiae biomass and substrate pH. Cu and Cd concentration in P. cornucopiae as well as the available Cu and Cd in substrate reduced for the presence of amendments, and the silicon fertilizer had the biggest inhibition on metal uptake. The smallest amount of Cu and Cd in P. cornucopiae was only 30.8 and 5.51% of control, respectively. Moreover, application of amendments also decreased malondialdehyde (MDA) and hydrogen peroxide (H2O2) level in metal-stressed mushroom by 4.38-53.74 and 8.90-58.42% relative to control, respectively. The decreased oxidative stress could well contribute to the growth of P. cornucopiae, and the elevated substrate pH might lead to the lower metal availability, thus resulting in the reduction of metal accumulation in mushroom. These above results suggest that application of combined amendments in mushroom substrate could be implemented in a general scheme aiming at controlling metal content in P. cornucopiae.

Benzoic acid derivatives with improved antifungal activity: Design, synthesis, structure-activity relationship (SAR) and CYP53 docking studies.

Previously, we identified CYP53 as a fungal-specific target of natural phenolic antifungal compounds and discovered several inhibitors with antifungal properties. In this study, we performed similarity-based virtual screening and synthesis to obtain benzoic acid-derived compounds and assessed their antifungal activity against Cochliobolus lunatus, Aspergillus niger and Pleurotus ostreatus. In addition, we generated structural models of CYP53 enzyme and used them in docking trials with 40 selected compounds. Finally, we explored CYP53-ligand interactions and identified structural elements conferring increased antifungal activity to facilitate the development of potential new antifungal agents that specifically target CYP53 enzymes of animal and plant pathogenic fungi.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus.

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

Potential of Pleurotus ostreatus mycelium for selenium absorption.

The aim of this study was to evaluate the effect of high selenium (Se) concentrations on morphophysiological and ultrastructural properties of Pleurotus ostreatus. Mycelium growth was good in media enriched with 5.0, 10.0, and 20.0 mg L(-1) of Se, concentration of 500.0 mg L(-1) strongly inhibited growth, and 1000.0 mg L(-1) was the minimum inhibitory concentration. Contrary to thin-walled, hyaline, branched, and anastomized hyphae with clamp-connections in the control, at Se concentrations of 100.0 and 500.0 mg L(-1), they were noticeably short, frequently septed and branched, with a more intensive extracellular matrix, and without clamp-connections. At high Se concentrations, hyphae with intact membrane, without cellular contents, with a high level of vacuolization, and with numerous proteinaceous bodies were observed. Biomass yield ranged between 11.8 g L(-1), in the control, and 6.8 g L(-1), at an Se concentration of 100.0 mg L(-1), while no production was detected at a concentration of 500.0 mg L(-1). Se content in the mycelia reached a peak (938.9 µg g(-1)) after cultivation in the medium enriched with Se at the concentration of 20.0 mg L(-1), while the highest absorption level (53.25%) was found in the medium enriched with 5.0 mg L(-1) Se.

Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii.

This study was conducted to evaluate the physiological effects of different selenium (Se) levels on the growth of white-rot fungus, Pleurotus eryngii, with special reference to the regulation of ligninolytic enzymes such as laccase and versatile peroxidase. The fungus was grown in medium supplemented with 1, 10, 100, 1,000 and 10,000 µM of sodium selenite. Mycelial growth was stronger at lower Se levels, but declined significantly at higher concentrations of 1,000 and 10,000 µM, highlighting its association in mediating toxic responses. Inhibition of fungal growth was accompanied with dense and entangled hyphae taking the shape of irregular short strips. Additionally, hyphal swellings and septation were noticed which lead to a reduction in the advancement of the mycelium. Along with the inhibition of fungal biomass, the reducing sugar and protein concentrations increased to about 30.2 and 3.5 mg/ml respectively in the growth medium. Additionally, the laccase gene expression showed a twofold upregulation at higher levels of Se, although the activity of the enzyme was compromised with an inverse relationship with increased gene transcripts. The versatile peroxidase transcript showed a complete downregulation at 10,000 µM after an upregulation at lower levels of Se. We also confirmed the direct relationship of different Se levels on laccase activity of Rhus vernicifera that showed similar behavior to the fungal laccase. The results of the present study suggest that Se supplementation regulates mRNA levels of laccase and versatile peroxidase depending on exposure and may play a role in the toxicity associated with Se.

Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH.

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn(2+) (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.