Silencing of the Laccase (lacc2) Gene from Pleurotus ostreatus Causes Important Effects on the Formation of Toxocyst-like Structures and Fruiting Body.

A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus.

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity.

Laccases are enzymes produced by plants and white rot fungi, such as Pleurotus ostreatus, with industrial applications. Fungal laccases have been widely studied, and investigations, such as those involving recombinant DNA technology or adding inducers, have been made to increase laccase production. On the other hand, it has been proposed that extracellular proteases could decrease laccase activity when both types of enzymes are produced by P. ostreatus. The aim of this work was to evaluate the effects of proteases on the activity of extracellular laccases produced by P. ostreatus PoB in submerged culture. Results showed that P. ostreatus PoB produced alkaline, acidic, and neutral proteases. Protease activity was quantified, and the highest activity at alkaline pH (9.0) was 5.63 IU/L (192 h), that at acidic pH (2.0) was 3.38 IU/L (192 h), and that at neutral pH (7.0) was 6.20 IU/L (312 h). The protease activity decreased in the presence of different protease inhibitors, as phenylmethylsulfonyl fluoride (PMSF), EDTA, pepstatin A, and a cocktail of protease inhibitors. Laccase activity was determined in cultures with and without protease inhibitors. In the control culture (without inhibitor), the highest laccase specific activity was 99.88 IU/mg protein. In cultures with PMSF, pepstatin A, or a cocktail of protease inhibitors, laccase activity increased by approximately 1.35-fold (138 IU/mg protein) with respect to the control culture. The inhibitor EDTA did not produce a positive effect on extracellular laccase activity. These results suggest that laccase activity is affected by the actions of acidic and neutral extracellular proteases.

Silencing of Amylomyces rouxii aspartic II protease by siRNA to increase tyrosinase activity.

Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43-90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity.

Laccase Production from Agrocybe pediades: Purification and Functional Characterization of a Consistent Laccase Isoenzyme in Liquid Culture.

Laccases are valuable enzymes as an excellent ecological alternative for bioremediation issues because they can oxidize persistent xenobiotic compounds. The production and characterization of extracellular laccases from saprotrophic fungi from disturbed environments have been scarcely explored, even though this could diversify their functional characteristics and expand the conditions in which they carry out their catalysis. Agrocybe pediades, isolated from a disturbed forest, produces an extracellular laccase in liquid culture. The enzyme was purified, identified and characterized. Copper and hexachlorobenzene do not function as inducers for the laccase produced. Partial amino acid sequences were obtained by LC-MS/MS that share similarity with laccases from other fungi. Purified laccase is a monomer with a molecular mass between 55-60 kDa and had an optimum activity at pH 5.0 and the optimum temperature at 45 °C using 2,6-dimethoxyphenol (2,6-DMP) as substrate. The Km and Vmax also determined with 2,6-DMP were 100 µM and 285 µmol∙min-1∙mg-1, respectively, showing that the laccase of A. pediades has a higher affinity for this substrate than that of other Agaricales. These features could provide a potential catalyst for different toxic substrates and in the future laccase could be used in environmental recovery processes.

Application of 2III7-3 fractional factorial experimental design to enhance enzymatic activities of Pleurotus ostreatus with high concentrations of polychlorinated biphenyls.

A 2(III)(7-3) fractional factorial experimental design was used to establish 16 culture media, with and without PCBs to enhance the activities of laccase (Lac), manganese peroxidase (MnP), and versatile peroxidase (VP) produced by the white rot fungus Pleurotus ostreatus. The culture was added to 10,000 mg L(-1) of transformer oil, containing 71% of the identified Arochlor 1242. The culture conditions were established with eight variables at two values (levels); pH (4 and 6), agitation (100 and 200 rpm), CuSO(4) (150 and 250 mg L(-1)), MnSO(4) (50 and 200 mg L(-1)), Tween 80 (13 and 3500 mg L(-1)), wheat straw (0 and 2.5 g L(-1)), sugarcane bagasse (0 and 2.5 g L(-1)),and Arochlor 1242 (0 and 7100 mg L(-1)) at 4, 8, 12, 16 and 20 days old culture. Laccase activity was enhanced at a high value of pH and low value of agitation (P<0.001) and correlated positively (R(2)= 0.9; α=0.05) with the removal of polychlorinated biphenyls (PCBs). VP activity was enhanced 27-fold with PCBs, Tween 80 and pH. The MnP activity was increased 1.2-fold with PCBs. The fractional factorial experimental design methodology allowed us to determine the P. ostreatus culture media conditions to enhance Lac and VP activities for efficient removal of Arochlor 1242 (one of the most recalcitrant organochloride pollutants). The factors that shown the greatest effect on Lac activity were: pH, agitation and high concentrations of Arochlor 1242.

A method for the extraction of high quality fungal RNA suitable for RNA-seq.

Transcriptomic analysis is an OMICs technology that is becoming indispensable to understand and get a complete picture of cell functioning and adaptation to the environmental cues the cell is continuously receiving. Among the techniques available to perform transcriptomics, RNA-seq is becoming the method of choice. The quality of the RNA used for the generation of cDNA libraries and subsequent sequencing is crucial for the success of the process. Good RNA-seq performance is often limited by problems such as low RNA yield and/or integrity, RNA stability, and contamination with DNA, salts or chemicals. RNA isolation from fungi usually faces these problems and is particularly sensitive to degradation due to the high RNase activity content present in many species. Here we describe the development of a robust, highly reproducible and simple RNA purification method for filamentous fungi, which combines various strategies to get fully DNA-free RNA samples of high purity and integrity without the need to use a DNase I digestion step. The obtained RNA samples complied with all required standards to be used for RNA-seq and showed an excellent performance when subjected to Illumina-HiSeq 2500.

Pentachlorophenol sorption in nylon fiber and removal by immobilized Rhizopus oryzae ENHE.

This study describes pentachlophenol (PCP) sorption in nylon fiber in which Rhizopus oryzae ENHE was immobilized to remove the chemical compound. The experimental sorption data were analyzed using the Langmuir, Freundlich, and Redlich-Peterson isotherm models using non-linear error functions to fit the experimental data to the three models. Results showed that the isotherm obtained from the data fitted the three models used. However, the g parameter from Redlich-Peterson model showed that the isotherm obtained approaches the Freundlich model. This support reached the sorption equilibrium concentration at 3mg PCPg(-1)nylon. To study PCP removal capability by R. oryzae ENHE and to eliminate the error caused by PCP sorbed by the nylon fiber during its quantification, nylon fiber at PCP equilibrium sorption concentration was used to immobilize R. oryzae ENHE. It was found that this fungus grew within nylon fiber cubes in presence or not of PCP, even when PCP caused growth inhibition. Maximum biomass accumulated into nylon cubes without PCP was of 32 mg biomass g(-1)nylon and into nylon cubes at PCP equilibrium concentration was of 18 mg g(-1)nylon. The results showed that R. oryzae ENHE immobilized into nylon fiber removed 88.6% and 92% of PCP in cultures with 12.5 and 25 mg PCPL(-1), as initial concentration, respectively. This is the first work to report that a zygomycete, such as R. oryzae ENHE, immobilized into nylon fiber kept its potential to remove PCP.

High lovastatin production by Aspergillus terreus in solid-state fermentation on polyurethane foam: an artificial inert support.

A novel solid-state fermentation (SSF) process, using high-density polyurethane foam (PUF) as an inert support, was developed for the production of lovastatin. Results indicated that forced aeration is not conducive to metabolite production since it reduces the solid medium's moisture content. The highest level of production was achieved in closed flasks (CF), in which 7.5 mg of lovastatin was generated per gram of dry culture, equivalent to 493 mug/mg dry mycelium. However, since mycelial growth is aeration-dependent, the CF cultures presented the lowest level of growth: 15.19 mg/gdc (milligrams per gram of dry culture). It was possible to increase the biomass concentration to 24.4 mg/gdc by increasing the culture medium concentration to 2.5x and the initial moisture content of the solid medium to 85%. Results also revealed that the density of the culture support is a key parameter in determining lovastatin production; high yields were only obtained on PUF at a density of 17 or 20 kg/m(3). SSF using the latter reached a lovastatin level of 19.95 mg/gdc, with specific production of 815 mug/mg dry mycelium. A comparative study showed that lovastatin production during PUF SSF was two-fold higher than that of the better-known system of bagasse SSF. Moreover, lovastatin yields on PUF were 30 times higher than those of liquid submerged fermentation (SmF; 0.57 mg/ml) and lovastatin biomass was almost 15 times more productive.

Cr(VI) reduction by an Aspergillus tubingensis strain: role of carboxylic acids and implications for natural attenuation and biotreatment of Cr(VI) contamination.

Experiments conducted in shake flask cultures, in minimal medium of pH 5.3 containing 50 microg mL(-1)Cr(VI) with glucose as a carbon source, indicated that the biomass of Aspergillus sp. strain Ed8, a chromate-tolerant fungal strain previously isolated from a chromium-polluted soil, responds to the presence of citrate in the medium by increasing the rate of Cr(VI) reduction; this effect required the use of live biomass and was not observed in medium with lactate. Other natural carboxylic acids or non-natural metal chelating agents showed a stimulatory effect of Cr(VI) reduction by Ed8 biomass; salicylate, tartrate and citrate were the stronger stimulators of the specific rate of Cr(VI) reduction, with about 12, 8 and 7-fold stimulatory effects, respectively, as compared to control cultures without additions. A procedure for Cr(VI) removal from a diluted electroplating effluent was devised, based on the use of growth medium amended with citrate or a mixture of salycilate-tartrate and cycles of recharge of growth medium-diluted effluent. In addition, conditions were adjusted in a 2-L bioreactor to reach a 20-fold increase in the volume of the reduction system with no loss of efficiency. Strain Ed8 was identified as an Aspergillus tubingensis isolate (included in Aspergillus niger species complex) on the basis of the ITS1-5.8s rDNA-ITS2 sequence similarity.