Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the gld1 ⁺ gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 ⁺ was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

A protective role of methionine-R-sulfoxide reductase against cadmium in Schizosaccharomyces pombe

The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.     

Polyethylene glycol-mediated transformation of fused egfp-hph gene under the control of gpd promoter in Pleurotus eryngii

Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90–210 transformants were produced per μg plasmid DNA per 10⁷ viable protoplasts.     

The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli

In order to increase the synthesis of bovine growth hormone (bGH) using T7 promoter system in E. coli, the artificial AT-rich block was introduced into the upstream region of a consensus Shine-Dalgarno (SD) sequence and the spacer region (between SD and ATG codon) was enriched with A and T nucleotides. The cells harboring pTAJ plasmids with AT-rich block produced bGH in the range of 3% to 25% and the cells harboring pTBJ plasmids with AT-rich sequence in the spacer region from 0.8% to 20% of total cell proteins. This result suggests that AT rich block and AT nucleotides in the spacer region destabilize mRNA secondary structure, depending on the downstream coding information of bGH gene and also, implying that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.     

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25–60 °C and pH 3.0–9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL^?1. In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.     

Development of orthogonal T7 expression system in Klebsiella pneumoniae

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.     

Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.     

Evaluation of Burma Reed as Substrate for Production of Pleurotus eryngii

Burma reed (Neyraudia reynaudiana), a giant C₄ grass, was included in substrate at the rates of 0, 20, 40 and 66 % to partially or wholly substitute sawdust and cottonseed hulls to evaluate its suitability for Pleurotus eryngii cultivation. Inclusion of 20 and 40 % Burma reed did not significantly affect linear mycelial growth, dry matter loss, spawn run period and fructification, and achieved high fruiting body yields and biological efficiency of 336.67 g/bag, 67.33 % and 342.15 g/bag, 68.43 %, respectively, which were not significantly different from 350.08 g/bag to 70.02 % obtained from the control substrate. Enzyme assay revealed that on the mixed substrates laccase and manganese peroxidase activity were significantly enhanced, but cellulase was significantly reduced in the middle stage of incubation as compared with the control substrate. Even on Burma reed substrate without sawdust and cottonseed hulls, fruiting body yield (313.56 g/bag) and biological efficiency (62.71 %) were satisfactory, although significantly lower than that on the control substrate. Therefore, Burma reed was a promising potential substrate for P. eryngii production to largely substitute sawdust and cottonseed hulls.     

High-level production of recombinant glutamic acid-specific protease from Bacilllus licheniformis in Bacillus subtilis expression system

To obtain large quantities of glutamic acid-specific protease isolated originally from Bacillus licheniformis (BLase), an expression plasmid was constructed by inserting the BLase gene into a plasmid vector (pUB110) for Bacillus subtilis. B. subtilis strain ISW1214 harboring the resultant recombinant plasmid containing the coding and 5′-promoter and 3′-terminator regions of BLase gene secreted approximately 0.25 g/l of BLase in a culture medium contained in a 90-l jar fermentor, corresponding to nearly 10 times the natural production level and resulting in a stable large-scale production. The amount of BLase in the culture medium accounted for roughly 60% of the total extracellular proteins secreted from the recombinant strain, simplifying enzyme purification.     

Are stag beetles fungivorous?

Stag beetle larvae generally feed on decaying wood; however, it was unknown whether they can use wood-rotting fungi alone as food. Here, to clarify this, newly hatched larvae of Dorcus rectus (Motschulsky) (Coleoptera: Lucanidae) were reared for 14 days on artificial diets containing a fixed amount of freeze-dried mycelia of the following fungi: Bjerkandera adusta, Trametes versicolor, Pleurotus ostreatus, and Fomitopsis pinicola. The mean incremental gain in larval body mass was greatest on diets containing B. adusta, followed by T. versicolor, P. ostreatus, and F. pinicola. The growth rate of body mass correlated positively with mycelial nitrogen content of the different fungi. It also correlated positively with the mycelial content of B. adusta in the diet. Addition of antibiotics to diets with mycelia nearly halved larval growth, indicating that larvae were able to use fungal mycelia as food without the assistance of associated microbes although the microbes positively affected larval growth. Four newly hatched larvae reared on artificial diets containing B. adusta mycelia developed to the second instar in 21-34 days; and one developed to the third (=final) instar. This study provides evidence that fungi may constitute the bulk of the diet of D. rectus larvae.     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

Anti-saprolegnia potency of some plant extracts against Saprolegnia diclina,the causative agent of saprolengiasis

Saprolegnosis of fresh water fishes caused by Saprolegnia diclina often results in serious economic losses to fish hatcheries. Despite the proven efficiency of malachite green as a potential fungicide in prevention and control of fish saprolegnosis, there is a strong debate about its safety aspects in use since it was documented to be responsible for many carcinogenic and teratogenic attributes. Bioactivity of four ethanolic plant extracts were assessed to attain a natural alternative to the traditional fungicide currently used in saprolegnosis control. Ethanolic extracts of Punica granatum and Thymus vulgaris exhibited a potential efficacy in suppressing mycelial growth of S. diclina at concentration of 0.5 mg/ml while extracts of Nigella sativa and Zingiber officinales were not effective respectively. The extract of pomegranate showed the highest antifungal potency with minimum inhibitory concentration (MIC) of 200 ppm while thyme extract was less effective and recorded MIC of 400 ppm against S. diclina. The acute fish toxicity of the plant extracts indicated the low toxicity of P. granatum and T. vulgaris extracts as no fish mortalities were detected at aquaria containing 200, 400 and 800 ppm of plant extracts respectively. Considering the low toxicity of these plant extracts, it may be concluded that 200 and 400 ppm of pomegranate and thyme extracts which suppressed the mycelial growth of the S. diclina could be safely used for saprolegniasis control.Both of pomegranate and thyme extracts which proved to possess a potential antifungal activity can be considered as a natural alternative fungicides to control saprolegniasis avoiding carcinogenic malachite green application.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.     

Clostridium beijerinckii Cells Expressing Neocallimastix patriciarum Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.     

Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK ⁺) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.     

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5–30 °C) and weakly basic conditions (pH 8.5–10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.