Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL^?1 initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu²⁺ and Triton X-100, at toxin concentration of 5 µg mL^?1. P. ostreatus GHBBF10 showed highest degradation in the presence of Zn²⁺ and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.     

Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white‐rot agaricomycete Pleurotus ostreatus

White‐rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next‐generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white‐rot fungus Pleurotus ostreatus. In this study, they identified two genes – chd1 and wtr1 – mutations in which cause an almost complete loss of Mn²⁺‐dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete‐specific protein with a putative DNA‐binding domain. The chd1‐1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.     

Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU‐P by ClpCP

The response regulator DegU and its cognate kinase DegS constitute a two‐component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU‐lacZ fusions, we showed that phosphorylated DegU (DegU‐P) binds to two direct repeats (DR1 and DR2) of the consensus DegU‐binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP‐dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild‐type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU‐P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU‐P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N‐terminal region of DegU is important for proteolysis.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.     

Pseudomonas azotoformans F77和Pseudomonas paracarnis P1风化黑云母的效应及机制比较

【目的】比较高效矿物风化固氮假单胞菌(Pseudomonas azotoformans) F77及其亲缘关系较近的假单胞菌(Pseudomonas paracarnis) P1风化黑云母的效应和机制。【方法】通过检测两株菌在不同时间点的发酵液中细胞数量、pH值、葡萄糖剩余量、葡萄糖酸浓度和可溶性Fe、Al释放量,比较它们对黑云母的风化效果与生理机制。采用RNA-seq技术研究这两株菌风化黑云母过程中出现差异的分子机制。【结果】在持续5 d的风化试验中,菌株F77发酵液中的细胞数量和pH值低于菌株P1,葡萄糖酸浓度是菌株P1的27.3-53.9倍,Fe和Al元素的释放量是菌株P1的3.3-23.3倍。比较转录组数据表明,菌株F77特有的基因数量(2 872)和差异基因数量(1 832)均多于菌株P1 (分别为1 903和1 258)。菌株F77在胞内物质跨膜转运与碳代谢、细胞运动、趋化与信号诱导等途径中基因数量也高于菌株P1。此外,菌株F77的超氧化物歧化酶和过氧化氢酶基因差异表达倍数、葡萄糖酸合成基因数量和差异表达倍数也明显高于菌株P1。【结论】菌株F77风化黑云母以及合成葡萄糖酸的能力显著高于菌株P1。菌株F77通过产生葡萄糖酸来促进黑云母的风化。添加黑云母显著促进了菌株F77胞内与矿物风化相关基因的表达,如物质跨膜转运、细胞运动与趋化、信号诱导、碳代谢及能量代谢等途径基因。此外,葡萄糖酸合成途径基因、超氧化物歧化酶基因以及过氧化氢酶基因在矿物风化中可能发挥重要作用。     

Molecular breeding of a novel <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> strain possessing a heterologous laccase gene, <Emphasis Type="Italic">lccK</Emphasis>, driven by a constitutive promoter

Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds.     

Degradation of styrene by white-rot fungi

Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [¹⁴C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO₂ in 24 h and no other volatile products were found. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2′,3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.     

糙皮侧耳和鞘脂菌NS7对多环芳烃污染土壤的生物强化与协同作用

[背景] 真菌和细菌被认为在多环芳烃污染土壤生物修复过程中发挥协同作用,目前在真实土壤体系中开展真菌-细菌协同降解研究较少。[目的] 研究真菌和细菌对不同种类多环芳烃降解的差异及对蒽和苯并[a]蒽的生物强化与协同作用。[方法] 选用多环芳烃降解真菌和细菌各一株,在液体纯培养体系下分析它们对不同种类多环芳烃降解的差异,在土壤体系中采用放射性同位素示踪技术研究2种微生物对蒽和苯并[a]蒽的生物强化与协同作用。[结果] 供试细菌鞘脂菌NS7能够很好地降解低环种类多环芳烃,以蒽作为唯一碳源时可以将其完全降解,在复合污染条件下对菲、蒽、荧蒽、芘等降解效果突出（>90%）,对苯并[a]芘降解效果较差（9.76%）。相比而言,供试真菌糙皮侧耳菌对苯并[a]芘具有更好的降解效果（21.18%）,对低环多环芳烃降解效果明显不如降解菌NS7。在自然土壤中,蒽和苯并[a]蒽具有明显不同的矿化效率,分别为18.61%和4.28%,在蒽污染土壤中加入鞘脂菌NS7并未显著提高蒽的矿化率（P>0.05）,相比而言,苯并[a]蒽污染土壤中加入糙皮侧耳显著提高了污染物矿化效率（2.24倍）,表明真菌和细菌在土壤环境中的定殖存活能力可能影响了生物强化效果。采用灭菌土壤排除土著微生物的竞争排斥作用,研究了真菌菌丝对生物强化降解的影响,发现在蒽污染土壤中,真菌菌丝的迁移作用显著提高了细菌鞘脂菌NS7对污染物的矿化率,从1.75%提高到5.91%;而在苯并[a]蒽灭菌污染土壤中,接种糙皮侧耳却没有发现苯并[a]蒽矿化率提高的现象,表明自然土壤中真菌强化降解苯并[a]蒽的作用可能是源于真菌菌丝促进污染物和土著降解菌的接触,而非直接来自真菌本身。[结论] 细菌能够很好地降解低环种类多环芳烃,而真菌对高环种类多环芳烃降解效果较好。真菌可能通过菌丝促进土著微生物在土壤中的迁移,增大多环芳烃和土著降解菌的接触,从而促进了多环芳烃降解。研究加深了对多环芳烃污染土壤生物强化修复的认识,对发展基于真菌-细菌协同作用的生物强化与调控技术提供理论指导。     

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN′

 Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-β-naphthylamide (APA-βNH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN′ in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-βNH-Nap substrate compared to their non-deleted parental strains. Received: 15 May 1995/Received last revision: 2 October 1995/Accepted: 4 October 1995     

Analysis of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles in <Emphasis Type="Italic">Bacillus Thuringiensis</Emphasis> Strains Isolated During Epizootics in <Emphasis Type="Italic">Cydia pomonella</Emphasis> L.

The crystal morphology and the profiles of genes encoding protein toxins (Cry and Cyt) were analyzed in 12 Bacillus thuringiensis strains isolated during epizootics in laboratory culture lines of Cydia pomonella, 2 isolates cultured from Leucoma salicis larvae, and 9 reference strains. Epizootic isolates produced crystals of the same bipyramidal shape; however, they revealed a variety of number and type of cry genes. Genes cry1I, cry2Ab, and cry9B were the most frequently observed in epizootic strains. Gene cry1I was noted in of 50% epizootic isolates. Eighty-three percent of them harbored gene cry2Ab. Gene cry9B was found for 42% of strains isolated during epizootics. Three isolates showed the largest number of cry genes and their variety; hence, they were chosen for the toxicity assay of their crystals and spores on C. pomonella larvae. One of them had approximately sixfold higher insecticidal activity than the reference strain B. thuringiensis subsp. kurstaki BTK STANDARD.     

Rationally engineered biotransformation of p‐nitrophenol

An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Improved pectinase production in Penicillium griseoroseum recombinant strains

Aims: To obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. Methods and Results: A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266‐ and 27‐fold greater, respectively, than the wild‐type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. Conclusions: This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. Significance and Impact of the Study: PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum.     

Toluene biodegradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1: targeting culture stability in long-term operation

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h⁻¹). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO₂ produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.     

Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

Decolourization of Municipal Effluent and Sludge by Pleurotus sajor-caju and Pleurotus ostreatus

Summary The Americana Municipal Treatment Station, S?o Paulo, Brazil, manages 400 l of effluent s⁻¹, from domestic and textile origin, which produces an average of 20 t of sludge per day. The decolourization of the effluent and sludge by three strains of Pleurotus (Pleurotus sajor-caju F2, F6 and Pleurotus ostreatus) was evaluated. The strains of P. sajor-caju F2 and F6 were able to decolourize the sludge, while P. ostreatus was less efficient. Detoxification was appraised with three bioassays comprising the cnidarian Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. After exposure to fungi, effluent toxicity decreased but not that of its sludge. Strain P. sajor-caju F6 presented signs of toxicity shown by electron microscopy in the presence of the effluent. The three strains produced high amounts of manganese-peroxidase (Mn–P) and laccase in the presence of the sludge. Although P. ostreatus produced large amount of Mn–P and laccase enzymes, these enzymes did not result in decolourization of the sludge, suggesting that other factors are likely to be involved. Carbon content decreased only in the treatment with P. ostreatus.