Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.     

Antioxidant Activity of Mycelia from Aspergillus candidus

The antioxidant activity of mycelia extracts produced from the submerged cultures of Aspergillus candidus CCRC 31543 was investigated. Inhibition of peroxidation (IP%) of mycelia acetone extracts (MAE) in linoleic acid peroxidation was equal to that of BHA and significantly (p < 0.05) higher than that of α-tocopherol. As measured by the Rancimat method in lard, MAE showed marked antioxidant activity with an induction time of 8.4 h at a concentration of 200 μg/mL. MAE also exhibited strong scavenging effect on α,α-diphenyl-β-picrylhydrazyl radicals and marked reducing power. HPLC-DAD analysis suggested that MAE possessed the antioxidant components 3,3"-dihydroxyterphenyllin, 3-hydroxyterphenyllin, and candidusin B.     

Microbiological, physicochemical and organoleptic changes in dry-salted olives of Thassos variety stored under different modified atmospheres at 4 and 20 °C

Microbiological, physico-chemical and organoleptic changes were studied in dry-salted olives, cv. Thassos, stored under different atmospheres (100% carbon dioxide and nitrogen, 40% CO₂/30%O₂/30%N₂ and air) at 4 and 20 °C for 180 days. The initial microbial flora comprised of yeasts, no lactic acid bacteria, enterobacteria, pseudomonads or Staphylococcus aureus were detected, as the low water activity/high salt content does not favour their growth. At 4 °C, the population of yeasts declined steadily throughout storage but to a different extent depending on the gaseous atmospheres. At 20 °C, there was an initial decline in yeast counts in all samples followed by a steady increase until the end of the storage period. The CO₂ atmosphere was most effective at keeping the number of yeasts low at both storage temperatures. All gas atmospheres prevented fungal growth at both temperatures apart from the samples stored in air. The pH, a_w and salt content of the olives did not change significantly throughout the storage period. The prevailing yeast species was the salt tolerant Candida famata . The organoleptic characteristics did not differ significantly among differently treated olives. However, increased rancidity and reduced fruit colour was observed in the samples stored at 20 °C.     

Biotransformation of isoflavones by Aspergillus niger as biocatalyst

Biotransformation of the isoflavones, 6,7,4′‐trimethoxyisoflavone ( 1 ) and 5,7,4′‐trimethoxyisoflavone ( 2 ) by Aspergillus niger was investigated. Compound 1 was transformed to 4′‐hydroxy‐6,7‐dimethoxyisoflavone ( 3 ) and 2 to 4′‐hydroxy‐5,7‐dimethoxyisoflavone ( 4 ). This suggested that 1 and 2 were demethylated at the C‐4′ position with regioselectivity by Aspergillus niger. Copyright © 2006 Society of Chemical Industry     

AstPT Catalyses both Reverse N1‐ and Regular C2 Prenylation of a Methylated Bisindolyl Benzoquinone

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti‐HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG_09980 and ATEG_00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG_09980, not in those with EAU39348 encoded by ATEG_00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1‐reversely and C2‐regularly monoprenylated derivatives, as well as N1′,N1′′reversely, N1′‐reversely, C2′′‐regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives.     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.     

Biodegradation of anthracene by Aspergillus fumigatus

An anthracene-degrading strain, identified as Aspergillus fumigatus, showed a favorable ability in degradation of anthracene. The degradation efficiency could be maintained at about 60% after 5d with initial pH of the medium kept between 5 and 7.5, and the optimal temperature of 30 °C. The activity of this strain was not affected significantly by high salinity. Exploration on co-metabolism showed that the highest degradation efficiency was reached at equal concentration of lactose and anthracene. Excessive carbon source would actually hamper the degradation efficiency. Meanwhile, the strain could utilize some aromatic hydrocarbons such as benzene, toluene, phenol etc. as sole source of carbon and energy, indicating its degradation diversity. Experiments on enzymatic degradation indicated that extracellular enzymes secreted by A. fumigatus could metabolize anthracene effectively, in which the lignin peroxidase may be the most important constituent. Analysis of ion chromatography showed that the release of anions of A. fumigatus was not affected by addition of anthracene. GC-MS analysis revealed that the molecular structure of anthracene changed with the action of the microbe, generating a series of intermediate compounds such as phthalic anhydride, anthrone and anthraquinone by ring-cleavage reactions.     

Uniform culture in solid-state fermentation with fungi and its efficient enzyme production

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.