BackgroundFilamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.ResultsVolatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation.Conclusions1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and β-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.

Aflatoxin is a mycotoxin and the most potent naturally occurring carcinogen in many animals. Aflatoxin contamination of food and feed crops causes a significant global burden on human and animal health. However, available methods to eliminate aflatoxin from food and feed are not fully effective. Our goal is to discover novel, efficient, and practical methods to control aflatoxin contamination in crops during storage. In the present study, we tested the effect of volatiles produced by willow (Salix acutifolia and Salix babylonica) and maple (Acer saccharinum) bark on fungal growth, development, and aflatoxin production by the fungus Aspergillus parasiticus, one economically important aflatoxin producer. S. acutifolia bark volatiles nearly eliminated aflatoxin accumulation (>90% reduction) by A. parasiticus grown on a minimal agar medium. The decrease in aflatoxin accumulation correlated with a twofold reduction in ver-1 (encodes a middle aflatoxin pathway enzyme) transcript level. Expression data also indicate that one histone H4 acetyltransferase, MYST3, may play a role in epigenetic control of aflatoxin gene transcription in response to volatile exposure. Volatiles derived from wood bark samples also increased fungal growth up to 20% and/or enhanced conidiospore development. Solid-phase microextraction-gas chromatographic-mass spectrometric analysis of bark samples identified sets of shared and unique volatile compounds that may mediate the observed regulatory effects on growth, development, and aflatoxin synthesis. This work provides an experimental basis for the use of willow industry by-products to control aflatoxin contamination in food and feed crops.

We performed comparative profiling of four specialized metabolites in the lichen Evernia prunastri, collected at three different geographic locations, California and Maine, USA, and Yoshkar Ola, Mari El, Russia. Among the compounds produced at high concentrations that were identified in all three specimens, evernic acid, usnic acid, lecanoric acid and chloroatranorin, evernic acid was the most abundant. Two depsidones, salazinic acid and physodic acid, were detected in the Yoshkar-Ola collection only. The crystalline structure of evernic acid (2hydroxy-4-[(2-hydroxy-4-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoate) (hmb) revealed two crystallographically and conformationally distinct hmb anions, along with two monovalent sodium atoms. One hmb moiety contained an exotetradentate binding mode to sodium, whereas the other exhibited an exohexadentate binding mode to sodium. Embedded edge-sharing {Na 2 O 8 } n sodium-oxygen chains connected the hmb anions into the full three-dimensional crystal structure of the title compound. The crystal used for single-crystal X-ray diffraction exhibited non-merohedral twinning. The data suggest the importance of the acetyl-polymalonyl pathway products to processes of maintaining integrity of the lichen holobiont community.[a] Data for one replicate.

Salicylate based phytopharmaceuticals provoke cellular pro-and anti-inflammatory CCN responses under non-stress conditions, which adapt to anti-inflammatory responses after LPS-stimulation. CCN-profiles of the single extracts are not additives in combination. A simultaneous activation of cellular pro- and anti-inflammatory cytokines might heighten the immunological reactivity status of a cell.

Lichens are symbiotic organisms formed by a fungus and one or more photosynthetic partners which are usually alga or cyanobacterium. Their diverse and scarcely studied metabolites facilitate adaptability to extreme living conditions. We investigated Evernia prunastri (L.) Ach., a widely distributed lichen, for its antimicrobial and antioxidant potential. E. prunastri was sequentially extracted by hexane (Hex), dichloromethane (DCM) and acetonitrile (ACN) that were screened for their antioxidant and antimicrobial (against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans) activities. The Hex extract possessed the highest antioxidant capacity (87 mg ascorbic acid/g extract) corresponding to the highest content of phenols (73 mg gallic acid/g extract). The DCM and Hex extracts were both active against S. aureus (MICs of 4 and 21 µg/ml, respectively) but were less active against Gram-negative bacteria and yeast. The ACN extract exhibited activity on both S. aureus (MIC 14 µg/ml) and C. albicans (MIC 38 µg/ml) and was therefore further fractionated by silica gel column chromatography. The active compound of the most potent fraction was subsequently characterized by 1H and 13C-NMR spectroscopy and identified as evernic acid. Structural similarity analyses were performed between compounds from E. prunastri and known antibiotics from different classes. The structural similarity was not present. Antioxidant and antimicrobial activities of E. prunastri extracts originate from multiple chemical compounds; besides usnic acid, most notably evernic acid and derivatives thereof. Evernic acid and its derivatives represent possible candidates for a new class of antibiotics. Graphic abstract