RESUMO
Fungal secondary metabolites constitute a rich source of yet undiscovered bioactive compounds. Their production is often silent under standard laboratory conditions, but the production of some compounds can be triggered simply by altering the cultivation conditions. The usage of an organic salt - ionic liquid - as growth medium supplement can greatly impact the biosynthesis of secondary metabolites, leading to higher diversity of compounds accumulating extracellularly. This study examines if such supplements, specifically cholinium-based ionic liquids, can support the discovery of bioactive secondary metabolites across three model species: Neurospora crassa, Aspergillus nidulans, and Aspergillus fumigatus. Enriched organic extracts obtained from medium supernatant revealed high diversity in metabolites. The supplementation led apparently to increased levels of either 1-aminocyclopropane-1-carboxylate or α-aminoisobutyric acid. The extracts where bioactive against two major foodborne bacterial strains: Staphylococcus aureus and Escherichia coli. In particular, those retrieved from N. crassa cultures showed greater bactericidal potential compared to control extracts derived from non-supplemented cultures. An untargeted mass spectrometry analysis using the Global Natural Product Social Molecular Networking tool enabled to capture the chemical diversity driven by the ionic liquid stimuli. Diverse macrolides, among other compounds, were putatively associated with A. fumigatus; whereas an unexpected richness of cyclic (depsi)peptides with N. crassa. Further studies are required to understand if the identified peptides are the major players of the bioactivity of N. crassa extracts, and to decode their biosynthesis pathways as well.
RESUMO
BACKGROUND: The inherent potential of filamentous fungi, especially of Ascomycota, for producing diverse bioactive metabolites remains largely silent under standard laboratory culture conditions. Innumerable strategies have been described to trigger their production, one of the simplest being manipulation of the growth media composition. Supplementing media with ionic liquids surprisingly enhanced the diversity of extracellular metabolites generated by penicillia. This finding led us to evaluate the impact of ionic liquids' stimuli on the fungal metabolism in Aspergillus nidulans and how it reflects on the biosynthesis of secondary metabolites (SMs). RESULTS: Whole transcriptional profiling showed that exposure to 0.7 M cholinium chloride or 1-ethyl-3-methylimidazolium chloride dramatically affected expression of genes encoding both primary and secondary metabolism. Both ionic liquids apparently induced stress responses and detoxification mechanisms but response profiles to each stimulus were unique. Primary metabolism was up-regulated by choline, but down-regulated by 1-ethyl-3-methylimidazolium chloride; both stimulated production of acetyl-CoA (key precursor to numerous SMs) and non proteinogenic amino acids (building blocks of bioactive classes of SMs). In total, twenty one of the sixty six described backbone genes underwent up-regulation. Accordingly, differential analysis of the fungal metabolome showed that supplementing growth media with ionic liquids resulted in ca. 40 differentially accumulated ion masses compared to control conditions. In particular, it stimulated production of monodictyphenone and orsellinic acid, otherwise cryptic. Expression levels of genes encoding corresponding polyketide biosynthetic enzymes (i.e. backbone genes) increased compared to control conditions. The corresponding metabolite extracts showed increased cell polarity modulation potential in an ex vivo whole tissue assay (The lial Live Targeted Epithelia; theLiTE™). CONCLUSIONS: Ionic liquids, a diverse class of chemicals composed solely of ions, can provide an unexpected means to further resolve the diversity of natural compounds, guiding discovery of fungal metabolites with clinical potential.