Transcriptomic and metabolomic profiling of ionic liquid stimuli unveils enhanced secondary metabolism in Aspergillus nidulans.

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.

Elucidating how the saprophytic fungus Aspergillus nidulans uses the plant polyester suberin as carbon source.

BACKGROUND: Lipid polymers in plant cell walls, such as cutin and suberin, build recalcitrant hydrophobic protective barriers. Their degradation is of foremost importance for both plant pathogenic and saprophytic fungi. Regardless of numerous reports on fungal degradation of emulsified fatty acids or cutin, and on fungi-plant interactions, the pathways involved in the degradation and utilisation of suberin remain largely overlooked. As a structural component of the plant cell wall, suberin isolation, in general, uses harsh depolymerisation methods that destroy its macromolecular structure. We recently overcame this limitation isolating suberin macromolecules in a near-native state. RESULTS: Suberin macromolecules were used here to analyse the pathways involved in suberin degradation and utilisation by Aspergillus nidulans. Whole-genome profiling data revealed the complex degrading enzymatic machinery used by this saprophytic fungus. Initial suberin modification involved ester hydrolysis and ω-hydroxy fatty acid oxidation that released long chain fatty acids. These fatty acids were processed through peroxisomal ß-oxidation, leading to up-regulation of genes encoding the major enzymes of these pathways (e.g. faaB and aoxA). The obtained transcriptome data was further complemented by secretome, microscopic and spectroscopic analyses. CONCLUSIONS: Data support that during fungal growth on suberin, cutinase 1 and some lipases (e.g. AN8046) acted as the major suberin degrading enzymes (regulated by FarA and possibly by some unknown regulatory elements). Suberin also induced the onset of sexual development and the boost of secondary metabolism.

Ex situ reconstitution of the plant biopolyester suberin as a film.

Biopolymers often have unique properties of considerable interest as a basis for new materials. It is however not evident how to extract them from plants without destroying their chemical skeleton and inherent properties. Here we report the ex situ reconstitution of the biopolyester suberin as a new waterproof and antimicrobial material. In plant cell walls, suberin, a cross-linked network of aromatic and aliphatic monomers, builds up a hydrophobic protective and antimicrobial barrier. Recently we succeeded in extracting suberin from the plant cell wall using the ionic liquid cholinium hexanoate. During extraction the native three-dimensional structure of suberin was partially preserved. In this study, we demonstrate that this preservation is the key for its ex situ reconstitution. Without any chemical additives or purification, the suberin composing macromolecules undergo self-association on the casting surface forming a film. Suberin films obtained show barrier properties similar to those of the suberin barrier in plants, including a potentially broad bactericidal effect.

Cholinium-based supported ionic liquid membranes: a sustainable route for carbon dioxide separation.

Aiming at full sustainability of CO2 separation processes, a series of supported ionic liquid membranes based on environmentally friendly cholinium carboxylate ionic liquids were successfully prepared. Their gas permeation properties were measured and high permselectivities were obtained for both CO2 /CH4 and CO2 /N2 .

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls.

Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE). Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (ß-glucosidases and endo-1,5-α-l-arabinosidase). Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of ß-glucosidase in cork secretome. BIOLOGICAL SIGNIFICANCE: Cork degradation by fungi remains largely overlook. Herein we aimed at understanding how A. nidulans colonise cork cell walls and how this relates to wood colonisation. To address this, the protein species consistently present in the secretome were analysed, as well as major alterations occurring in the substrate, including lignin degradation compounds being released. The obtained data demonstrate that this fungus has superficially attacked the cork cell walls apparently by using both enzymatic and Fenton-like reactions. Only a few polysaccharide degrading enzymes could be detected in the secretome which was dominated by protein species associated with autolysis. Lignin degradation was corroborated by the identification of some degradation products, but the suberin barrier in the cell wall remained virtually intact. Comparative proteomics revealed that cork and wood colonisation share a common set of enzymatic mechanisms.

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota.

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork-colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the significance of these fungi to have an interesting potential to be used in PCP bioremediation processes.