Cutin-derived oligomers induce hallmark plant immune responses.

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix-cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and mitogen-activated protein kinase activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of pattern-triggered immunity. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitor compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses. Further investigation is needed to understand how cutin breakdowns are perceived and to explore their potential use in agriculture.

An Ionic Liquid Extraction That Preserves the Molecular Structure of Cutin Shown by Nuclear Magnetic Resonance.

The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plant's outermost defensive barrier, the cuticle. Cutin influences many biological processes in planta; however, due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterization of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymeric materials are highly esterified, amorphous, and have a typical monomeric composition as demonstrated by solid-state NMR, complemented by spectroscopic, thermal, and x-ray scattering analyses. We performed a systematic study by solution-state NMR of cryogenically milled cutins extracted from tomatoes (Solanum lycopersicum 'Micro-Tom'; the wild type and the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE [GPAT6] and CUTIN SYNTHASE [CUS1] mutants). We resolved their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters. Our data demonstrate the existence of free hydroxyl groups in cutin and provide insight into how the mutations affect the esterification arrangement of cutin. The usage of ionic liquids for studying plant polyesters has advantages over conventional approaches, since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes essential tools to fingerprint the multifunctionality and the structure of cutin in planta.

Unveiling Concealed Functions of Endosymbiotic Bacteria Harbored in the Ascomycete Stachylidium bicolor.

Among the plethora of unusual secondary metabolites isolated from Stachylidium bicolor are the tetrapeptidic endolides A and B. Both tetrapeptides contain 3-(3-furyl)-alanine residues, previously proposed to originate from bacterial metabolism. Inspired by this observation, we aimed to identify the presence of endosymbiotic bacteria in S. bicolor and to discover the true producer of the endolides. The endobacterium Burkholderia contaminans was initially detected by 16S rRNA gene amplicon sequencing from the fungal metagenome and was subsequently isolated. It was confirmed that the tetrapeptides were produced by the axenic B. contaminans only when in latency. Fungal colonies unable to produce conidia and the tetrapeptides were isolated and confirmed to be free of B. contaminans A second endosymbiont identified as related to Sphingomonas leidyi was also isolated. In situ imaging of the mycelium supported an endosymbiotic relationship between S. bicolor and the two endobacteria. Besides the technical novelty, our in situ analyses revealed that the two endobacteria are compartmentalized in defined fungal cells, prevailing mostly in latency when in symbiosis. Within the emerging field of intracellular bacterial symbioses, fungi are the least studied eukaryotic hosts. Our study further supports the Fungi as a valuable model for understanding endobacterial symbioses in eukaryotes.IMPORTANCE The discovery of two bacterial endosymbionts harbored in Stachylidium bicolor mycelium, Burkholderia contaminans and Sphingomonas leidyi, is described here. Production of tetrapeptides inside the mycelium is ensured by B. contaminans, and fungal sporulation is influenced by the endosymbionts. Here, we illustrate the bacterial endosymbiotic origin of secondary metabolites in an Ascomycota host.

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.

Understanding fungal functional biodiversity during the mitigation of environmentally dispersed pentachlorophenol in cork oak forest soils.

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

The old 3-oxoadipate pathway revisited: new insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans.

Aspergilli play major roles in the natural turnover of elements, especially through the decomposition of plant litter, but the end catabolism of lignin aromatic hydrocarbons remains largely unresolved. The 3-oxoadipate pathway of their degradation combines the catechol and the protocatechuate branches, each using a set of specific genes. However, annotation for most of these genes is lacking or attributed to poorly- or un-characterised families. Aspergillus nidulans can utilise as sole carbon/energy source either benzoate or salicylate (upstream aromatic metabolites of the protocatechuate and the catechol branches, respectively). Using this cultivation strategy and combined analyses of comparative proteomics, gene mining, gene expression and characterisation of particular gene-replacement mutants, we precisely assigned most of the steps of the 3-oxoadipate pathway to specific genes in this fungus. Our findings disclose the genetically encoded potential of saprophytic Ascomycota fungi to utilise this pathway and provide means to untie associated regulatory networks, which are vital to heightening their ecological significance.

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.

Bringing up to date the toolkit for the catabolism of aromatic compounds in fungi: The unexpected 1,2,3,5-tetrahydroxybenzene central pathway.

Saprophytic fungi are able to catabolize many plant-derived aromatics, including, for example, gallate. The catabolism of gallate in fungi is assumed to depend on the five main central pathways, i.e., of the central intermediates' catechol, protocatechuate, hydroxyquinol, homogentisate and gentisate, but a definitive demonstration is lacking. To shed light on this process, we analysed the transcriptional reprogramming of the growth of Aspergillus terreus on gallate compared with acetate as the control condition. Surprisingly, the results revealed that the five main central pathways did not exhibit significant positive regulation. Instead, an in-depth analysis identified four highly expressed and upregulated genes that are part of a conserved gene cluster found in numerous species of fungi, though not in Aspergilli. The cluster comprises a monooxygenase gene and a fumarylacetoacetate hydrolase-like gene, which are recognized as key components of catabolic pathways responsible for aromatic compound degradation. The other two genes encode proteins with no reported enzymatic activities. Through functional analyses of gene deletion mutants in Aspergillus nidulans, the conserved short protein with no known domains could be linked to the conversion of the novel metabolite 5-hydroxydienelatone, whereas the DUF3500 gene likely encodes a ring-cleavage enzyme for 1,2,3,5-tetrahydroxybenzene. These significant findings establish the existence of a new 1,2,3,5-tetrahydroxybenzene central pathway for the catabolism of gallate and related compounds (e.g. 2,4,6-trihydroxybenzoate) in numerous fungi where this catabolic gene cluster was observed.

Fungi: friends or foes-an outreach science initiative for the collection of airborne fungal spores by high school students.

Fungi mostly reproduce through spores that are adapted for airborne dispersal; hence, fungal spores (and fungi) are found virtually everywhere. Fungi can be "friends or foes." Our friends include fungi used in the food and biotech industries, fungi that contribute to the cycling of carbon and nutrients, and those involved in the decontamination of polluted soils and/or water, to mention just a few examples. Many species, however, are foes-they are detrimental to plants, animals, and/or humans. Annually, >1.5 million people die due to invasive fungal infections. With the aim of enhancing microbiology literacy and the understanding of microbial concepts, we set up a project for the collection of airborne spores (the principal agent through which human airways are exposed to fungi). Students from five high schools in the Oeiras municipality partnered with us as citizen scientists; they carried out sampling by collecting fungal spores on adhesive stickers. The fungal spores collected by the students were subsequently processed in the schools and our research laboratory. Results obtained by the students themselves revealed a large variety of fungal species capable of growing in a rich medium at 30°C. In the research laboratory, using selective isolation conditions, 40 thermotolerant fungi were isolated, 32 of which were taxonomically identified as aspergilla, mostly from within the Aspergillus fumigatus taxa, yet exhibiting high genetic heterogeneity. The protocols and results were presented to the students, who were made aware of the local dispersal of airborne fungal spores, including some from potentially pathogenic fungi. Through carrying out scientific activities, the students developed both the interest and the self-confidence needed to implement future environmental investigations.

NmrB (AN9181) expression is activated under oxidative stress conditions acting as a metabolic repressor of Aspergillus nidulans.

Aspergilli comprise a diversity of species that have been extensively studied due to their catabolic diversity, biotechnological and ecological value, and pathogenicity. An impressive level of structural and functional conservation has been shown for aspergilli, regardless of many (yet) cryptic genomic elements. We have hypothesized the existence of conserved genes responsive to stress in aspergilli. To test the hypothesis of such conserved stress regulators in aspergilli, a straightforward computational strategy integrating well-established bioinformatic tools was used as the starting point. Specifically, five transcriptome-based datasets on exposure to organic compounds were used, covering three distinct Aspergillus species. Among the identified up-regulated genes, only one gene showed the same response in all conditions, AN9181. This gene encodes a protein containing a phenylcoumaran benzylic ether reductase-like domain and a Nitrogen metabolite repressor regulator domain (NmrA). Deletion of this gene caused significant phenotypic alterations compared to that of the parental strain across diverse conditions. Specifically, the deletion of AN9181 raised the mutant's metabolic activity in different nitrogen sources. The acquired data supports that AN9181 acts by repressing (slowing down) A. nidulans growth when exposed to aromatic compounds in a concentration dependent manner. The same phenotype was observed for amphotericin B. Finally, AN9181 underwent differential upregulation under oxidative stress conditions. Collectively, the data suggest that AN9181, herein assigned as NmrB (Nitrogen Metabolite Repression Regulator B), builds up the genetic machinery of perception of oxidative stress by negatively regulating growth under such conditions.

Purification of archetypal soybean root suberin mostly comprising alka(e)noic acids using an ionic liquid catalyst.

Soybean (Glycine max) is an increasingly relevant crop due to its economic importance and also a model plant for the study of root symbiotic associations with nodule forming rhizobia. Plant polyesters mediate plant-microbe interactions with both pathogenic and beneficial microbes; suberin has been hypothesized to play a key role during the early steps of rhizobia attachment to the root. The downside is that suberin chemistry in soybean root is still scarcely studied. This study addresses this outstanding question by reporting a straightforward workflow for a speedy purification of suberin from soybean root and for its subsequent detailed chemical analysis. To purify suberin, cholinium hexanoate (an ionic liquid) was used as the catalyst. The ensuing suberin is highly esterified as observed by a precise Nuclear Magnetic Resonance quantification of each ester type, discriminating between primary and acylglycerol esters. Moreover, the composing hydrolysable monomers detected through GC-MS revealed that hexadecanoic acid is the most abundant monomer, similar to that reported before by others. Overall, this study highlights the adequacy of the ionic liquid catalyst for the isolation of suberin from soybean roots, where the polymer natural abundance is low, and builds new knowledge on the specificities of its chemistry; essential to better understand the biological roles of suberin in roots.

Rosin from Pinus pinaster Portuguese forests shows a regular profile of resin acids.

Pinus pinaster forestry occupies >20% of the forest ecosystem area in the continental territory of Portugal with a high impact on the national economy. This species' major derived non-wood product is oleoresin, the raw material for rosin production. Rosin comprises mainly a blend of resin acids and has broad industrial and pharmaceutical applications. Oleoresin production in Portugal has been progressively reduced due to low-cost producers in other countries; currently, it reaches only 2% of the existing P. pinaster trees. To support this value chain, the chemical fingerprint of rosin derived from the national forest requires focused analysis. In the present study, we collected oleoresin within seven geographically distinct pure P. pinaster forests in two consecutive collection years. A high-resolution nuclear magnetic resonance (NMR) method was used to quantify the diversity of resin acids in the corresponding rosin samples. Overall, the acquired data highlighted that the profile of resin acids in P. pinaster rosin produced in Portugal is highly regular, regardless of the forest location, having as the major constituents abietic acid and dehydroabietic acid. The diversity of resin acids is possibly influenced, to a minor extent, by some edaphoclimatic factors.

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

Ionic liquids: a pathway to environmental acceptability.

Ionic liquids were initially proposed as replacements for conventional organic solvents; however, their chemistry has developed remarkably and offers unexpected opportunities in numerous fields, ranging from electrochemistry to biology. As a consequence of ionic liquids advancing towards potential and actual applications, a comprehensive determination of their environmental, health and safety impact is now required. This critical review aims to present an overview of the current understanding of the toxicity and environmental impact of the principal ionic liquid groups, and highlights some emerging concerns. Each cation type is considered separately, examining the significance of the biological data, and identifying the most critical questions, some yet unresolved. The need for more, and more detailed, studies is highlighted (176 references).

Untargeted Metabolomics Sheds Light on the Secondary Metabolism of Fungi Triggered by Choline-Based Ionic Liquids.

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.

Finding a Needle in a Haystack: Producing Antimicrobial Cutin-Derived Oligomers from Tomato Pomace.

Agro-industrial residues comprise a rich diversity of plant polymers and bioactive compounds, constituting promising sources for the development of materials, including bioplastics, and food supplements, among other applications. In particular, the polyester cutin is abundant in fruit peel, a plentiful constituent of pomace agro-industrial residues. The potential of diverse fruit pomaces as a source for the development of cutin-derived materials/products has been extensively sought out. This study expands the established knowledge: it sets proof of concept for the production of antimicrobial oligomers from cutin-rich materials isolated in a single step from tomato pomaces generated by two remote agro-industries. Specifically, it first analyzed how the chemical signature (nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS)) of a pomace (and of its major constituents) mirrors that of the corresponding cutin-rich material isolated using an ionic liquid extractant. The cutin-rich materials were then deconstructed (using mild hydrolyses), and the resultant mixtures were chemically characterized and screened for bactericidal activity against Escherichia coli and Staphylococcus aureus. The presence of esterified structures, linear and/or branched, likely comprising dioic acids as a major building block (but not exclusively) is a prerequisite for activity against E. coli but not against S. aureus that was susceptible to monomers as well. Further studies are required to optimize the production of broad bactericidal oligomers from any cutin-rich pomace source, moving ahead toward their circular usage.

Twists and Turns in the Salicylate Catabolism of Aspergillus terreus, Revealing New Roles of the 3-Hydroxyanthranilate Pathway.

In fungi, salicylate catabolism was believed to proceed only through the catechol branch of the 3-oxoadipate pathway, as shown, e.g., in Aspergillus nidulans However, the observation of a transient accumulation of gentisate upon the cultivation of Aspergillus terreus in salicylate medium questions this concept. To address this, we have run a comparative analysis of the transcriptome of these two species after growth in salicylate using acetate as a control condition. The results revealed the high complexity of the salicylate metabolism in A. terreus with the concomitant positive regulation of several pathways for the catabolism of aromatic compounds. This included the unexpected joint action of two pathways-3-hydroxyanthranilate and nicotinate-possibly crucial for the catabolism of aromatics in this fungus. Importantly, the 3-hydroxyanthranilate catabolic pathway in fungi is described here for the first time, whereas new genes participating in the nicotinate metabolism are also proposed. The transcriptome analysis showed also for the two species an intimate relationship between salicylate catabolism and secondary metabolism. This study emphasizes that the central pathways for the catabolism of aromatic hydrocarbons in fungi hold many mysteries yet to be discovered.IMPORTANCE Aspergilli are versatile cell factories used in industry for the production of organic acids, enzymes, and pharmaceutical drugs. To date, bio-based production of organic acids relies on food substrates. These processes are currently being challenged to switch to renewable nonfood raw materials-a reality that should inspire the use of lignin-derived aromatic monomers. In this context, aspergilli emerge at the forefront of future bio-based approaches due to their industrial relevance and recognized prolific catabolism of aromatic compounds. Notwithstanding considerable advances in the field, there are still important knowledge gaps in the central catabolism of aromatic hydrocarbons in fungi. Here, we disclose a novel central pathway, 3-hydroxyanthranilate, defying previously established ideas on the central metabolism of the aromatic amino acid tryptophan in Ascomycota We also observe that the catabolism of the aromatic salicylate greatly activated the secondary metabolism, furthering the significance of using lignin-derived aromatic hydrocarbons as a distinctive biomass source.

Tailoring amphotericin B as an ionic liquid: an upfront strategy to potentiate the biological activity of antifungal drugs.

Aspergillus species are the primary cause of invasive aspergillosis, which afflicts hundreds of thousands of patients yearly, with high mortality rates. Amphotericin B is considered the gold standard in antifungal drug therapy, due to its broad-spectrum activity and rarely reported resistance. However, low solubility and permeability, as well as considerable toxicity, challenge its administration. Lipid formulations of amphotericin B have been used to promote its slow release and diminish toxicity, but these are expensive and adverse health effects of their prolonged use have been reported. In the past decades, great interest emerged on converting biologically active molecules into an ionic liquid form to overcome limitations such as low solubility or polymorphisms. In this study, we evaluated the biological activity of novel ionic liquid formulations where the cholinium, cetylpyridinium or trihexyltetradecylphosphonium cations were combined with an anionic form of amphotericin B. We observed that two formulations increased the antifungal activity of the drug, while maintaining its mode of action. Molecular dynamics simulations showed that higher biological activity was due to increased interaction of the ionic liquid with the fungal membrane ergosterol compared with amphotericin B alone. Increased cytotoxicity could also be observed, probably due to greater interaction of the cation with cholesterol, the main sterol in animal cells. Importantly, one formulation also displayed antibacterial activity (dual functionality), likely preserved from the cation. Collectively, the data set ground for the guided development of ionic liquid formulations that could improve the administration, efficacy and safety of antifungal drugs or even the exploitation of their dual functionality.

Quantification of Structure-Property Relationships for Plant Polyesters Reveals Suberin and Cutin Idiosyncrasies.

Polyesters, as they exist in planta, are promising materials with which to begin the development of "green" replacements. Cutin and suberin, polyesters found ubiquitously in plants, are prime candidates. Samples enriched for plant polyesters, and in which their native backbones were largely preserved, were studied to identify "natural" structural features; features that influence critical physical properties. Quantitative nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X-ray scattering methods were used to quantify structure-property relationships in these polymeric materials. The degree of esterification, namely, the presence of acylglycerol linkages in suberin and of secondary esters in cutin, and the existence of mid-chain epoxide groups defining the packing of the aliphatic chains were observed. This packing determines polymer crystallinity, the resulting crystal structure, and the melting temperature. To evaluate the strength of this rule, tomato cutin from the same genotype, studying wild-type plants and two well-characterized mutants, was analyzed. The results show that cutin's material properties are influenced by the amount of unbound aliphatic hydroxyl groups and by the length of the aliphatic chain. Collectively, the acquired data can be used as a tool to guide the selection of plant polyesters with precise structural features, and hence physicochemical properties.