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.

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.

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.

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.

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.

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.

Studies of benzothiazole and benzoselenazole squaraines as fluorescent probes for albumins detection.

Series of squaraine benzothiazole and benzoselenazole dyes were studied as possible fluorescent probes for the detection of proteins, particularly albumins. It was shown that majority of the studied squaraines give significant fluorescent response on the human serum albumin (HSA) and bovine serum albumin presence. For squaraine dyes with N-hexyl pendent groups (P-1, P-2, P-3, P-5) about 100-540-fold fluorescence intensity increase upon albumins addition was observed. At the same time in presence of other proteins, namely insulin, avidin from hen egg white, immunoglobulin G (IgG), carbonic anhydrase fluorescence enhancement values were considerably lower -up to 43 times in IgG presence. It was noted that generally, squaraines with long N-hexyl pendent groups demonstrate higher emission increase values upon proteins addition comparing with their analogues with short N-ethyl tails. It was shown that fluorescence intensity enhancement for benzothiazole squaraine dye P-3, relates linearly to the HSA concentration over the wide range-from 0.2 to 500 microg/ml. Together with noticeable selectivity of this dye to albumins, existence of wide dynamic range gives possibility to propose P-3 dye as probe for HSA quantification.