The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimXCDK1 as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

Many small molecules of food origin may effect human health but lack an adequate description of their biological activity. To fill this knowledge gap, a first-line workflow is needed to assign putative functions, rank the endpoints for testing and guide wet-lab experiments. In this framework, the identification of potential biological targets can be used to probe the activity of orphan compounds using a so-called "target fishing" approach. Here, we present a proof of concept study using an in silico/in vitro target fishing approach on the fungal secondary metabolite atromentin. The procedure relies on a computational screening for activity identification coupled with experimental trials for dose-response characterization. Computational results identified estrogen receptors and 17-β-hydroxysteroid dehydrogenase as potential targets. Experiments confirmed a weak estrogenic activity, supporting the reliability of the procedure. Despite limited estrogenicity of atromentin, the proposed inhibition of 17-β-hydroxysteroid dehydrogenase should be considered as a source for endocrine disruptive effects.

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli ∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome.

The chemical synthesis of new lipophilic polyphenols with improved properties presents technical difficulties. Here we describe the selection, isolation and identification of lipolytic bacteria from food-processing industrial wastes, and their use for tailoring a new set of compounds with great interest in the food industry. These bacteria were employed to produce lipolytic supernatants, which were applied without further purification as biocatalysts in the chemoselective and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols. The chemoselectivity of polyphenols acylation/deacylation was analyzed, revealing the preference of the lipases for phenolic hydroxyl groups and phenolic esters. In addition, the alcoholysis of peracetylated 3,4-dihydroxyphenylglycol resulted in a series of lipophilic 2-alkoxy-2-(3,4-dihydroxyphenyl)ethyl acetate through an unexpected lipase-mediated etherification at the benzylic position. These new compounds are more lipophilic and retained their antioxidant properties. This approach can provide access to unprecedented derivatives of 3,4-dihydroxyphenylglycol with improved properties.

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