Biology subjects

Schittmayer, M.

Publications and source records attributed to Schittmayer, M..

3 recordsLinked to original sources

bioRxiv2025

In vivo activation of the dia BGC allows consolidation of the biosynthetic pathways of diaporthin, dichlorodiaporthin, diaporthinic acid, and diaporthinol

Fungal secondary metabolites exhibit remarkable chemical diversity and biological activity, making them valuable sources of bioactive compounds. A group of polyketides, with a similar core structure, have been isolated previously from different fungi - the phytotoxins diaporthin and orthosporin, the mediocre antimicrobial dichlorodiaporthin, as well as diaporthinol and diaporthinic acid. Previous studies in Aspergillus oryzae suggest that diaporthin and orthosporin originate from a different biosynthetic gene cluster than dichlorodiaporthin, while the biosynthetic routes for diaporthinic acid and diaporthinol have not yet been identified. In this study, we successfully activated the dia biosynthetic gene cluster in Trichoderma reesei via transcription factor overexpression, leading to the identification of diaporthinic acid as the primary metabolite. Structural elucidation using NMR confirmed its identity, while bioactivity assays revealed no significant antimicrobial effects. Further, diaporthin, orthosporin, dichlorodiaporthin, and diaporthinol, as well as an isomer of alternariol were attributed to the same cluster. Gene deletion experiments demonstrated that Dia1, Dia4, and Dia5 are essential for diaporthinic acid biosynthesis, with Dia4 likely catalyzing the oxidation of dichlorodiaporthin yielding diaporthinic acid. Dia2 and Dia3 were found to be dispensable, challenging previous in vitro findings for the biosynthesis of dichlorodiaporthin. These findings provide new insights into the biosynthetic network of the dia BGC, expanding our understanding of natural product biosynthesis in filamentous fungi.

biochemistry↗

bioRxiv2024

Discovery of a novel antifungal compound, ilicicolin K, through genetic activation of the ilicicolin biosynthetic pathway in Trichoderma reesei

In the quest to discover novel antifungal agents and new antifungal production processes, we investigated the biosynthetic gene cluster (BGC) for ilicicolin H in the fungus Trichoderma reesei. While the BGC is silent under standard cultivation conditions, we achieved to activate it by over-expressing its transcription factor TriliR. Successful BGC activation was confirmed by RT-qPCR, proteomic and metabolomic analyses. Metabolomic profiling upon BGC expression revealed high-yield production of the supposed main product ilicicolin H. To elucidate the functionality of this BGC, we employed a combination of overexpression and deletions of individual biosynthetic gene cluster constituents. Deletion of triliA, encoding for the core polyketide synthase TriliA, completely ceased product formation, as expected. In contrast to previous heterologous expression experiments, we could demonstrate that the epimerase TriliE is necessary for the formation of ilicicolin H in the native host. While we hardly observed any of the previously reported side- or shunt products associated with heterologous ilicicolin H expression, we discovered a novel member of the ilicicolin family using a metabolomic molecular networking approach. This new compound, which we termed ilicicolin K, is expressed in substantial amounts in the genetically engineered Trichoderma reesei, enabling us to elucidate its structure by NMR. The structure of ilicicolin K is similar to that of ilicicolin H but differs by an additional hydroxylation and an intramolecular etherification of the hydroxyl group at the pyridone towards the tyrosine moiety of the molecule. Initial tests of ilicicolin K showed antifungal activity against Saccharomyces cerevisiae and Aspergillus nidulans with a similar minimum inhibitory concentration as ilicicolin H.

biochemistry↗

bioRxiv2020

Plasma glutathione status as indicator of pre-analytical centrifugation delay

Prolonged incubation of blood prior to plasma preparation can significantly influence the quality of the resulting data. Different markers for this pre-clinical variability have been proposed over the years but with limited success. In this study we explored the usefulness of glutathione (GSH) status, namely ratio of reduced to oxidized glutathione (GSH/GSSG), as potential marker of plasma preparation delay. For that purpose, blood from 20 healthy volunteers was collected into tubes with a cysteine quencher (N-ethylmaleimide; NEM) for GSH stabilization. Plasma preparation was delayed at room temperature for up to 3 hours and every hour, a plasma sample was prepared and the GSH/GSSG ratio measured. We report that over the course of the investigation, plasma concentrations of both GSH and GSSG increased linearly (R2 = 0.99 and 0.98, respectively). Since GSH increased at a much faster rate compared to GSSG, the GSH/GSSG ratio also increased linearly in a time dependent manner (R2 = 0.99). As GSH is an intracellular antioxidant, we speculated that this might stem from ongoing blood hemolysis, which was confirmed by the time dependent rise in lactate dehydrogenase (LDH) activity in the plasma samples. Moreover, we demonstrate that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not seem to influence downstream analysis of clinical parameters. In conclusion we propose that the glutathione status could be used as an indicator of the centrifugation delay prior to plasma preparation.

pathology↗