title: Elucidation of the Biosynthesis of Echinulin and Other Fungal Secondary Metabolites in Ascomycetes
title: Aufklärung der Biosynthese von Echinulin und anderer pilzlicher Sekundärmetabolite in Ascomyceten
titleType: AlternativeTitle
Creators: name: Wohlgemuth, Viola
Publisher: name: Philipps-Universität Marburg
Year: 2025
Subjects: subject: Chemoenzymatische Synthese
subject: Neoechinulin
subject: Chemoenzymatic Synthesis
subject: Pharmacology &amp; therapeutics, prescription drugs
subject: Pharmakologie, Therapeutik
subject: Aspergillus ruber
subject: Fungal natural products
subject: Microbiology
subject: FOS: Biological sciences
subject: Prenyltransferase
subject: Secondary metabolites
subject: Naturstoffchemie
subject: Mikrobiologie
subject: Echinulin
subject: Pharmazeutische Biologie
subject: Indole alkaloids
subject: Biochemistry
subject: Natural Product Chemistry
subject: Pharmaceutical Biology
subject: Biotechnology
subject: Biochemie
subject: Biotechnologie

description: Fungi exemplify extraordinary structural diversity, enabling them to thrive across virtually every ecologi-cal niche on Earth. This remarkable adaptability is driven by their unparalleled metabolic flexibility, particularly their ability to biosynthesize secondary metabolites (SMs). While these compounds are not essential for immediate survival, they confer significant ecological advantages, enabling fungi to endure extreme conditions and compete most effectively within their ecological niche. For humanity, fungal SMs are both a curse, e.g. as harmful mycotoxins, and a blessing, offering immense pharmaceutical potential. Many fungal SMs, refined through microbial competition, display remarkable specificity for pharmacological receptors. Their discovery and medical application once heralded the "Golden Age of Antibiotics," with up to half of FDA-approved drugs now stemming from secondary metabolites or derivatives.
Despite decades of research, the explored knowledge of fungal metabolism represents only a fraction of their genomic potential. Alarmingly, the anthropogenic climate crisis is causing an irreversible loss of this untapped diversity and its pharmaceutical potential every day. This underscores the critical role of fungi in addressing major clinical challenges, making both the research and harnessing of fungal me-tabolism more urgent than ever, particularly for developing therapeutics against multidrug-resistant pathogens, neurodegenerative diseases, and for other pressing challenges of our time, including adapta-tion to climate and pollution crises.

This thesis aims to expand our understanding of fungal biosynthetic pathways and enrich the portfolio of native and biochemically modified SMs, as well as enzymes for further chemoenzymatic synthesis of those. In particular, the research focuses on the structures of indole diketopiperazine (DKP) alkaloids and lipophilicity-enhancing prenylation reactions, which can significantly enhance bioactivity of ad-dressed structures. Genome mining in A. ruber CBS135680 revealed several new biosynthetic gene clusters (BGCs). Further bioinformatics predicted two of them potentially involved in the echinulin and flavoglaucin group biosynthesis, both of high pharmaceutical interest due to their diverse biological activities. Surprisingly, only three prenyltransferases were found in the fungal genome, which stands in stark contrast to the highly prenylated structures of echinulin and neoechinulin. Their synthesis would typically require a significantly higher number of PTs, as, according to current research, each enzyme catalyzes only one specific prenylation reaction. Biochemical studies confirmed that the echinulin biosynthetic cascade is controlled by just two prenyltransferases. EchPT1, a reverse C2-prenyltransferase, catalyzes the first prenylation step on the DKP scaffold, forming preechinulin. The unique prenyltransferase EchPT2 catalyzes up to three consecutive dimethylallyl additions to preech-inulin and its dehydro forms, neoechinulins A and B, producing at least 23 native derivatives with two to four prenyl groups, exhibiting diverse prenylation patterns but a common C7-prenylation endpoint. This confirms EchPT2’s unique structural configuration, which enables it to accept and catalyze prenylation on its own mono-, di-, and triprenylated intermediates revealing a previously uncharacterized in vivo enzymatic potential for a fungal prenyltransferase. Detailed MS and NMR analyses of fungal extracts and in vitro enzyme-generated intermediates provided critical insights into the dynamics of this novel prenylation cascade. Furthermore, this work also marked the first documentation of two tetraprenylat-ed echinulin derivatives in A. ruber. 


Probing all eight reversely C2-prenylated stereoisomers of cyclo-Trp-Ala and cyclo-Trp-Pro variants with EchPT2 revealed a significant influence of substrate stereochemistry on the consecutive prenylation cascade. While triprenylated products were detected in all reactions, tetraprenylated derivatives emerged as the second most predominant products with (R,S)-configured isomers, resulting in the structural elucidation of four new triprenylated and four new tetraprenylated compounds. These findings ultimately highlight EchPT2’s remarkable potential as a biocatalyst for generating a novel generation of polyprenylated DKPs with potential pharmaceutical applications, especially as its precision in regio- and chemo selective catalysis enables access to difficult-to-alter ring positions on substrate scaffolds that are almost unattainable through conventional synthetic approaches.

In a collaborative projects with Dr. Nies and Dr. Ran, the biosynthesis of salicylaldehyde flavoglaucin in A. ruber was elucidated. The heterologous expression of the remaining third prenyltransferase in E. coli and the full flavoglaucin cluster in A. nidulans, combined with deletion and feeding experiments and detailed NMR structure analysis, confirmed the functions of the associated enzymes. These studies also established the role of the third A.ruber PT, FogH, in catalyzing the prenylation of a salicyl alcohol intermediate, which is subsequently oxidized to salicylaldehyde. Dr. Nies conducted the genetic experi-ments, Dr. Ran performed the isolation and structure elucidation of the compounds, while I contributed the overexpressed FogH prenyltransferase for biochemical characterization.
A further research project with Dr. Ran explored advanced structural modifications and ring expansion of initially prenylated DKPs through the activity of redox enzymes. A new non-heme FeII/2-oxoglutarate (FeII/2-OG)-dependent oxygenase, EAW25734, was shown to catalyze as double bond migration and hydroxylation within the dimethylallyl moiety of tryprostatin B. This enzymatic activity significantly expanded the chemical diversity of prenylated DKPs, highlighting its potential for generating novel, cyclic bioactive compounds. The characterized and overproduced enzyme was handed over to Dr. Ran, who conducted the subsequent analysis.

Overall, this work contributed to the exploration of the echinulin and flavoglaucin biosynthesis in A.ruber and expanded the biocatalytic toolbox with the three A. ruber prenyltransferases and one FeII/2-OG-dependent oxygenases. These enzymes enable complex regio- and stereospecific tailoring reactions on prenylated natural and synthetic DKP scaffolds. The findings lay the foundation for future studies aimed at engineering novel bioactive DKP derivatives and highlight the potential of combinatorial biosynthesis for producing structurally complex molecules that would be challenging to synthesize through conventional chemical methods.