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In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research. ISSN:2160-1836

Fungi are competent producers of a large variety of natural products, including peptides. Peptides can be non-ribosomally encoded, referred to as non-ribosomal peptides (NRPs), or ribosomally encoded, referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). While many fungal NRPs have been studied to date, including prominent representatives such as penicillin and cyclosporin, the first fungal RiPP was not discovered until 2007. RiPPs have several properties that make them interesting targets for research and biotechnological applications: Their precursors are genetically encoded and can be genetically modified, they are processed by promiscuous enzymes, and they can adapt a high degree of complexity through various posttranslational modifications. In recent years, another chapter was added to research on RiPPs when it was discovered that a class of peptide-encoding precursor proteins, called KEX2-processed repeat proteins (KEPs), are widely distributed in the fungal kingdom and their functions are largely unknown. The aim of this thesis was to expand our understanding of ribosomally produced fungal peptides, with a special focus on peptides derived from KEPs. Chapter 1 provides an introduction to fungal RiPPs and their biosynthesis. First, the RiPP family of amatoxins/phallotoxins is discussed, which includes famous lethal toxins present in, e.g., the death cap, followed by the most recently discovered RiPP family, the borosins, which carry backbone N-methylations. Finally, the dikaritins are examined. These are functionally diverse cyclic peptides processed from precursor proteins with a KEP-like structure. In addition, the potential of RiPP biosynthetic pathways for the development of peptide therapeutics is discussed, both in terms of the identification of new fungal RiPPs and in terms of a synthetic biology approach using biosynthetic enzymes to generate new-to-nature peptide libraries that could be screened for bioactivity. Chapter 2 describes the structural and functional characterization of KEP-derived peptides in the agaricomycetes Coprinopsis cinerea, Lentinula edodes, Pleurotus ostreatus and Pleurotus eryngii. Genome mining identified several KEPs in C. cinerea. A selection of these proteins was successfully expressed in Pichia pastoris and processed by the yeast’s enzymatic machinery, resulting in an accumulation of KEP-derived peptides in the culture supernatant. This experimental setup was used to establish a protocol for the extraction and detection of KEP-derived peptides in fungal tissues by mass spectrometry. Using this KEP detection protocol, multiple KEP-derived peptides were confirmed in culture supernatant and tissue samples of C. cinerea, L. edodes, P. ostreatus and P. eryngii. In addition, we established a CRISPR-mediated knockout protocol for C. cinerea and generated knockout strains of six different KEPs, three KEX2 homologs and one KEX1 homolog. We found that the kep knockouts resulted in no discernible phenotype, while the kex knockouts revealed defects in mycelial growth and fruiting body formation. These results suggest that the investigated KEP-derived peptides do not have an organism-intrinsic role but rather function in the interaction with the biotic environment. The KEX proteases, on the other hand, apparently target a wide variety of substrates, including some with roles in mycelial growth and differentiation. 6 Chapter 3 presents the new draft genome sequences of four fungal species: Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria and Sphaerostilbella broomeana. The genomes were screened for precursor proteins or gene clusters involved in the synthesis of the peptide macrocycles mariannamides and the backbone N-methylated macrocycles verrucamides and broomeanamides. We performed BLAST searches to find potential RiPP precursor proteins of these peptides and included a general screen for borosin-producing precursor proteins. While this search was unsuccessful, we found non-ribosomal peptide synthase (NRPS) gene clusters in an antiSMASH screening that may be responsible for the biosynthesis of mariannamides, verrucamides and broomeanamides. In addition, we discovered several gene clusters encoding putative peptaibols, an antimicrobial class of peptides. Finally, Chapter 4 summarizes the findings of this thesis and gives an outlook on future experiments

Laut Studien in dem Vereinigten Königreich und Dänemark haben 73% – 79% der Technologie-KMUs nur unzureichenden oder keinen Zugang zu wissenschaftlichen Publikationen, die sie benötigen würden ! Das führt eine Politik ad absurdum, die der Wirtschaft hohe staatliche Förderungen für Forschung bereitstellt, aber öffentlich geförderte Forschungsergebnisse nicht zugänglich sind. Daher ist eine Strategie zur Umsetzung einer weitgehenden Öffnung wissenschaftlicher Beiträge vorgesehen, essentiell ist zu diesem Zweck, die Urheberrechtsreform 2015 zu erweitern: Der österreichische Gesetzgeber sollte das Urheberrecht so anpassen, dass etwa Autor/innen von wissenschaftlichen Publikationen unabhängig von Publikationsform und -ort das Recht haben, nach einer Embargofrist von maximal zwölf Monaten die Originalversion ihrer Publikation in einem Repositorium frei zugänglich zu machen. Zudem sollten beispielsweise große Datenbestände für wissenschaftliche Zwecke ohne Restriktionen für Suche, Vernetzung und Weiterverwendung (Content Mining) genutzt werden können.

Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a basic and applied research point of view, due to the discoveries of several novel posttranslational modifications of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics., Applied Microbiology and Biotechnology, 103 (14), ISSN:0175-7598