Combining reverse genetics and peptidomics to characterize ribosomal peptides in fungi

Abstract

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