Discovery of novel ribosomally synthesized and post-translationally modified peptides (RiPPs) originating from unusual two-domain precursors

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Secondary metabolites (natural products) represent nature’s “chemical playground of evolution”, as these compounds show an exceptional structural diversity. Often, these metabolites have potent activities and many of them are harnessed for pharmaceutical applications. The identification of novel secondary metabolites and the characterization of the corresponding biosynthetic enzymes are thus of great importance to understand the genetic and biochemical mechanisms that evolved this great chemical diversity. Further, the discovery of novel compounds with biological activities enables the development of new anti-infectives, which are urgently needed in light of increased antibiotic resistance. This project focuses on a unique group of ribosomally synthesized and post-translationally modified peptides (RiPPs) from bacteria. RiPPs are a large class of secondary metabolites that are derived from genetically encoded precursor peptides that undergo post-translation modification. The precursor peptides usually consist of a leader sequence and a core sequence. Different enzymes recognize the leader and install post-translational modifications on the core peptide, which is released as the mature natural product after proteolytic cleavage. Leader sequences are often short and lack clear structural features. As an exception, the family of the “nitrile hydratase leader peptides” (NHLP) are characterized by unusually long leader sequences that show similarity to the enzyme nitrile hydratase. In certain species of the order Burkholderiales, these NHLP precursors further appear as tandem genes, i.e., two copies of the precursor are present in a row. Intriguingly, in a few strains, these genes are fused into a single two-domain precursor, resulting in a large ~270 amino acid precursor protein. The function of the two leader domains, as well as the nature of the resulting RiPPs is currently unclear. The unique domain architecture of these precursors likely affects the maturation of the resulting peptides and represents an unexplored and exciting opportunity for the discovery of RiPPs with novel structures and functions. In this project, we aim to investigate these so-far uncharacterized RiPP gene clusters to identify the produced metabolite(s), characterize the biosynthetic enzymes, and explore the role of the two leader domains in the precursors. We will employ top-down approaches, including metabolomics and proteomics, in order to identify and isolate the RiPPs from the producing organisms. We will further complement these methods with a bottom-up approach where we will iteratively reconstitute the biosynthetic genes in Escherichia coli to monitor the successive installment of modifications in the precursor. To understand the function of the two NHLP domains in the precursor, we plan to crystallize the protein and solve its structure. Lastly, isolated RiPPs will be assessed for their bioactivities to evaluate their anti-infective potential.