Charakterzation of the prenylated peptide natural product nanmandolamid and the key biosynthetic prenyltransferase NcbP

• Applicant: Dr. Florian Hubrich
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 576514428

The project aims to expand the potential of peptide prenyltransferases and their biosynthesized natural products of ribosomal origin. After intensive and targeted genome mining, we selected a cyanobacterial biosynthetic gene cluster. Based on our bioinformatic analyses, the biosynthetic gene cluster encodes a peptide prenyltransferase from a recently characterized peptide prenyltransferase family that diversifies the spectrum of prenylated amino acid side chains for this family, beyond the prenylation of tryptophan. Our preliminary work with heterologous expression of the biosynthetic pathway in Escherichia coli confirms our hypothesis and provides a good starting point for the project. To realize the project, we propose an approach with four subprojects: In the first subproject, we will optimize the production of the final natural product (termed nanmadolamide) in E. coli to obtain sufficient quantities for its structural characterization and bioactivity testing. Accordingly, the two additional milestones of the first subproject are the structural characterization of the natural product and the bioactivity testing of the natural product against pathogenic bacteria. The subprojects two and three, we will investigate the biosynthetic and biocatalytic potential of the peptide prenyltransferase NcbP, which plays a key role in the biosynthesis of nanmadolamide. The second subproject, we will generate different variants of the NcbP peptide substrate via site-directed mutagenesis and aim to prenylate them by co-production with NcbP in E. coli. The results obtained will provide information on the accepted substrate spectrum of NcbP. Subproject three, we will develop and optimize a enzymatic prenylation assay, test the dependence of the prenyltransferase NcbP on a peptide substrate recognition sequence and, if necessary, circumvent this by mutagenesis experiments. Based on this work and the results from subproject two, we will test non-natural peptide substrates in the biocatalytic assay to evaluate the biocatalytic potential of NcbP for the prenylation of various peptide substrates. Finally, we will investigate the nanmadolamid biosynthesis in the cyanobacterial host and/or via heterologous production in a cyanobacterial host. Overall, the project will expand the diversity of peptide prenyltransferases and the prenylated peptides accessible with them to harness their potential for drug candidate optimization and development.

• DFG Programme: Research Grants