Discovery of origamins: extensively modified ribosomal peptides with up to 43 post-translational modifications Christoph Martin Meier, Clarissa C. Forneris, Lida Vadakumchery, Alessandro Lott, Lucas Paoli and Jörn Piel ETH Zurich, Switzerland The discovery of polytheonamide showcased the diversity of chemical transformations combined in one ribosomally synthesized and post-translationally modified peptide (RiPP) natural product. 1 50 posttranslational modification catalyzed by only seven enzymes, including, among others, an epimerase, an N -methyltransferase, and a C -methyltransferase, illustrate the unprecedented biochemistry of a single RiPP biosynthetic gene cluster. This fundamental tailoring machinery enables folding of polytheonamide into the activity-conferring β-helical conformation with its picomolar cytotoxicity. 2,3 Isolated from the Entotheonella factor , a bacterial symbiont of the marine sponge Theonella swinhoei , polytheonamides were believed to be unique in nature. Genome mining efforts resulted in the discovery of origamins, a new class of RiPPs that putatively convergently evolved the same polytheonamide-like β-helix tailoring machinery. This new class of natural products is widely distributed among well-characterized and culturable bacterial genera. The basic features of origamin biosynthetic gene clusters are a precursor peptide with the polytheonamide-like NX 5 N pattern, an epimerase, epimerizing alternating amino acids in the peptide-backbone, and a SAM-dependent N -methyltransferase. The gene clusters further contain peptidase or a transport machinery and an immunity protein. Depending on additional maturases, the origamins were divided in subtypes. An additional maturase annotated as B 12 -dependent SAM C -methyltransferase distinguish type III origamin from types I and II. We utilized synthetic biology tools to study a type III origamin cluster possessing two precursor peptides. Heterologous expression of the cluster in the host Microvirgula aerodenitrificans , a host that contains all biosynthetic pathways to make cobalamin, produced a hypermodified RiPP. For the first precursor, the predicted 40 amino acid long core contains 43 modifications corresponding to more than 14 modifications per enzyme. The highly processive enzymes yield 18 epimerization, 13 N -methylations and 12 C -methylations. For the second precursor, the same types of modifications are observed. Concurrence of N -methylation and C -methylations at asparagine produce an as-yet uncharacterized amino acid. Heterologous expression of the precursors with all tailoring enzymes yielded inefficient transformations but co-expression of the immunity protein aided in reaching the fully modified compound and suggests potential cytotoxicity of the final product. Altogether, we characterized a full cluster of a new RiPP natural product class, which produces hypermodified peptides with the highest number of modifications per tailoring enzyme. With isotope-labeling, Marfrey's analysis and NMR, characterization of the unknown amino acid and full structure elucidation of the fully modified peptide is currently being pursued. The fully-modified peptides will also be tested for their bioactivity and cytotoxicity. References 1. F. Freeman et al. Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium. Nat. Chem. 9, 387-395 (2017) 2. Hamada et al. Solution structure of polytheonamide B, a highly cytotoxic nonribosomal polypeptide from marine sponge. JACS 132, 12941-12945 (2010) 3. Bhushan et al. Genome mining- and synthetic biology-enabled production of hypermodified peptides. Nat. Chem. 11, 931- 939 (2019)
P47
© The Author(s), 2022
Made with FlippingBook Learn more on our blog