{"doi":"10.1101/2025.10.31.685832","title":"Co-opting the bacterial lipoprotein pathway for the biosynthesis of lipidated macrocyclic peptides","abstract":"Abstract Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally diverse natural products that possess a range of bioactivities, often acting as antibiotics, antifungals, or metallophores. In RiPP biosynthesis, different modifying enzymes install an array of chemical motifs onto a precursor peptide. A recently described RiPP-modifying enzyme, ChrH, catalyzes a remarkably complex reaction on its precursor peptide that results in a macrocycle, heterocycle, and S- methyl group. By leveraging comparative genomics, we demonstrate that the products from a subfamily of enzymes related to ChrH display unexpected structural diversity, including the production of unmethylated macrocyclic congeners and C-terminally modified proteins over 30 kDa in size. Several of these precursors contain a signal peptide, sending them for downstream maturation by the bacterial lipoprotein biosynthetic pathway. Like bacterial lipoproteins, such peptides are modified by addition of a diacylglycerol (DAG) group to the N-terminal cysteine residue along with acylation of the N-terminal amine. Genome mining reveals that these RiPP-lipoprotein hybrids, which we term DAG-RiPPs, are widespread across bacterial phyla and are likely involved in different biological roles. Together, these results highlight a novel maturation paradigm for membrane-bound RiPPs and lay the foundation for the discovery and bioengineering of other RiPP-lipoprotein hybrids. Significance Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of natural products that display antibiotic, antifungal, anticancer, and metal-binding activities. Their biosynthesis typically follows a common logic in which modifying enzymes install chemical motifs onto a precursor peptide, followed by proteolytic processing and export from the cell. Herein, we describe the discovery and biochemical characterization of a new class of lipid-RiPP hybrid products. These RiPPs contain a signal peptide that exploits the endogenous bacterial lipoprotein biosynthesis pathway for lipidation, membrane localization, and potential secretion. Genome mining shows that these lipid-peptide hybrids are widespread across bacterial phyla.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555800,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9522,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":469874,"name":"Lingyang Zhu","orcid":"0000-0002-6657-271X","position":1,"is_corresponding":false},{"id":1454649,"name":"K. Zhang","orcid":"0009-0000-3646-2395","position":2,"is_corresponding":false},{"id":1454650,"name":"Deborah A. Berthold","orcid":"0000-0003-3043-5462","position":3,"is_corresponding":false},{"id":256350,"name":"Wilfred A. van der Donk","orcid":"0000-0002-5467-7071","position":4,"is_corresponding":false},{"id":1028140,"name":"Jeff Y. Chen","orcid":"0000-0002-8507-8215","position":0,"is_corresponding":true}],"reference_count":49,"raw_metadata":null,"created_at":"2026-07-19T02:55:03.976486Z","pmid":"41279403","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}