{"doi":"10.1101/2025.10.30.685158","title":"Integrated metabolomic and genomic insights into amino acid incorporation within the hybrid polyketide-alkaloid antibiotic TLN-05220","abstract":"Abstract Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus . The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety, yet the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 containing both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes ( tln 1 and tln 5) that putatively encode pyridoxal 5’-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS), identified L-alanine, L-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective β-substitution reaction of O -phospho-L-serine with a preferential D-alanine nucleophile. Alanine racemization and Tln5 pseudodipeptide L-serine-Cβ- N -D-alanine (D,L-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics. Graphical Abstract","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":579688,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.951,"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":1316633,"name":"Rachel A. Johnson","orcid":"0000-0003-1958-2218","position":1,"is_corresponding":false},{"id":1202832,"name":"Andrew E. Whiteley","orcid":"0000-0002-0165-8395","position":2,"is_corresponding":false},{"id":1490218,"name":"Calista A. Horta","orcid":"0000-0002-2036-6400","position":3,"is_corresponding":false},{"id":1028229,"name":"Jayna Nicholas","orcid":"0000-0001-9821-6384","position":4,"is_corresponding":false},{"id":1490219,"name":"Diana M. Owen","orcid":"0009-0009-6954-9863","position":5,"is_corresponding":false},{"id":1396754,"name":"Laura Rodriguez-Velandia","orcid":"0009-0001-5675-6291","position":6,"is_corresponding":false},{"id":412231,"name":"Gordon T. Luu","orcid":"0000-0001-5937-7855","position":7,"is_corresponding":false},{"id":554834,"name":"Valentina Petukhova","orcid":null,"position":8,"is_corresponding":false},{"id":1490220,"name":"Jackson T. Calhoun","orcid":"0009-0000-5486-2130","position":9,"is_corresponding":false},{"id":263087,"name":"Laura M. Sanchez","orcid":"0000-0001-9223-7977","position":10,"is_corresponding":false},{"id":305305,"name":"Shaun M. K. McKinnie","orcid":"0000-0001-6776-6455","position":11,"is_corresponding":false},{"id":1490221,"name":"Katharine R. Watts","orcid":"0000-0002-6421-8370","position":12,"is_corresponding":false},{"id":356291,"name":"Jennifer L. Cordoza","orcid":"0000-0001-8589-9200","position":0,"is_corresponding":true}],"reference_count":35,"raw_metadata":null,"created_at":"2026-07-19T02:58:34.718602Z","pmid":"41279854","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":[]}