{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:04:52Z","timestamp":1785409492062,"version":"3.56.0"},"posted":{"date-parts":[[2025,10,31]]},"group-title":"Biochemistry","reference-count":36,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["CBE 2300890"],"award-info":[{"award-number":["CBE 2300890"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["CBE 2300891"],"award-info":[{"award-number":["CBE 2300891"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["MCB 2128044"],"award-info":[{"award-number":["MCB 2128044"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]},{"award":["NIGMS RO1 GM125943"],"award-info":[{"award-number":["NIGMS RO1 GM125943"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,31]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry.\n                  <jats:italic>Micromonospora echinospora<\/jats:italic>\n                  subsp.\n                  <jats:italic>challisensis<\/jats:italic>\n                  NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens including vancomycin-resistant\n                  <jats:italic>Enterococcus faecalis<\/jats:italic>\n                  and methicillin-resistant\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  . 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\n                  <jats:italic>M. echinospora<\/jats:italic>\n                  ATCC 15837 containing both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (\n                  <jats:italic>tln<\/jats:italic>\n                  1 and\n                  <jats:italic>tln<\/jats:italic>\n                  5) that putatively encode pyridoxal 5\u2019-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/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 \u03b2-substitution reaction of\n                  <jats:italic>O<\/jats:italic>\n                  -phospho-L-serine with a preferential D-alanine nucleophile. Alanine racemization and Tln5 pseudodipeptide L-serine-C\u03b2-\n                  <jats:italic>N<\/jats:italic>\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.\n                <\/jats:p>\n                <jats:sec id=\"s5\">\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"685158v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.10.30.685158","type":"posted-content","created":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T00:10:47Z","timestamp":1761955847000},"source":"Crossref","is-referenced-by-count":0,"title":["Integrated metabolomic and genomic insights into amino acid incorporation within the hybrid polyketide-alkaloid antibiotic TLN-05220"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8589-9200","authenticated-orcid":false,"given":"Jennifer L.","family":"Cordoza","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1958-2218","authenticated-orcid":false,"given":"Rachel A.","family":"Johnson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0165-8395","authenticated-orcid":false,"given":"Andrew E.","family":"Whiteley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2036-6400","authenticated-orcid":false,"given":"Calista A.","family":"Horta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9821-6384","authenticated-orcid":false,"given":"Jayna C.","family":"Nicholas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6954-9863","authenticated-orcid":false,"given":"Diana M.","family":"Owen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5675-6291","authenticated-orcid":false,"given":"Laura","family":"Rodriguez-Velandia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5937-7855","authenticated-orcid":false,"given":"Gordon T.","family":"Luu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina Z.","family":"Petukhova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5486-2130","authenticated-orcid":false,"given":"Jackson T.","family":"Calhoun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9223-7977","authenticated-orcid":false,"given":"Laura M.","family":"Sanchez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6776-6455","authenticated-orcid":false,"given":"Shaun M. 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