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The product template (PT) domain of fungal NRPKSs catalyzes the regioselective aldol cyclization of the reactive poly-\u03b2-ketone chain during polyketide maturation and is essential for product fidelity of the NRPKSs. Herein, we discovered the fungal NRPKS MecA of which the polyketide cyclization steps do not involve a functional PT domain. MecA is encoded in the biosynthetic gene cluster (BGC) responsible for the formation of 5-hydroxy-7-methoxy-2,3-dimethylchromone (1) from the biocontrol fungus Trichoderma afroharzianum T22. Heterologous expression and in vitro enzymatic assays fully established a biosynthetic pathway of 1 that involves three enzymes: MecA, dehydratase MecB, and methyltransferase MecC. The PT domain of MecA does not contain the conserved catalytic histidine that is essential for aldol cyclization activity. Consistent with the predicted inactive PT domain, MecA was demonstrated to catalyze a C1\u2013C6 Claisen condensation as the first cyclization step of the pentaketide precursor, instead of the canonical aldol cyclization. Reconstitution of dissected MecA domains further demonstrated that the activity of MecA was not affected when the PT domain was removed. A Claisen Cyclase (CLC) domain that typically catalyzes Claisen cyclization in NRPKSs is also absent in MecA, implying the observed C1\u2013C6 cyclization step is catalyzed by the remaining domains. This work contributes to the diversity of NRPKS cyclization chemistry and enables further genome mining and engineering of NRPKSs that may lead to biosynthesis of new aromatic polyketides.<\/jats:p>","DOI":"10.1021\/acschembio.6c00730","type":"journal-article","created":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T15:02:15Z","timestamp":1792681335000},"source":"Crossref","is-referenced-by-count":0,"title":["Characterization of Chromone Biosynthetic Pathway Reveals a Noncanonical Mode of Fungal Polyketide Cyclization"],"prefix":"10.1021","author":[{"given":"Sydney","family":"Choi","sequence":"first","affiliation":[{"name":"University of California Los Angeles , , , ,","place":["Los Angeles, California, United States, 90095"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brandon","family":"Wong","sequence":"additional","affiliation":[{"name":"University of California Los Angeles , , , ,","place":["Los Angeles, California, United States, 90095"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zuodong","family":"Sun","sequence":"additional","affiliation":[{"name":"University of California Los Angeles , , , ,","place":["Los Angeles, California, United States, 90095"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1597-0141","authenticated-orcid":true,"given":"Yi","family":"Tang","sequence":"additional","affiliation":[{"name":"University of California Los Angeles , , , ,","place":["Los Angeles, California, United States, 90095"]},{"name":"University of California Los Angeles , , , ,","place":["Los Angeles, California, United States, 90095"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,22]]},"reference":[{"key":"2026102211020495800_cit1","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1039\/b911518h","article-title":"Cyclization of Aromatic Polyketides from Bacteria and Fungi","volume":"27","author":"Zhou","year":"2010","journal-title":"Nat. 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