{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T14:07:39Z","timestamp":1789567659641,"version":"build-2803163510"},"reference-count":29,"publisher":"American Chemical Society (ACS)","issue":"36","license":[{"start":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T00:00:00Z","timestamp":1787961600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP22gm1610007"],"award-info":[{"award-number":["JP22gm1610007"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP25ama121001"],"award-info":[{"award-number":["JP25ama121001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP25ama121023"],"award-info":[{"award-number":["JP25ama121023"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP25ama121038"],"award-info":[{"award-number":["JP25ama121038"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP25ama121040"],"award-info":[{"award-number":["JP25ama121040"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000646","name":"Japan Society for the Promotion of Science London","doi-asserted-by":"publisher","award":["JP25KJ0585"],"award-info":[{"award-number":["JP25KJ0585"]}],"id":[{"id":"10.13039\/501100000646","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["23K24038"],"award-info":[{"award-number":["23K24038"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["25H01566"],"award-info":[{"award-number":["25H01566"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP23K07918"],"award-info":[{"award-number":["JP23K07918"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP25K01957"],"award-info":[{"award-number":["JP25K01957"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJAN23D4"],"award-info":[{"award-number":["JPMJAN23D4"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJFR205W"],"award-info":[{"award-number":["JPMJFR205W"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJSP2114"],"award-info":[{"award-number":["JPMJSP2114"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Flavonoids are polyphenolic natural products predominantly isolated from plants and exhibit a diverse array of biological activities. Because they serve as important pharmaceuticals and nutraceuticals, there is a strong demand for their sustainable supply. Chlorflavonin is a rare fungal flavonoid with potent antitubercular activity. While its biosynthetic enzymes are expected to be valuable tools for application in fungal production of flavonoids, the biosynthetic pathway remains unknown. Here, we elucidate the complete biosynthetic pathway for chlorflavonin through detailed functional analysis of each biosynthetic enzyme. Previously, stepwise and straightforward introduction of 3-, 7-, and 8-methoxy; 2\u2032-hydroxy; and 3\u2032-chloro functionalities have been proposed. In contrast to this proposal, we uncovered an intricate biosynthetic route involving a dynamic interconversion between the 6- and 8-methoxy forms of flavonoid skeletons mediated by the chalcone isomerase CfvF and the flavin-dependent oxygenase CfvI. CfvF interconverted the 6- and 8-methoxyflavanones, likely via a chalcone intermediate. CfvI oxidized the chemically inert 2,3-double bond of flavanone, giving the hemiacetal product. Although these enzymes generate products existing in equilibrium states, respective downstream enzymes catalyze selective conversion of one specific species, facilitating smooth progression to the final product. We also solved the crystal structure of CfvK, the dehydratase that selectively converts the 8-methoxy form of the CfvI product. Through analyzing the structure complexed with its substrate and product and site-directed mutagenesis, key residues determining the substrate selectivity were identified. Our comprehensive analysis established a rational framework for preparing 46 flavonoids, including unnatural ones, setting the stage for fungal production of structurally diverse flavonoids.<\/jats:p>","DOI":"10.1021\/jacs.6c13856","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T12:20:38Z","timestamp":1788351638000},"page":"39199-39210","source":"Crossref","is-referenced-by-count":0,"title":["The Biosynthesis of\na Fungal Flavonoid, Chlorflavonin,\nPrecisely Controlled by Two Types of Equilibrium States"],"prefix":"10.1021","volume":"148","author":[{"given":"Sho","family":"Furumura","sequence":"first","affiliation":[{"name":"Tohoku University , , ,","place":["Sendai, Japan, 980-8578"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4673-4478","authenticated-orcid":true,"given":"Taro","family":"Ozaki","sequence":"additional","affiliation":[{"name":"Tohoku University , , ,","place":["Sendai, Japan, 980-8578"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuya","family":"Hasegawa","sequence":"additional","affiliation":[{"name":"Japan Synchrotron Radiation Research Institute , , 1-1-1 Kouto, Sayo , , ,","place":["Sayo, Hyogo, Japan, 679-5198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hironori","family":"Hayashi","sequence":"additional","affiliation":[{"name":"Tohoku University , , 2-1, Seiryo-Machi, Aoba-Ku , , ,","place":["Sendai, Miyagi, Japan, 980-8575"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masao","family":"Inoue","sequence":"additional","affiliation":[{"name":"Yokohama City University , , 1-7-29, Suehiro-Cho, Tsurumi-Ku , ,","place":["Yokohama, Japan, 230-0045"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaori","family":"Fujimoto","sequence":"additional","affiliation":[{"name":"Hokkaido University , , North 21 West 11 , ,","place":["Sapporo, Japan, 001-0021"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8442-8642","authenticated-orcid":true,"given":"Yohei","family":"Morishita","sequence":"additional","affiliation":[{"name":"Tohoku University , , ,","place":["Sendai, Japan, 980-8578"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3199-6931","authenticated-orcid":true,"given":"Mitsunori","family":"Ikeguchi","sequence":"additional","affiliation":[{"name":"Yokohama City University , , 1-7-29, Suehiro-Cho, Tsurumi-Ku , ,","place":["Yokohama, Japan, 230-0045"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Kumasaka","sequence":"additional","affiliation":[{"name":"Japan Synchrotron Radiation Research Institute , , 1-1-1 Kouto, Sayo , , ,","place":["Sayo, Hyogo, Japan, 679-5198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6622-2752","authenticated-orcid":true,"given":"Eiichi N.","family":"Kodama","sequence":"additional","affiliation":[{"name":"Tohoku University , , 2-1, Seiryo-Machi, Aoba-Ku , , ,","place":["Sendai, Miyagi, Japan, 980-8575"]},{"name":"Tohoku University , , 2-1, Seiryo-Machi, Aoba-Ku , , ,","place":["Sendai, Miyagi, Japan, 980-8575"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4965-7383","authenticated-orcid":true,"given":"Tohru","family":"Taniguchi","sequence":"additional","affiliation":[{"name":"Hokkaido University , , North 21 West 11 , ,","place":["Sapporo, Japan, 001-0021"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8903-392X","authenticated-orcid":true,"given":"Teigo","family":"Asai","sequence":"additional","affiliation":[{"name":"Tohoku University , , ,","place":["Sendai, Japan, 980-8578"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,29]]},"reference":[{"key":"2026091609125382300_cit1","doi-asserted-by":"publisher","first-page":"943","DOI":"10.3389\/fpls.2019.00943","article-title":"The Origin and Evolution of Plant\nFlavonoid Metabolism","volume":"10","author":"Yonekura-Sakakibara","year":"2019","journal-title":"Front. Plant Sci."},{"key":"2026091609125382300_cit2","doi-asserted-by":"publisher","first-page":"12824","DOI":"10.3390\/ijms222312824","article-title":"The Flavonoid Biosynthesis Network in Plants","volume":"22","author":"Liu","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"2026091609125382300_cit3","doi-asserted-by":"publisher","first-page":"485","DOI":"10.1104\/pp.126.2.485","article-title":"Flavonoid\nBiosynthesis. A Colorful Model for Genetics, Biochemistry, Cell Biology,\nand Biotechnology","volume":"126","author":"Winkel-Shirley","year":"2001","journal-title":"Plant Physiol."},{"key":"2026091609125382300_cit4","doi-asserted-by":"publisher","first-page":"634","DOI":"10.1016\/j.biotechadv.2016.02.012","article-title":"Microbial Production\nof Natural and Non-Natural Flavonoids: Pathway Engineering, Directed\nEvolution and Systems\/Synthetic Biology","volume":"34","author":"Pandey","year":"2016","journal-title":"Biotechnol.\nAdv."},{"key":"2026091609125382300_cit5","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1080\/09168451.2019.1690976","article-title":"Reconstitution\nof Biosynthetic Machinery of Fungal Natural Products in Heterologous\nHosts","volume":"84","author":"Oikawa","year":"2020","journal-title":"Biosci., Biotechnol., Biochem."},{"key":"2026091609125382300_cit6","doi-asserted-by":"publisher","first-page":"2484","DOI":"10.1021\/acs.jnatprod.2c00487","article-title":"Total\nHeterologous Biosynthesis of\nFungal Natural Products in Aspergillus nidulans","volume":"85","author":"Chiang","year":"2022","journal-title":"J. Nat. Prod."},{"key":"2026091609125382300_cit7","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202214609","article-title":"Conversion\nof\nPolyethylenes into Fungal Secondary Metabolites","volume":"62","author":"Rabot","year":"2023","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091609125382300_cit8","doi-asserted-by":"publisher","first-page":"5222","DOI":"10.1021\/jacs.2c12285","article-title":"Polystyrene\nUpcycling into Fungal Natural Products and a Biocontrol Agent","volume":"145","author":"Rabot","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"2026091609125382300_cit9","doi-asserted-by":"publisher","first-page":"388","DOI":"10.7164\/antibiotics.22.388","article-title":"Chlorflavonin, A New Antifungal Antibiotic","volume":"22","author":"Richards","year":"1969","journal-title":"J. Antibiot."},{"key":"2026091609125382300_cit10","doi-asserted-by":"publisher","first-page":"718","DOI":"10.1128\/am.19.5.718-720.1970","article-title":"Production\nof Chlorflavonin,\nan Antifungal Metabolite of Aspergillus candidus","volume":"19","author":"Munden","year":"1970","journal-title":"Appl. Microbiol."},{"key":"2026091609125382300_cit11","doi-asserted-by":"publisher","first-page":"1031","DOI":"10.7164\/antibiotics.54.1031","article-title":"CJ-19,784, a New Antifungal\nAgent from a Fungus, Acanthostigmella sp","volume":"54","author":"Watanabe","year":"2001","journal-title":"J. Antibiot."},{"key":"2026091609125382300_cit12","doi-asserted-by":"publisher","first-page":"401","DOI":"10.1016\/S0167-4838(98)00083-1","article-title":"Biosynthesis\nof 2-aceto-2-hydroxy\nacids: acetolactate synthases and acetohydroxyacid synthases","volume":"1385","author":"Chipman","year":"1998","journal-title":"Biochim. Biophys. Acta"},{"key":"2026091609125382300_cit13","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1007\/s00726-005-0297-3","article-title":"Acetohydroxyacid synthase and its\nrole in the biosynthetic\npathway for branched-chain amino acids","volume":"31","author":"McCourt","year":"2006","journal-title":"Amino\nAcids"},{"key":"2026091609125382300_cit14","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1021\/acsinfecdis.7b00055","article-title":"Chlorflavonin\nTargets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic\nKilling of Mycobacterium tuberculosis","volume":"4","author":"Rehberg","year":"2018","journal-title":"ACS Infect. Dis."},{"key":"2026091609125382300_cit15","doi-asserted-by":"publisher","first-page":"522","DOI":"10.1021\/acs.jnatprod.4c01374","article-title":"Screening,\nDiscovery,\nand Optimization of the Natural Antitubercular Chlorflavonin from\na Marine-Derived Fungal Library","volume":"88","author":"Wang","year":"2025","journal-title":"J. Nat. Prod."},{"key":"2026091609125382300_cit16","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1021\/acs.jnatprod.2c01027","article-title":"Identification\nand Functional Characterization of Fungal Chalcone Synthase and Chalcone\nIsomerase","volume":"86","author":"Furumura","year":"2023","journal-title":"J. Nat. Prod."},{"key":"2026091609125382300_cit17","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1715954115","article-title":"Linking Secondary Metabolites to Gene Clusters through Genome Sequencing\nof Six Diverse Aspergillus Species","volume":"115","author":"Kj\u00e6rb\u00f8lling","year":"2018","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026091609125382300_cit18","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202215529","article-title":"Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by\nGenome Mining","volume":"62","author":"Zhang","year":"2023","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091609125382300_cit19","doi-asserted-by":"publisher","first-page":"941","DOI":"10.1104\/pp.004820","article-title":"A Cluster of Genes Encodes the Two\nTypes of Chalcone\nIsomerase Involved in the Biosynthesis of General Flavonoids and Legume-Specific\n5-Deoxy(Iso)Flavonoids in Lotus japonicus","volume":"131","author":"Shimada","year":"2003","journal-title":"Plant Physiol."},{"key":"2026091609125382300_cit20","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate Structure Prediction\nof Biomolecular Interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"2026091609125382300_cit21","doi-asserted-by":"publisher","first-page":"1439","DOI":"10.1002\/anie.201306952","article-title":"Enzymatic Conversion\nof Flavonoids Using Bacterial Chalcone Isomerase and Enoate Reductase","volume":"53","author":"Gall","year":"2014","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091609125382300_cit22","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1002\/(SICI)1097-0134(19990601)35:4&lt;425::AID-PROT6&gt;3.0.CO;2-1","article-title":"High-Resolution Structures of Scytalone\nDehydratase-Inhibitor Complexes Crystallized at Physiological PH","volume":"35","author":"Wawrzak","year":"1999","journal-title":"Proteins"},{"key":"2026091609125382300_cit23","doi-asserted-by":"publisher","first-page":"17926","DOI":"10.1074\/jbc.M109.009258","article-title":"The C-Glycosylation\nof Flavonoids in Cereals","volume":"284","author":"Brazier-Hicks","year":"2009","journal-title":"J. Biol. Chem."},{"key":"2026091609125382300_cit24","doi-asserted-by":"publisher","first-page":"7292","DOI":"10.1021\/jo00026a020","article-title":"Epoxidation of Flavones\nby Dimethyldioxirane","volume":"56","author":"Adam","year":"1991","journal-title":"J. Org. Chem."},{"key":"2026091609125382300_cit25","doi-asserted-by":"publisher","first-page":"882","DOI":"10.1104\/pp.104.056747","article-title":"Molecular\nand Biochemical Characterization\nof 2-Hydroxyisoflavanone Dehydratase. Involvement of Carboxylesterase-Like\nProteins in Leguminous Isoflavone Biosynthesis","volume":"137","author":"Akashi","year":"2005","journal-title":"Plant Physiol."},{"key":"2026091609125382300_cit26","doi-asserted-by":"publisher","first-page":"15519","DOI":"10.1021\/jacs.9b08935","article-title":"Efficient Reconstitution of Basidiomycota\nDiterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences","volume":"141","author":"Liu","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"2026091609125382300_cit27","doi-asserted-by":"publisher","first-page":"1372","DOI":"10.1080\/09168451.2019.1606699","article-title":"Production of the Plant Polyketide Curcumin in Aspergillus oryzae: Strengthening Malonyl-CoA Supply for\nYield Improvement","volume":"83","author":"Kan","year":"2019","journal-title":"Biosci., Biotechnol., Biochem."},{"key":"2026091609125382300_cit28","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00409-19","article-title":"Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production\nof Basidiomycete\nTerpenes by Using Genomic DNA Sequences","volume":"85","author":"Nagamine","year":"2019","journal-title":"Appl.\nEnviron. Microbiol."},{"key":"2026091609125382300_cit29","doi-asserted-by":"publisher","first-page":"116001","DOI":"10.1016\/j.bmc.2021.116001","article-title":"The Subgroup\nof 2\u2019-Hydroxy-Flavonoids: Molecular Diversity, Mechanism of\nAction, and Anticancer Properties","volume":"32","author":"Bailly","year":"2021","journal-title":"Bioorg. Med.\nChem."}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c13856","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/39199\/67739441\/jacs.6c13856.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/39199\/67739441\/jacs.6c13856.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T13:31:03Z","timestamp":1789565463000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/36\/39199\/5361405\/The-Biosynthesis-of-a-Fungal-Flavonoid"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,29]]},"references-count":29,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,8,29]]},"published-print":{"date-parts":[[2026,9,16]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c13856","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,29]]}}}