Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation

Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation Frank Hahn, Lisa Nguyen, Luca Schlotte, Julian Hoffmann, Dominik Betz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7262642/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract α,β-Unsaturated δ-lactones (AUDLs) are key pharmacophores found in various reduced polyketides exhibiting potent biological activity. Fostriecin has attracted interest as an anticancer agent, but its structural characteristics have limited its development and motivated investigations into biosynthesis-based production strategies. In this study, we reconstitute the enzymatic steps responsible for AUDL formation in fostriecin biosynthesis. We demonstrate that the terminal polyketide synthase (PKS) module FosMod8 produces a 3-O-malonylthioester intermediate using the unusual bifunctional thioesterase FosTE, which catalyses O-malonylation and subsequent lactonisation. Structural modelling revealed two arginines mediating malonyl-CoA binding and transesterification analogously to PKS-acyltransferases. Backbone phosphorylation by the kinase FosH primes the compound for site-specific demalonylation by FosM. This highly regulated pathway ensures optimised formation of bioactive phosphorylated AUDL metabolites and represents a blueprint for others, like cytostatin or phoslactomycin. Understanding this unique enzymatic logic will support new synthetic strategies to AUDL polyketides using chemoenzymatic synthesis or engineered biosynthesis. Physical sciences/Chemistry/Biosynthesis Biological sciences/Chemical biology/Enzymes Biological sciences/Chemical biology/Natural products Biological sciences/Biochemistry/Biocatalysis Biological sciences/Chemical biology/Enzyme mechanisms Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformationfinal.pdf Supporting information Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7262642","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500124448,"identity":"00ebc2cf-c592-438c-84e2-dc810b1bd035","order_by":0,"name":"Frank Hahn","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0603-3482","institution":"Universität Bayreuth","correspondingAuthor":true,"prefix":"","firstName":"Frank","middleName":"","lastName":"Hahn","suffix":""},{"id":500124449,"identity":"c706d7ee-3795-41ae-8a8e-1fcd2714bafe","order_by":1,"name":"Lisa Nguyen","email":"","orcid":"","institution":"Universität Bayreuth","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Nguyen","suffix":""},{"id":500124450,"identity":"e1d39aeb-052e-46b7-9977-ab5365213461","order_by":2,"name":"Luca Schlotte","email":"","orcid":"","institution":"Universität Bayreuth","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Schlotte","suffix":""},{"id":500124451,"identity":"8f0e84de-c08f-42a2-a2b5-d0de514dd574","order_by":3,"name":"Julian Hoffmann","email":"","orcid":"","institution":"Universität Bayreuth","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Hoffmann","suffix":""},{"id":500124452,"identity":"632d5816-36d6-4d9d-8eb9-2346cfd63075","order_by":4,"name":"Dominik Betz","email":"","orcid":"","institution":"Universität Bayreuth","correspondingAuthor":false,"prefix":"","firstName":"Dominik","middleName":"","lastName":"Betz","suffix":""},{"id":500124453,"identity":"c34248b8-8100-4194-9a94-78c91ef5c97e","order_by":5,"name":"Marius Schröder","email":"","orcid":"","institution":"Universität Bayreuth","correspondingAuthor":false,"prefix":"","firstName":"Marius","middleName":"","lastName":"Schröder","suffix":""}],"badges":[],"createdAt":"2025-07-31 13:20:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7262642/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7262642/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-70144-5","type":"published","date":"2026-03-10T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":104469240,"identity":"e205e760-5e03-4baa-a1e9-c5c9782d6f47","added_by":"auto","created_at":"2026-03-12 07:07:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3717883,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuskriptfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7262642/v1_covered_594fa26e-4ec4-43be-87f4-e6e1774f9982.pdf"},{"id":90553607,"identity":"7ad4ad9e-d546-4794-83dd-f024f17ebe87","added_by":"auto","created_at":"2025-09-04 03:51:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12473388,"visible":true,"origin":"","legend":"Supporting information","description":"","filename":"SupportingInformationfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7262642/v1/b602f1aba08d25787d98580c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7262642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7262642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"α,β-Unsaturated δ-lactones (AUDLs) are key pharmacophores found in various reduced polyketides exhibiting potent biological activity. Fostriecin has attracted interest as an anticancer agent, but its structural characteristics have limited its development and motivated investigations into biosynthesis-based production strategies. In this study, we reconstitute the enzymatic steps responsible for AUDL formation in fostriecin biosynthesis. We demonstrate that the terminal polyketide synthase (PKS) module FosMod8 produces a 3-O-malonylthioester intermediate using the unusual bifunctional thioesterase FosTE, which catalyses O-malonylation and subsequent lactonisation. Structural modelling revealed two arginines mediating malonyl-CoA binding and transesterification analogously to PKS-acyltransferases. Backbone phosphorylation by the kinase FosH primes the compound for site-specific demalonylation by FosM. This highly regulated pathway ensures optimised formation of bioactive phosphorylated AUDL metabolites and represents a blueprint for others, like cytostatin or phoslactomycin. Understanding this unique enzymatic logic will support new synthetic strategies to AUDL polyketides using chemoenzymatic synthesis or engineered biosynthesis.","manuscriptTitle":"Covalent Warhead Assembly in Fostriecin Biosynthesis: Malonylation-Lactonisation by a Bifunctional Thioesterase and Enzymatic Demalonylation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 03:51:53","doi":"10.21203/rs.3.rs-7262642/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1d333e24-a03a-430c-a816-ae3655e541aa","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53129537,"name":"Physical sciences/Chemistry/Biosynthesis"},{"id":53129538,"name":"Biological sciences/Chemical biology/Enzymes"},{"id":53129539,"name":"Biological sciences/Chemical biology/Natural products"},{"id":53129540,"name":"Biological sciences/Biochemistry/Biocatalysis"},{"id":53129541,"name":"Biological sciences/Chemical biology/Enzyme mechanisms"}],"tags":[],"updatedAt":"2026-03-12T07:07:10+00:00","versionOfRecord":{"articleIdentity":"rs-7262642","link":"https://doi.org/10.1038/s41467-026-70144-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-03-10 04:00:00","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2025-09-04 03:51:53","video":"","vorDoi":"10.1038/s41467-026-70144-5","vorDoiUrl":"https://doi.org/10.1038/s41467-026-70144-5","workflowStages":[]},"version":"v1","identity":"rs-7262642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7262642","identity":"rs-7262642","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}