nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale

nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale | 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 nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale Gilles van Wezel, Isabel Nunez Santiago, Nataliia V. Machushynets, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3507422/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Microbial natural products form the basis of most of the antibiotics used in the clinic. The vast majority has not yet been discovered, among others because the hidden chemical space is obscured by previously identified (and typically abundant) antibiotics in culture extracts. Thus, efficient metabolic dereplication is key to the discovery of those molecules that may represent our future medicines. Here we present an analytical platform for the efficient identification and prioritization of low abundance bioactive compounds at nanoliter scale, called nanoRAPIDS. NanoRAPIDS encompasses analytical scale separation and nanofractionation of natural extracts, followed by high-throughput bioassay, automated mass spectrometry identification, and Global Natural Products Social molecular networking (GNPS) for dereplication. As little as 10 μL crude extract is thereby fractionated into 384 fractions. First, bioactive congeners of iturins and surfactins were identified in Bacillus, based on their activity against Gram-positive bacteria, Gram-negative bacteria and/or fungi. Subsequently, bioactive molecules were identified in an extensive network of angucyclines elicited by catechol in cultures of Streptomyces sp. This allowed the discovery of a highly unusual N-acetylcysteine conjugate of saquayamycin, despite low production levels in an otherwise abundant molecular family. These data underline the utility and broad application of the technology for the prioritization of minor bioactive compounds in complex extracts. Biological sciences/Drug discovery/Drug screening Biological sciences/Biotechnology/Metabolomics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files NunezSI.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Communications Chemistry → 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-3507422","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":252705435,"identity":"539c385a-1744-454b-a6e7-c1e3474ff501","order_by":0,"name":"Gilles van Wezel","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0341-1561","institution":"Leiden University","correspondingAuthor":true,"prefix":"","firstName":"Gilles","middleName":"van","lastName":"Wezel","suffix":""},{"id":252705436,"identity":"d595b286-917f-4690-b782-9771be5c2bb9","order_by":1,"name":"Isabel Nunez Santiago","email":"","orcid":"","institution":"Institute of Biology, Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"Nunez","lastName":"Santiago","suffix":""},{"id":252705437,"identity":"f861bfa6-ecab-4463-8029-4cb489fb1de1","order_by":2,"name":"Nataliia V. Machushynets","email":"","orcid":"","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Nataliia","middleName":"V.","lastName":"Machushynets","suffix":""},{"id":252705438,"identity":"16fd2292-e9d7-428f-821e-27119016fe52","order_by":3,"name":"Marija Mladic","email":"","orcid":"","institution":"DSM-Firmenich","correspondingAuthor":false,"prefix":"","firstName":"Marija","middleName":"","lastName":"Mladic","suffix":""},{"id":252705439,"identity":"5c7d514c-271b-4f02-ba97-c2478eab01ac","order_by":4,"name":"Doris van Bergeijk","email":"","orcid":"https://orcid.org/0000-0002-2316-920X","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Doris","middleName":"van","lastName":"Bergeijk","suffix":""},{"id":252705440,"identity":"83bbb518-4ed2-4e6b-910e-d278429a0b94","order_by":5,"name":"Somayah Elsayed","email":"","orcid":"https://orcid.org/0000-0003-3837-6137","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Somayah","middleName":"","lastName":"Elsayed","suffix":""},{"id":252705441,"identity":"550eaf71-b85c-4a49-b757-131115af644b","order_by":6,"name":"Thomas Hankemeier","email":"","orcid":"","institution":"Leiden Academic Centre for Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Hankemeier","suffix":""}],"badges":[],"createdAt":"2023-10-29 13:37:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3507422/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3507422/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-024-01153-y","type":"published","date":"2024-04-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53913247,"identity":"03aa1115-fdfd-44a3-b942-9dadd6712b35","added_by":"auto","created_at":"2024-04-02 07:09:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1100076,"visible":true,"origin":"","legend":"","description":"","filename":"Nunezmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3507422/v1_covered_1643f83b-54c9-4d47-9e78-064d6ddc62f6.pdf"},{"id":47180326,"identity":"10334439-5b7a-42fb-885f-e854416d5894","added_by":"auto","created_at":"2023-11-28 06:45:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4829100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"NunezSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3507422/v1/0d9795bd44f7a9b31b92116e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale","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-3507422/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3507422/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Microbial natural products form the basis of most of the antibiotics used in the clinic. The vast majority has not yet been discovered, among others because the hidden chemical space is obscured by previously identified (and typically abundant) antibiotics in culture extracts. Thus, efficient metabolic dereplication is key to the discovery of those molecules that may represent our future medicines. Here we present an analytical platform for the efficient identification and prioritization of low abundance bioactive compounds at nanoliter scale, called nanoRAPIDS. NanoRAPIDS encompasses analytical scale separation and nanofractionation of natural extracts, followed by high-throughput bioassay, automated mass spectrometry identification, and Global Natural Products Social molecular networking (GNPS) for dereplication. As little as 10 μL crude extract is thereby fractionated into 384 fractions. First, bioactive congeners of iturins and surfactins were identified in Bacillus, based on their activity against Gram-positive bacteria, Gram-negative bacteria and/or fungi. Subsequently, bioactive molecules were identified in an extensive network of angucyclines elicited by catechol in cultures of Streptomyces sp. This allowed the discovery of a highly unusual N-acetylcysteine conjugate of saquayamycin, despite low production levels in an otherwise abundant molecular family. These data underline the utility and broad application of the technology for the prioritization of minor bioactive compounds in complex extracts.","manuscriptTitle":"nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-28 06:44:56","doi":"10.21203/rs.3.rs-3507422/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ae28964-86a8-4034-a1c9-a4369cb970fa","owner":[],"postedDate":"November 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":26686720,"name":"Biological sciences/Drug discovery/Drug screening"},{"id":26686721,"name":"Biological sciences/Biotechnology/Metabolomics"}],"tags":[],"updatedAt":"2024-04-02T07:08:53+00:00","versionOfRecord":{"articleIdentity":"rs-3507422","link":"https://doi.org/10.1038/s42004-024-01153-y","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2024-04-01 04:00:00","publishedOnDateReadable":"April 1st, 2024"},"versionCreatedAt":"2023-11-28 06:44:56","video":"","vorDoi":"10.1038/s42004-024-01153-y","vorDoiUrl":"https://doi.org/10.1038/s42004-024-01153-y","workflowStages":[]},"version":"v1","identity":"rs-3507422","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3507422","identity":"rs-3507422","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}