A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids

A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids | 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 A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids Fernando López-Gallego, Alejandro Orrego, Idania López, Daniel Andrés-Sanz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7196754/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Enantiomerically pure β-hydroxy acids are valuable building blocks in polymer and fine chemical industries. However, biosynthetic routes to these compounds are limited by narrow feedstock availability. Here, we report a confined, cell-free biosynthetic pathway that converts primary alcohols into β-hydroxy acids using a multifunctional heterogeneous biocatalyst. Five enzymes were co-immobilized and spatially organized on aldehyde-functionalized porous supports. The system efficiently transforms ethanol into 3-hydroxybutyric acid, achieving a sevenfold yield increase over soluble enzymes through a design-build-test-learn (DBTL) approach. Enzyme confinement promotes optimal cofactor gradients, supporting redox balance and driving this thermodynamically unfavorable cascade. The biocatalyst exhibits broad substrate scope, converting esters, aldehydes, and diols (i.e., ethylene glycol, potentially derived from plastic waste) into β-hydroxy acids. Operated in a packed-bed flow reactor, it retained over 50% activity after three weeks. This work advances in vitro biocatalytic cascades using immobilized enzymes to convert simple, sustainable feedstocks into high-value chiral molecules. Biological sciences/Biotechnology/Metabolic engineering Physical sciences/Chemistry/Biosynthesis Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SIManuscriptPduPV4.pdf A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids Cite Share Download PDF Status: Posted 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-7196754","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489777918,"identity":"201ecd74-0b61-4633-a130-fa9cd27bd6a6","order_by":0,"name":"Fernando López-Gallego","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBAC9nYwZcPAxsDDcIAoLTyHwVQa6VpAJA+RDuNhZj724MOf8/J87GcPHviYY2e34XbzAYYPf/BpYUs3nMFz27CNJy/h4Mxtyckb7hxLYJzZhluLPTOPmTSPxO0ENgkeg8O825iTDW7kGDDzNuCzBajlj8E5mJZ6oJb8D8x/8DoMqIUh4QBMy2E7oC0MzMAAxO+XngPJML8cT5C8kWZwsBePX3jYm489+PHHTl6+/ezhDx+3Vdvz3Uh+CBTBrQUIUN2Q2AAkDuDVgK7FnoDqUTAKRsEoGIEAABXPT1jkmCWWAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0031-1880","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":true,"prefix":"","firstName":"Fernando","middleName":"","lastName":"López-Gallego","suffix":""},{"id":489777919,"identity":"986a34c1-970c-41c9-9095-25df63ff537c","order_by":1,"name":"Alejandro Orrego","email":"","orcid":"","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Orrego","suffix":""},{"id":489777920,"identity":"33895646-1d57-40a9-8942-c93e43fea112","order_by":2,"name":"Idania López","email":"","orcid":"","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":false,"prefix":"","firstName":"Idania","middleName":"","lastName":"López","suffix":""},{"id":489777921,"identity":"25cef79a-2226-4c8d-8739-bee8cec41eec","order_by":3,"name":"Daniel Andrés-Sanz","email":"","orcid":"","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Andrés-Sanz","suffix":""},{"id":489777922,"identity":"1e15dfdf-9e81-48c3-aee8-555976926fa7","order_by":4,"name":"Uxoa Fernández-Pelayo","email":"","orcid":"","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":false,"prefix":"","firstName":"Uxoa","middleName":"","lastName":"Fernández-Pelayo","suffix":""},{"id":489777923,"identity":"044ee25d-8ca7-4efe-9612-ea15424df5ab","order_by":5,"name":"Maialen Iturralde","email":"","orcid":"","institution":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE)","correspondingAuthor":false,"prefix":"","firstName":"Maialen","middleName":"","lastName":"Iturralde","suffix":""}],"badges":[],"createdAt":"2025-07-23 13:11:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7196754/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7196754/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89824333,"identity":"b318742b-cc63-4a9b-a019-cbb205703594","added_by":"auto","created_at":"2025-08-25 12:18:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":997302,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptPduPV4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7196754/v1_covered_eb49805b-45f7-43a4-b4d8-1784046450fa.pdf"},{"id":89451246,"identity":"719e539f-5965-4e29-a07d-84708682e90c","added_by":"auto","created_at":"2025-08-20 06:15:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1265838,"visible":true,"origin":"","legend":"A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into \u0026#x03B2;-Hydroxy Acids","description":"","filename":"SIManuscriptPduPV4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7196754/v1/ebf3394de5b4e008c06ac10a.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7196754/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7196754/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Enantiomerically pure β-hydroxy acids are valuable building blocks in polymer and fine chemical industries. However, biosynthetic routes to these compounds are limited by narrow feedstock availability. Here, we report a confined, cell-free biosynthetic pathway that converts primary alcohols into β-hydroxy acids using a multifunctional heterogeneous biocatalyst. Five enzymes were co-immobilized and spatially organized on aldehyde-functionalized porous supports. The system efficiently transforms ethanol into 3-hydroxybutyric acid, achieving a sevenfold yield increase over soluble enzymes through a design-build-test-learn (DBTL) approach. Enzyme confinement promotes optimal cofactor gradients, supporting redox balance and driving this thermodynamically unfavorable cascade. The biocatalyst exhibits broad substrate scope, converting esters, aldehydes, and diols (i.e., ethylene glycol, potentially derived from plastic waste) into β-hydroxy acids. Operated in a packed-bed flow reactor, it retained over 50% activity after three weeks. This work advances in vitro biocatalytic cascades using immobilized enzymes to convert simple, sustainable feedstocks into high-value chiral molecules.","manuscriptTitle":"A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 06:15:43","doi":"10.21203/rs.3.rs-7196754/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68e21deb-e219-4e07-b4b0-0aca8e0742de","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51999211,"name":"Biological sciences/Biotechnology/Metabolic engineering"},{"id":51999212,"name":"Physical sciences/Chemistry/Biosynthesis"}],"tags":[],"updatedAt":"2025-09-08T12:46:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 06:15:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7196754","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7196754","identity":"rs-7196754","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}