Evaluation of Vibrio natriegens as a fast-growing alternative host for plasmid DNA production

Evaluation of Vibrio natriegens as a fast-growing alternative host for plasmid DNA production | 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 Research Article Evaluation of Vibrio natriegens as a fast-growing alternative host for plasmid DNA production Lara S. Möller, Gabriel A. Monteiro, Duarte M.F. Prazeres, A. Rita Silva-Santos This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9108382/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Plasmid DNA (pDNA) is a key biomolecule for gene therapies, DNA vaccines, and mRNA-based applications. Although Escherichia coli is the established host for pDNA production, limitations such as moderate growth rates and endotoxin-associated challenges motivate the exploration of alternative production hosts. Vibrio natriegens is a fast-growing, non-pathogenic bacterium with a doubling time below 10 minutes and high metabolic versatility. While it has been adopted for cloning and protein expression, its potential for pDNA production remains largely unexplored. Results We evaluated pDNA production by V. natriegens in comparison to E. coli in shake flask cultivations across four complex media (LB, BHI, 2xYT, TB) and one defined minimal medium (MSM). Under all tested conditions, V. natriegens exhibited faster biomass accumulation and enabled earlier pDNA isolation. Quantitative analysis of triplicate experiments revealed that V. natriegens achieved comparable or higher volumetric pDNA yields in shorter cultivation times than E. coli . Agarose gel electrophoresis confirmed plasmid integrity, and functional validation using a cell-free expression system demonstrated efficient eGFP expression from V. natriegens -derived plasmids. Conclusions This proof-of-concept study demonstrates that V. natriegens is a promising alternative host for rapid pDNA production. Its combination of accelerated growth, robust volumetric pDNA yields, and functional plasmid quality highlights its potential for further development as a pDNA production platform. bioprocess intensification Escherichia coli microbial cell factory plasmid DNA production rapid biomanufacturing Vibrio natriegens volumetric productivity Full Text Additional Declarations No competing interests reported. Supplementary Files PaperVibrioSupFinal.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviews received at journal 27 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 14 Mar, 2026 Submission checks completed at journal 14 Mar, 2026 First submitted to journal 12 Mar, 2026 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-9108382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606147666,"identity":"cd54f73b-3964-492f-9cd5-f01edb2b6453","order_by":0,"name":"Lara S. 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Although \u003cem\u003eEscherichia coli\u003c/em\u003e is the established host for pDNA production, limitations such as moderate growth rates and endotoxin-associated challenges motivate the exploration of alternative production hosts. \u003cem\u003eVibrio natriegens\u003c/em\u003e is a fast-growing, non-pathogenic bacterium with a doubling time below 10 minutes and high metabolic versatility. While it has been adopted for cloning and protein expression, its potential for pDNA production remains largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003cbr\u003e\nWe evaluated pDNA production by \u003cem\u003eV. natriegens\u003c/em\u003e in comparison to \u003cem\u003eE. coli\u003c/em\u003e in shake flask cultivations across four complex media (LB, BHI, 2xYT, TB) and one defined minimal medium (MSM). Under all tested conditions,\u003cem\u003e V. natriegens\u003c/em\u003e exhibited faster biomass accumulation and enabled earlier pDNA isolation. Quantitative analysis of triplicate experiments revealed that \u003cem\u003eV. natriegens\u003c/em\u003e achieved comparable or higher volumetric pDNA yields in shorter cultivation times than \u003cem\u003eE. coli\u003c/em\u003e. Agarose gel electrophoresis confirmed plasmid integrity, and functional validation using a cell-free expression system demonstrated efficient eGFP expression from\u003cem\u003e V. natriegens\u003c/em\u003e-derived plasmids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003cbr\u003e\nThis proof-of-concept study demonstrates that \u003cem\u003eV. natriegens\u003c/em\u003eis a promising alternative host for rapid pDNA production. Its combination of accelerated growth, robust volumetric pDNA yields, and functional plasmid quality highlights its potential for further development as a pDNA production platform.\u003c/p\u003e","manuscriptTitle":"Evaluation of Vibrio natriegens as a fast-growing alternative host for plasmid DNA production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 04:10:04","doi":"10.21203/rs.3.rs-9108382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-07T11:34:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T15:01:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T20:05:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177757095239296084002213124985928071140","date":"2026-03-23T11:48:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284295056317478145126008927699360478900","date":"2026-03-17T11:31:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T05:37:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T13:50:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-14T13:49:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-03-12T21:35:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70c96431-f1a0-400b-8948-d1ea2241340a","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T13:10:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 04:10:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9108382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9108382","identity":"rs-9108382","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}