Enzymatic production of deoxyguanosine triphosphate from waste DNA by a recombinant guanylate kinase expressed in Escherichia coli BL21(DE3)

Enzymatic production of deoxyguanosine triphosphate from waste DNA by a recombinant guanylate kinase expressed in Escherichia coli BL21(DE3) | 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 Enzymatic production of deoxyguanosine triphosphate from waste DNA by a recombinant guanylate kinase expressed in Escherichia coli BL21(DE3) Mrs.Zehra Özdemir, Mr.Ercan Arıcan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8714799/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 The ability to overcome industrial scalability and environmental sustainability constraints largely depends on the use of recombinant enzymes with high catalytic efficiency that can be produced at low cost. Such enzymes enable the valorization of biological waste or low-value biomass, forming the basis of biocatalytic production processes that enhance resource efficiency and align with circular bioeconomy principles. In this context, deoxyguanosine triphosphate (dGTP), a fundamental building block of DNA synthesis, represents a particularly valuable target molecule due to its essential role in GC-rich DNA synthesis, high demand in molecular biology applications, and the economic and environmental drawbacks associated with its conventional chemical synthesis. In this study, a novel enzymatic process based on a “waste-to-value” approach was developed to convert laboratory-derived genomic and waste DNA into dGTP. DNA was enzymatically fragmented using S1 nuclease and a Benzonase/Exonuclease III combination to generate nucleotide mixtures enriched in dGMP. These intermediates were subsequently phosphorylated using Escherichia coli BL21(DE3) cell lysates overexpressing GUK1. Owing to the endogenous kinase activities present in ZYM5052 autoinduced lysates, dGMP was efficiently converted into dGTP without the requirement for externally added nucleoside diphosphate kinases. The phosphorylation steps were supported by an ATP regeneration system comprising phosphoenolpyruvate and pyruvate kinase. dGTP production was confirmed and quantified by HPLC analysis, revealing a characteristic retention time of 5–7 minutes. A five-point external calibration curve (0.5–2.0 µM) exhibited excellent linearity ( R² = 0.9999). The comparative analysis of dGTP production revealed that using laboratory-derived waste DNA as a substrate for biocatalytic reaction resulted in a highly efficient process, producing yielding 1.416 ± 0.025 µM of dGTP, which significantly surpassed the yield from purified genomic DNA (1.155 ± 0.034 µM; p = 0.0004). The maximum biocatalytic output was achieved through a nuclease cocktail treatment (Exonuclease III/Benzonase), resulting in a yield of 1.719 ± 0.178 µM, representing a significant increase over standard S1 nuclease digestion ( p = 0.0057). These findings confirm that utilizing crude biological waste can effectively replace expensive purified substrates for sustainable nucleotide biosynthesis. In conclusion, this study demonstrates that waste DNA can be transformed into high-value nucleotides through an environmentally friendly, efficient, and scalable biocatalytic approach compatible with circular bioeconomy frameworks. dGTP biosynthesis waste DNA recycling E. coli BL21(DE3) ZYM5052 autoinduction enzymatic phosphorylation circular bioeconomy Full Text Additional Declarations No competing interests reported. Tables are available in the Supplementary Files section. Supplementary Files Table1.docx Table2.docx GraphicAbstract.tiff 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-8714799","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584662467,"identity":"f61f5ff3-9a61-4fb2-ab14-41ae2c39fc8c","order_by":0,"name":"Mrs.Zehra 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BL21(DE3)","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"dGTP biosynthesis, waste DNA recycling, E. coli BL21(DE3), ZYM5052 autoinduction, enzymatic phosphorylation, circular bioeconomy","lastPublishedDoi":"10.21203/rs.3.rs-8714799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8714799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ability to overcome industrial scalability and environmental sustainability constraints largely depends on the use of recombinant enzymes with high catalytic efficiency that can be produced at low cost. Such enzymes enable the valorization of biological waste or low-value biomass, forming the basis of biocatalytic production processes that enhance resource efficiency and align with circular bioeconomy principles. In this context, deoxyguanosine triphosphate (dGTP), a fundamental building block of DNA synthesis, represents a particularly valuable target molecule due to its essential role in GC-rich DNA synthesis, high demand in molecular biology applications, and the economic and environmental drawbacks associated with its conventional chemical synthesis.\u003c/p\u003e \u003cp\u003eIn this study, a novel enzymatic process based on a \u0026ldquo;waste-to-value\u0026rdquo; approach was developed to convert laboratory-derived genomic and waste DNA into dGTP. DNA was enzymatically fragmented using S1 nuclease and a Benzonase/Exonuclease III combination to generate nucleotide mixtures enriched in dGMP. These intermediates were subsequently phosphorylated using \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) cell lysates overexpressing GUK1. Owing to the endogenous kinase activities present in ZYM5052 autoinduced lysates, dGMP was efficiently converted into dGTP without the requirement for externally added nucleoside diphosphate kinases. The phosphorylation steps were supported by an ATP regeneration system comprising phosphoenolpyruvate and pyruvate kinase.\u003c/p\u003e \u003cp\u003edGTP production was confirmed and quantified by HPLC analysis, revealing a characteristic retention time of 5\u0026ndash;7 minutes. A five-point external calibration curve (0.5\u0026ndash;2.0 \u0026micro;M) exhibited excellent linearity (\u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.9999). The comparative analysis of dGTP production revealed that using laboratory-derived waste DNA as a substrate for biocatalytic reaction resulted in a highly efficient process, producing yielding 1.416\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 \u0026micro;M of dGTP, which significantly surpassed the yield from purified genomic DNA (1.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034 \u0026micro;M; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004). The maximum biocatalytic output was achieved through a nuclease cocktail treatment (Exonuclease III/Benzonase), resulting in a yield of 1.719\u0026thinsp;\u0026plusmn;\u0026thinsp;0.178 \u0026micro;M, representing a significant increase over standard S1 nuclease digestion (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0057). These findings confirm that utilizing crude biological waste can effectively replace expensive purified substrates for sustainable nucleotide biosynthesis.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that waste DNA can be transformed into high-value nucleotides through an environmentally friendly, efficient, and scalable biocatalytic approach compatible with circular bioeconomy frameworks.\u003c/p\u003e","manuscriptTitle":"Enzymatic production of deoxyguanosine triphosphate from waste DNA by a recombinant guanylate kinase expressed in Escherichia coli BL21(DE3)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 07:26:31","doi":"10.21203/rs.3.rs-8714799/v1","editorialEvents":[{"type":"communityComments","content":0}],"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":"36a4ee88-fa23-4b4b-8239-57d2f53c4182","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-14T04:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 07:26:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8714799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8714799","identity":"rs-8714799","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}