Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui

Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui | 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 Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui Angeliki Sitara, Rémi Hocq, Alexander Jiwei Lu, Stefan Pflügl This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5676099/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 9 You are reading this latest preprint version Abstract Background Due to its ability to grow fast on CO 2 , CO and H 2 at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g. ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate development of bioproduction scenarios. Currently, the genome editing tools available for T. kivui present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications. Results In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of T. kivui . Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 x 10 4 ± 8.7 x 10 3 CFU µg − 1 . Subsequently, Hi-TARGET was used to demonstrate gene knock-outs ( pyrE , rexA , hrcA ), a knock-in ( ldh ::pFAST), a single nucleotide mutation corresponding to PolC C629Y , and knock-down of the fluorescent protein pFAST. Analysis of the ∆ rexA deletion mutant created with Hi-TARGET revealed that the transcriptional repressor rexA is likely involved in the regulation of the expression of lactate dehydrogenase ( ldh ). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering T. kivui . Conclusions The CRISPR-based genome editing tool Hi-TARGET developed for T. kivui can be used for scarless deletion, insertion, point mutation and gene knock-down assays, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology. Genome editing endogenous CRISPR/Cas system thermophilic acetogen gas fermentation metabolic engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Additionalfile2.docx Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted Editorial decision: Revision requested 27 Feb, 2025 Reviews received at journal 26 Feb, 2025 Reviews received at journal 24 Feb, 2025 Reviewers agreed at journal 01 Feb, 2025 Reviewers agreed at journal 31 Jan, 2025 Reviewers invited by journal 31 Jan, 2025 Editor assigned by journal 19 Dec, 2024 Submission checks completed at journal 19 Dec, 2024 First submitted to journal 19 Dec, 2024 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. 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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-5676099","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392537552,"identity":"aed4b3a5-aee3-4809-bea7-7b8f491508cd","order_by":0,"name":"Angeliki Sitara","email":"","orcid":"","institution":"Technische Universität Wien","correspondingAuthor":false,"prefix":"","firstName":"Angeliki","middleName":"","lastName":"Sitara","suffix":""},{"id":392537553,"identity":"103f5bc8-956c-4cc8-aee7-8f79035a911c","order_by":1,"name":"Rémi Hocq","email":"","orcid":"","institution":"Technische Universität Wien","correspondingAuthor":false,"prefix":"","firstName":"Rémi","middleName":"","lastName":"Hocq","suffix":""},{"id":392537554,"identity":"f270f116-65a3-4369-b875-8702069f5e14","order_by":2,"name":"Alexander Jiwei Lu","email":"","orcid":"","institution":"Technische Universität Wien","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"Jiwei","lastName":"Lu","suffix":""},{"id":392537555,"identity":"657b344d-618c-422f-ba57-4c0410a2a5a9","order_by":3,"name":"Stefan Pflügl","email":"data:image/png;base64,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","orcid":"","institution":"Technische Universität Wien","correspondingAuthor":true,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Pflügl","suffix":""}],"badges":[],"createdAt":"2024-12-19 10:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5676099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5676099/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-025-02647-0","type":"published","date":"2025-04-30T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73358991,"identity":"83ed2ec4-5809-4b14-a8ee-59a598fb2ad3","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvestigation of the functionality of a type I-B CRISPR locus in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. kivui.\u003c/strong\u003e\u003c/em\u003e A) Genetic architecture of the CRISPR systems found in \u003cem\u003eT. kivui\u003c/em\u003e. Squares represent spacer sequences; diamonds represent repeat regions. Spacer and repeat sequences are provided in Additional File 1, Table S1. B) Sequence alignment of repeat regions from type I-B_4 CRISPR cluster of \u003cem\u003eT. kivui \u003c/em\u003eidentified with CRISPRCasFinder [33]\u003cem\u003e. \u003c/em\u003eNon-conserved bases are highlighted in red (bold).\u003cem\u003e \u003c/em\u003eThe selected repeat sequence is highlighted with a box. CRISPRTarget [38] was used to find native protospacer targets in MGE databases. A representative example is shown, CRISPR4 S2 (spacer 2 from Type I-B_4 in blue, putative PAM sequence in yellow). C) Putative PAM motif based on protospacer matches in CRISPRTarget [38]. Multiple sequence alignment of the reverse complement 3’ flank of protospacers visualized with Weblogo [52]). Source data are provided in Additional File 1, Table S2. D) Plasmid interference assay design. The putative PAMs are inserted in a plasmid upstream of a spacer identified in \u003cem\u003eT. kivui\u003c/em\u003e CRISPR cluster, and functional PAM sequences are determined by antibiotic challenge. E) Results from plasmid interference assay. Data represent three biological replicates (average ± standard deviation).\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/700e07a13d14394a86b1e2e8.jpg"},{"id":73358997,"identity":"cbdf877f-c20c-43b2-822d-cc6398bd1471","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":590368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment of a proof-of-concept for genome editing in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. kivui\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with its endogenous type I-B CRISPR system. \u003c/strong\u003eA) Cloning design with hierarchical Golden Gate assembly to generate the CRISPR plasmids. B) Genome interference assay design. A plasmid bearing a synthetic mini array with crRNAs targeting the \u003cem\u003epyrE \u003c/em\u003egene is transformed in \u003cem\u003eT. kivui\u003c/em\u003e. Functionality of the CRISPR system is determined by antibiotic challenge. C) Genome interference assay shows strain transformation is impaired for plasmids with \u003cem\u003epyrE\u003c/em\u003e crRNAs (CCA or CCC PAM). Controls: empty vector, mini array without cRNA. Data represent three biological replicates (average ± standard deviation). Statistical significance was determined using a two-tailed t-test for independent samples (*p \u0026lt; 0.05). D) Design of CRISPR-based \u003cem\u003epyrE\u003c/em\u003e deletion. The functionality of the CRISPR system is determined by antibiotic challenge and PCR. E) Transformation efficiencies of type I-B CRISPR vectors and editing efficiency of \u003cem\u003epyrE \u003c/em\u003edeletion. Controls: empty vector, mini array without crRNA. Transformation efficiency was measured for four independent experiments (average ± standard deviation), and editing efficiency is shown as the fraction of edited colonies (ten colonies, average ± standard deviation). F) Gel electrophoresis of PCR results from 2E. Data are representative of ten colonies per sample. G) Effect of homology arm length and plasmid nature (purified from \u003cem\u003eE. coli\u003c/em\u003e or Golden Gate assembly mixture) on \u003cem\u003epyrE\u003c/em\u003e deletion. Control: mini array without crRNA plasmid. Transformation efficiency was measured for four independent experiments (average ± standard deviation), and editing efficiency is shown as the fraction of edited colonies (ten colonies, average ± standard deviation).\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/aba31a5472404ce98003c73f.jpg"},{"id":73358992,"identity":"cb36aeea-a560-4db7-9f6b-5cc1712e1064","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":519311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the\u003c/strong\u003e \u003cstrong\u003eΔ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003erexA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant in heterotrophic batch fermentation\u003c/strong\u003e. A, B) Growth and product formation of wild type (A) and Δ\u003cem\u003erexA \u003c/em\u003e(B)\u003cem\u003e \u003c/em\u003ein batch fermentation with excess glucose (50 g L\u003csup\u003e-1\u003c/sup\u003e). Data represent three biological replicates (average ± standard deviation). The maximum specific growth rate µ\u003csub\u003emax\u003c/sub\u003e was calculated from time point 43 and 53 for the wild type and from time point 31 and 40 for Δ\u003cem\u003erexA\u003c/em\u003e. C) Results of RNA-seq analysis are shown as heatmap of the ten most differentially expressed genes in Δ\u003cem\u003erexA\u003c/em\u003e compared to the wild type. Log2 fold change differences (\u0026gt; ±1.5-fold) between wild type and Δ\u003cem\u003erexA\u003c/em\u003e (blue: downregulation, red: upregulation) as well as TPM values from triplicates are shown.\u003c/p\u003e\n\u003cp\u003eLocus tags are from the G-1 reference genome (OZ020628) [9]. Genes without formal gene name are displayed per the first word of the product name (LytTR = LytTR family DNA-binding domain-containing protein, GHLK = GHKL domain-containing protein). A grey line separates the genes found in the \u003cem\u003erexA\u003c/em\u003e locus from those involved in sugar metabolism. D) Top: Sequence logo generated with the consensus sequence for \u003cem\u003eThermoanaerobacterales \u003c/em\u003efrom the RegPrecise database [53]. Bottom: Schematic representation of the genetic architecture of a subset of differentially expressed genes in \u003cem\u003eT.\u0026nbsp;kivui\u003c/em\u003e and \u003cem\u003eT.\u0026nbsp;kivui\u003c/em\u003e Δ\u003cem\u003erexA\u003c/em\u003e. Colors are taken from Fig. 3C for log2-fold expression. Yellow diamond: Rex recognition motif. Arrow: Promoter. Source data can be found at Additional File 1, Table S4 and Table S5.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/37deffd5bce68f218358a8ae.jpg"},{"id":73358993,"identity":"7af611f2-4f77-49e6-8425-2be37b850ce3","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":211896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnock-down of the integrated fluorescent protein pFAST in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. kivui\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e A) Fluorescence quantification of \u003cem\u003eT. kivui \u003c/em\u003ecells\u003cem\u003e \u003c/em\u003eexpressing pFAST from a plasmid or the genome. Data represent three biological replicates (average ± standard deviation). B) Design of CRISPR-based knock-down of pFAST. Plasmids expressing short crRNAs are introduced in \u003cem\u003eT. kivui\u003c/em\u003e. The short crRNA interferes with pFAST transcription, which should result in reduced fluorescence. C) Results of fluorescence measurements obtained with short spacers in the crRNA (S11: 11 bp, S15: 15 bp, S20: 20 bp) targeting pFAST in \u003cem\u003eT. kivui\u003c/em\u003e. Data represent 5-7 biological replicates and are displayed as percentage average ± standard error. Statistical significance was determined using a two-tailed t-test for independent samples (*p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/598b496b3c00c49f041b0ded.jpg"},{"id":81987808,"identity":"9b7408bb-0211-4eea-988a-690811ce5fc2","added_by":"auto","created_at":"2025-05-05 16:06:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3075099,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript20241219.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1_covered_7b43a1d6-ba89-42d0-aaec-32c06572ad2a.pdf"},{"id":73358994,"identity":"88795a0f-54a0-4dde-807c-fb62063ed855","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":668041,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/b59a5984acbea5c6d0a67751.xlsx"},{"id":73358995,"identity":"a9893b40-7246-4e79-b91a-6585ac0e7903","added_by":"auto","created_at":"2025-01-09 08:31:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":289879,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5676099/v1/ff28240355eeed62d837c507.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genome editing, endogenous CRISPR/Cas system, thermophilic acetogen, gas fermentation, metabolic engineering","lastPublishedDoi":"10.21203/rs.3.rs-5676099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5676099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDue to its ability to grow fast on CO\u003csub\u003e2\u003c/sub\u003e, CO and H\u003csub\u003e2\u003c/sub\u003e at high temperatures and with high energy efficiency, the thermophilic acetogen \u003cem\u003eThermoanaerobacter kivui\u003c/em\u003e could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g. ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate development of bioproduction scenarios. Currently, the genome editing tools available for \u003cem\u003eT. kivui\u003c/em\u003e present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of \u003cem\u003eT. kivui\u003c/em\u003e. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 8.7 x 10\u003csup\u003e3\u003c/sup\u003e CFU \u0026micro;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (\u003cem\u003epyrE\u003c/em\u003e, \u003cem\u003erexA\u003c/em\u003e, \u003cem\u003ehrcA\u003c/em\u003e), a knock-in (\u003cem\u003eldh\u003c/em\u003e::pFAST), a single nucleotide mutation corresponding to PolC\u003csup\u003eC629Y\u003c/sup\u003e, and knock-down of the fluorescent protein pFAST. Analysis of the ∆\u003cem\u003erexA\u003c/em\u003e deletion mutant created with Hi-TARGET revealed that the transcriptional repressor \u003cem\u003erexA\u003c/em\u003e is likely involved in the regulation of the expression of lactate dehydrogenase (\u003cem\u003eldh\u003c/em\u003e). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering \u003cem\u003eT. kivui\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe CRISPR-based genome editing tool Hi-TARGET developed for \u003cem\u003eT. kivui\u003c/em\u003e can be used for scarless deletion, insertion, point mutation and gene knock-down assays, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.\u003c/p\u003e","manuscriptTitle":"Hi-TARGET: A fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 08:31:21","doi":"10.21203/rs.3.rs-5676099/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-27T05:09:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-26T20:01:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-25T03:22:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25619095461629292877607646144592056175","date":"2025-02-01T10:54:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14277024544712930833278996646299008204","date":"2025-02-01T03:51:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-31T18:37:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-19T11:20:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-19T11:20:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2024-12-19T10:42:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fbc02e2a-6959-4a7e-b78b-99a26b86eb6f","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:01:37+00:00","versionOfRecord":{"articleIdentity":"rs-5676099","link":"https://doi.org/10.1186/s13068-025-02647-0","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2025-04-30 15:57:09","publishedOnDateReadable":"April 30th, 2025"},"versionCreatedAt":"2025-01-09 08:31:21","video":"","vorDoi":"10.1186/s13068-025-02647-0","vorDoiUrl":"https://doi.org/10.1186/s13068-025-02647-0","workflowStages":[]},"version":"v1","identity":"rs-5676099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5676099","identity":"rs-5676099","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}