Co-Production of Hydrogen and Ethyl Acetate in E. Coli

Co-Production of Hydrogen and Ethyl Acetate in E. Coli | 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 Co-Production of Hydrogen and Ethyl Acetate in E. Coli Anna C. Bohnenkamp, Rene H. Wijffels, Servé W. M. Kengen, Ruud A. Weusthuis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-631018/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Ethyl acetate and hydrogen are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products render the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H 2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO 2 . Results: We engineered Escherichia coli , towards improved conversion of formate into hydrogen and CO 2 by inactivating the formate hydrogen lyase repressor ( hycA ), both uptake hydrogenases ( hyaAB , hybBC ) and/or overexpressing the hydrogen formate lyase activator ( fhlA ). Initially 10 strains were evaluated in anaerobic serum bottles with respect to growth, after which four strains were further analyzed. Anaerobic co-production of hydrogen and ethyl acetate via heterologous ethanol acyltransferase (Eat1) was achieved in 1.5-L pH controlled bioreactors. Conclusions: We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70 % of the pathway maximum for ethyl acetate and hydrogen, and propose in-situ product removal via gas stripping as efficient technique to isolate the products of interest. General Biochemistry Molecular Biology Immunology ethyl acetate hydrogen co-production fermentation Escherichia coli Eat1 formate hydrogen lyase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Supplementary Files SupplementaryFigure1.pdf Supplementary Figure 1 SupplementaryFigure2Rates.pdf Supplementary Figure 2 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revision 18 Jul, 2021 Review # 2 received at journal 16 Jul, 2021 Review # 1 received at journal 10 Jul, 2021 Reviewer # 2 agreed at journal 02 Jul, 2021 Reviewers invited by journal 30 Jun, 2021 Reviews received at journal 30 Jun, 2021 Reviewer # 1 agreed at journal 30 Jun, 2021 Submission checks completed at journal 18 Jun, 2021 Editor assigned by journal 16 Jun, 2021 Editor invited by journal 16 Jun, 2021 First submitted to journal 16 Jun, 2021 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-631018","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":35260053,"identity":"bb0a807f-226e-4016-a524-64b98330eff1","order_by":0,"name":"Anna C. Bohnenkamp","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYLACxgaGBCDFDGLLQYQKSNBiDBEyIEFLYgMhLfyzew9+YNxhk2fO3nvY4GfOnfQNN3IfMBfg0SJx51yyBOOZtGLLnnPJib3bnuVuuJFuwDwDn8Nu5BhIMLYdTtxwI8f4AO+2w7nbbqQxMPPg0SIPVPkDrOX+G+ODf7cdTjcjpMXgRo4Z1BYe42SgLQkEtRgCtVgknklL3HAmx9hYdtszw/1nnjEcxqdFDuiwGx932CRuOH7GWPLttjvyku1pjI95KvB4HwQSEMwDSCSRgCTFo2AUjIJRMEIAAEZ1VqtjqLjbAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1781-3983","institution":"Wageningen University and Research","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"C.","lastName":"Bohnenkamp","suffix":""},{"id":35260054,"identity":"13b20a58-857f-4d01-a6bc-5571dba5fda5","order_by":1,"name":"Rene H. Wijffels","email":"","orcid":"","institution":"Wageningen University and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rene","middleName":"H.","lastName":"Wijffels","suffix":""},{"id":35260055,"identity":"6727d27c-c4f8-47ce-8c87-30bf6226772e","order_by":2,"name":"Servé W. M. Kengen","email":"","orcid":"","institution":"Wageningen University and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Servé","middleName":"W. M.","lastName":"Kengen","suffix":""},{"id":35260056,"identity":"89e5f678-2e90-4adb-b524-57d667cb6d83","order_by":3,"name":"Ruud A. Weusthuis","email":"","orcid":"","institution":"Wageningen University and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruud","middleName":"A.","lastName":"Weusthuis","suffix":""}],"badges":[],"createdAt":"2021-06-16 19:34:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-631018/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-631018/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":10856317,"identity":"98397133-cdc1-4644-9d3e-aa107bb4c409","added_by":"auto","created_at":"2021-06-28 11:46:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74761,"visible":true,"origin":"","legend":"Schematic representation of anaerobic ethyl acetate production from glucose in E. coli via the Embden-Meyerhof-Parnas (EMP) pathway with hydrogen co-production. Lactate and acetate formation is limited by ack and ldh inactivation. Heterologous alcohol acetyltransferase Eat1 generates ethyl acetate from ethanol and acetyl-CoA. Hydrogen co-production is achieved via formate hydrogen lyase (Fhl). Ack – acetate kinase, DHAP – dihydroxyacetone phosphate, eat1 – ethanol acetyltransferase, FBR – fructose 1,6-bisphosphate, F6P – fructose 6-phosphate, G6P – glucose 6-phosphate, PEP – phosphoenolpyruvate, GAP – glyceraldehyde 3-phosphate, pta – phosphate acetyltransferase","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/a77b19b60dea8c79a905bd0f.png"},{"id":10856321,"identity":"ea0ae802-7273-44fd-997c-7070bac9cd68","added_by":"auto","created_at":"2021-06-28 11:46:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56007,"visible":true,"origin":"","legend":"OD600 after 72h of cultivation under anaerobic conditions with glucose as carbon source of a BW25113 ΔldhA ΔackA background strain containing additional KOs and/or overexpressing fhlA for improved hydrogen production. Initial OD600 was 0.2. Data and error bars indicate averages and standard deviations among duplicates ","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/de9398d6ba4effc04f9f9894.png"},{"id":10856155,"identity":"2cc3bcfa-d616-4aaf-a795-dc77c82e491c","added_by":"auto","created_at":"2021-06-28 11:43:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62329,"visible":true,"origin":"","legend":"Concentrations of glucose and products after 72 h of anaerobic cultivation for strains with a ΔldhA ΔackA (ΔΔ) background and further modifications for improved hydrogen production, from left to right: inactivation of hycA, hyaAB and hybBC (ΔΔΔΔ), overexpression of fhlA (ΔΔ p3-fhlA) and a combination of knockouts and overexpression (ΔΔΔΔ p3-fhlA). For CO2 and H2, data represent calculated concentrations. Data shows average values and standard deviations from biological duplicates.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/a0342cf2a62f5468db60f68b.png"},{"id":10856319,"identity":"79d3662a-a590-4cb3-ac8d-844045367c8d","added_by":"auto","created_at":"2021-06-28 11:46:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44500,"visible":true,"origin":"","legend":"Product yield on glucose on selected products after 72h of anaerobic fermentation for strains based on ΔldhA ΔackA (ΔΔ) with further modifications for improved hydrogen production, from left to right: inactivation of hycA, hyaAB and hybBC (ΔΔΔΔΔ), overexpression of fhlA (ΔΔ p3-fhlA) and a combination of knockouts and overexpression (ΔΔΔΔΔ p3-fhlA). Values are averages of two biological replicates and error bars represent standard deviations.","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/ea9afc5c34de69cd289c738b.png"},{"id":10856161,"identity":"a059cfd8-ab6e-4168-ad9c-1c17d591935c","added_by":"auto","created_at":"2021-06-28 11:43:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166549,"visible":true,"origin":"","legend":"Fermentation profile of four strains engineered for ethyl acetate and hydrogen co-production in pH-controlled bioreactors with continuous gas stripping. Strains based on ΔldhA ΔackA (ΔΔ) with further modifications for improved hydrogen production, from left to right: inactivation of hycA, hyaAB and hybBC (ΔΔΔΔΔ), overexpression of fhlA (ΔΔ p3-fhlA) and a combination of knockouts and overexpression (ΔΔΔΔΔ p3-fhlA) producing trEat1 Wan N-13 were induced by 0.01 mM IPTG and cultivated under anaerobic conditions in minimal medium with 55 mM glucose as carbon source.. Experiments were performed as biological duplicates; error bars represent the standard deviation. Circles – compounds in liquid broth, triangle – compounds in off-gas.","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/df6d9270d16c68abaf238e53.png"},{"id":10856322,"identity":"802ba58b-491d-42fa-a3e1-ec3b93b56c10","added_by":"auto","created_at":"2021-06-28 11:46:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":67633,"visible":true,"origin":"","legend":"Effect of modifications towards improved hydrogen production on product yields and productivities for main fermentation products, with from left to right: inactivation of hycA, hyaAB and hybBC, overexpression of fhlA and a combination of knockouts and overexpression. Strains producing trEat1 Wan N-13 were induced by 0.01 mM IPTG and grown under anaerobic conditions in minimal medium containing 55 mM glucose using pH-controlled bioreactors with 0.5 L working volume. (a) Product yields for ethyl acetate, hydrogen and formate in molproduct/molglucose after glucose depletion. The numbers above the bars represent the carbon recovery of the fermentations. (b) Volumetric productivities for ethyl acetate, hydrogen, and formate in mmol/L/h. Experiments were performed as biological duplicates or triplicates; error bars represent the standard deviation. Abbreviations: trEat1 – truncated Eat1 Wan N-13.","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/8d1ea018f2828a230c89272e.png"},{"id":13651481,"identity":"7405b080-3731-4c91-a3d8-cbab107456c0","added_by":"auto","created_at":"2021-09-17 09:45:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":699558,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscripthydrogenandethylacetatecoproduction.pdf","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1_covered.pdf"},{"id":10856442,"identity":"ef67d404-e977-487e-b462-d8d8708b1e6f","added_by":"auto","created_at":"2021-06-28 11:49:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":696010,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscripthydrogenandethylacetatecoproduction.pdf","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1_covered.pdf"},{"id":10856440,"identity":"d33e5214-404e-484e-ad3c-d5b5b2f32347","added_by":"auto","created_at":"2021-06-28 11:49:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50272,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/e0f7cec1362bda2a9a3c7a05.pdf"},{"id":10856441,"identity":"60824890-75b9-4d12-ad25-3529c2611377","added_by":"auto","created_at":"2021-06-28 11:49:40","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":79127,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"SupplementaryFigure2Rates.pdf","url":"https://assets-eu.researchsquare.com/files/rs-631018/v1/e32424582b7cd6c3e1a6ccd5.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCo-Production of Hydrogen and Ethyl Acetate in \u003cem\u003eE. Coli\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"This preprint is available for \u003ca href='/article/rs-631018/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e."}],"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":true,"isPdf":false,"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":"ethyl acetate, hydrogen, co-production, fermentation, Escherichia coli, Eat1, formate hydrogen lyase","lastPublishedDoi":"10.21203/rs.3.rs-631018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-631018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eEthyl acetate and hydrogen are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by \u003cem\u003eEscherichia coli\u003c/em\u003e before. However, relatively low yields for hydrogen are obtained and a mix of by-products render the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H\u003csub\u003e2\u003c/sub\u003e may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO\u003csub\u003e2\u003c/sub\u003e. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe engineered \u003cem\u003eEscherichia coli\u003c/em\u003e, towards improved conversion of formate into hydrogen and CO\u003csub\u003e2\u003c/sub\u003e by inactivating the formate hydrogen lyase repressor (\u003cem\u003ehycA\u003c/em\u003e), both uptake hydrogenases (\u003cem\u003ehyaAB\u003c/em\u003e, \u003cem\u003ehybBC\u003c/em\u003e) and/or overexpressing the hydrogen formate lyase activator (\u003cem\u003efhlA\u003c/em\u003e). Initially 10 strains were evaluated in anaerobic serum bottles with respect to growth, after which four strains were further analyzed. Anaerobic co-production of hydrogen and ethyl acetate via heterologous ethanol acyltransferase (Eat1) was achieved in 1.5-L pH controlled bioreactors. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70 % of the pathway maximum for ethyl acetate and hydrogen, and propose \u003cem\u003ein-situ\u003c/em\u003e product removal via gas stripping as efficient technique to isolate the products of interest.\u003c/p\u003e","manuscriptTitle":"Co-Production of Hydrogen and Ethyl Acetate in E. 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