Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in Corynebacterium glutamicum

Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in Corynebacterium glutamicum | 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 Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in Corynebacterium glutamicum Saori Kosono, Makoto Nagaoka, Junko Yamamoto, Makoto Nishiyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6691977/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Studies suggest that protein condensation regulates cellular metabolism. The overproduction of extracellular glutamate in Corynebacterium glutamicum causes a global increase in metabolic flux toward glutamate synthesis without increasing metabolic enzyme levels. This suggests a post-translational mechanism of metabolic regulation. In this study, we determined that citrate synthase (CgCS) formed droplet-like condensates in C. glutamicum cells. CgCS condensation was observed in growing cells containing catalytically active CgCS. In vivo and in vitro studies revealed that oxaloacetate, a substrate of CS, regulated condensate formation and suggested that substrate-assisted conformational change in CgCS was important for condensation, i.e. catalysis-dependent condensation. Notably, the impairment of CgCS condensation was correlated with defective extracellular glutamate production. In glutamate production-deficient strains, intracellular amino acid profiles fluctuated, although the metabolism to supply 2-oxoglutarate was active. Our results suggest that CgCS condensation plays a distinct role in glutamate overproduction-related metabolism. These findings support the development of engineered condensate-based regulatory modules. Biological sciences/Microbiology/Bacteriology Biological sciences/Biochemistry/Proteins Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation2.pdf Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in Corynebacterium glutamicum Cite Share Download PDF Status: Under Review 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. 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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-6691977","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":461081770,"identity":"2c2c3aa8-4e27-4f01-8976-7baaa232a28c","order_by":0,"name":"Saori Kosono","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIie2QMUvDQBTH31FIHF7NelnsV7hw0EmuXyXhIFO2gpNIoJApdW7QLyF+gUAgXbRZC11SXB0OBKegJhqlQy84Ct4P7t57w4/3vwMwGP4mo/acH8zYVxIPKuHPRH+rFEcUHawqnphqKhHbi70iCVw5+ODVcClgdHN8DduGPMiSnYyx5LRVqLtMOYNSArnNNQrwYhzvJNDIApK8U1bhlIKVA1n5mmDrlwKbjYTJc6dAr7wNKHnEJVq5AIq98phO24R6xd1Gcy9LpG9hyKm/ATdLyzkLriXq3nJare+pasTMsYu9UhfgOCjvavUqzjzNj30TJN39leSEdQ16q0EDZge9XX+WCR1WDAaD4d/wAak3VTT88+lxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7108-5142","institution":"The University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Saori","middleName":"","lastName":"Kosono","suffix":""},{"id":461081771,"identity":"36672a02-bbb0-4561-acae-6de40b7518c1","order_by":1,"name":"Makoto Nagaoka","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Nagaoka","suffix":""},{"id":461081772,"identity":"8df1e4fc-c67c-4a43-ade8-d719e2b870c9","order_by":2,"name":"Junko Yamamoto","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Junko","middleName":"","lastName":"Yamamoto","suffix":""},{"id":461081773,"identity":"10ea934f-85c8-4ff5-9f75-a3e2fa255089","order_by":3,"name":"Makoto Nishiyama","email":"","orcid":"https://orcid.org/0000-0001-8143-8052","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Nishiyama","suffix":""}],"badges":[],"createdAt":"2025-05-18 13:35:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6691977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6691977/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83530983,"identity":"31f7e7b5-f43b-4068-9a85-13ffa6627494","added_by":"auto","created_at":"2025-05-28 04:55:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2824670,"visible":true,"origin":"","legend":"Article File","description":"","filename":"manuscript2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6691977/v1_covered_c97312a8-f39a-4c6a-98b9-57fc6ff29c8e.pdf"},{"id":83530579,"identity":"fa5c4133-c094-4f74-8335-4171049ea4ab","added_by":"auto","created_at":"2025-05-28 04:46:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6192783,"visible":true,"origin":"","legend":"\u003cp\u003eCatalysis-dependent condensation of citrate synthase involved in glutamate overproduction in \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Supplementaryinformation2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6691977/v1/43178d174d92ca246c4d76ef.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in \u003ci\u003eCorynebacterium glutamicum\u003c/i\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6691977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6691977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Studies suggest that protein condensation regulates cellular metabolism. The overproduction of extracellular glutamate in Corynebacterium glutamicum causes a global increase in metabolic flux toward glutamate synthesis without increasing metabolic enzyme levels. This suggests a post-translational mechanism of metabolic regulation. In this study, we determined that citrate synthase (CgCS) formed droplet-like condensates in C. glutamicum cells. CgCS condensation was observed in growing cells containing catalytically active CgCS. In vivo and in vitro studies revealed that oxaloacetate, a substrate of CS, regulated condensate formation and suggested that substrate-assisted conformational change in CgCS was important for condensation, i.e. catalysis-dependent condensation. Notably, the impairment of CgCS condensation was correlated with defective extracellular glutamate production. In glutamate production-deficient strains, intracellular amino acid profiles fluctuated, although the metabolism to supply 2-oxoglutarate was active. 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