Mechanistic understanding of the "gatekeeper" phosphofructokinase of the glycolytic pathway on the central carbon metabolism

Mechanistic understanding of the "gatekeeper" phosphofructokinase of the glycolytic pathway on the central carbon metabolism | 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 Mechanistic understanding of the "gatekeeper" phosphofructokinase of the glycolytic pathway on the central carbon metabolism Lingyun Li, Ning Qin, Xin Chen, Yu Chen, Xu Ji, Yijie Zhang, Jens Nielsen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3418319/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 Background: Glycolysis is one of the oldest and most fundamental metabolic pathways responsible for glucose breakdown and energy production in cells. The key regulatory enzyme in glycolysis is phosphofructokinase (Pfks), which catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate. The regulation of Pfks plays a crucial role in fine-tuning glycolytic flux to meet the cellular energy demands, making it a "gatekeeper" enzyme of glycolysis. In Saccharomyces cerevisiae, Pfks has evolved into a hetero-octameric phosphofructokinase, consisting of the Alpha and Beta subunits encoded by PFK1 and PFK2, respectively. Deletion of either PFK1 or PFK2 could significantly impair the organism's fitness, whereas the underlying metabolic mechanisms remain unclear. In this study, we investigated the specific effects of PFK1 and PFK2 on the cellular metabolism of S. cerevisiae. Results: Deletion of either PFK1 or PFK2 significantly reduced the cell growth, decreased ethanol and glycerol production and increased acetate production. Furthermore, through Flux Balance Analysis (FBA) and transcriptome analysis, we observed a significant upregulation of the Tricarboxylic Acid (TCA) cycle and the electron transfer chain in the PFK2 deletion strain. This upregulation indicated a shift towards enhanced oxidative respiration and energy production under the absence of the Beta subunit of Pfks. Furthermore, we demonstrated that the deletion of PFK2 led to a 33% improvement in the free fatty acid production, indicating its effectiveness as a key factor in regulation of lipid metabolism as well as bioproduction. Conclusions: Our physiological and transcriptomic data suggested that, as an isoenzyme, Pfk1 has a relatively limited influence on the metabolism of S. cerevisiae, whereas Pfk2 plays significant and vital roles in cellular metabolism. Deletion of PFK2 has the potential to greatly enhance respiration in yeast under aerobic conditions. These observations underscore the pivotal role of PFK2 in governing cellular energy metabolism and suggest that manipulation of PFK2 expression could hold promising implications for bioproduction processes. PFK1 PFK2 glycolysis oxidative respiration free fatty acid Full Text Additional Declarations No competing interests reported. Supplementary Files supplymentalfiles.zip 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. 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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-3418319","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":238894931,"identity":"804919e4-c884-42bc-917c-2c685a05fbf9","order_by":0,"name":"Lingyun Li","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Li","suffix":""},{"id":238894932,"identity":"461fe1a1-7f46-405e-a032-e0a8dfbe4a14","order_by":1,"name":"Ning Qin","email":"","orcid":"","institution":"Beijing University of Chemical 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The key regulatory enzyme in glycolysis is phosphofructokinase (Pfks), which catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate. The regulation of Pfks plays a crucial role in fine-tuning glycolytic flux to meet the cellular energy demands, making it a \"gatekeeper\" enzyme of glycolysis. In Saccharomyces cerevisiae, Pfks has evolved into a hetero-octameric phosphofructokinase, consisting of the Alpha and Beta subunits encoded by PFK1 and PFK2, respectively. Deletion of either PFK1 or PFK2 could significantly impair the organism's fitness, whereas the underlying metabolic mechanisms remain unclear. In this study, we investigated the specific effects of PFK1 and PFK2 on the cellular metabolism of S. cerevisiae.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: \u0026nbsp;Deletion of either PFK1 or PFK2 significantly reduced the cell growth, decreased ethanol and glycerol production and increased acetate production. Furthermore, through Flux Balance Analysis (FBA) and transcriptome analysis, we observed a significant upregulation of the Tricarboxylic Acid (TCA) cycle and the electron transfer chain in the PFK2 deletion strain. This upregulation indicated a shift towards enhanced oxidative respiration and energy production under the absence of the Beta subunit of Pfks. Furthermore, we demonstrated that the deletion of PFK2 led to a 33% improvement in the free fatty acid production, indicating its effectiveness as a key factor in regulation of lipid metabolism as well as bioproduction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions: Our physiological and transcriptomic data suggested that, as an isoenzyme, Pfk1 has a relatively limited influence on the metabolism of S. cerevisiae, whereas Pfk2 plays significant and vital roles in cellular metabolism. Deletion of PFK2 has the potential to greatly enhance respiration in yeast under aerobic conditions. 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