Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation

Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation | 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 Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation Richard van Kranenburg, Maria Martin-Pascual, Stijn Kral, Adrian Garcia-Martinez, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6821618/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 The biotechnological synthesis of chemicals presents a greener and more sustainable alternative to traditional petrochemical processes. However, achieving titers, rates, and yields (TRY) allowing for economic competitiveness remains a significant challenge. A promising strategy to address this bottleneck is the increase of intracellular NADPH levels, a crucial reducing cofactor essential for the biosynthesis of numerous compounds. Here, we have engineered Pseudomonas putida with enhanced NADPH levels by redirecting flux towards the Pentose Phosphate pathway, introducing a phosphoketolase shunt and overexpressing key NADPH-generating enzymes. This strategy resulted in strains with enhanced NADPH pools, increasing TRY levels of the NADPH-dependent biosynthesis of furan dicarboxylic acid (FDCA) and violacein. Furthermore, we address the growth deficiencies typically associated with cofactor level alterations by integrating gntZ, rpe, and xfpk into the genome of P. putida ΔgcdΔeddΔhexR. This genomic integration, coupled to optimal ribosome binding site (RBS) sequences selected from a library, ensures stable gene expression and optimal growth rates without the need for plasmid maintenance. Lastly, we assessed the FDCA production using 1-L bioreactors, demonstrating the superior capabilities of the engineered strains in batch and fed-batch conditions. This strategic redesign of the central metabolism has yielded an NADPH-enhanced P. putida strains with improved product formation and a robust defense against oxidative stress. Biological sciences/Biotechnology/Metabolic engineering Biological sciences/Microbiology/Applied microbiology Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFile1.pdf Supplementary file 1: Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation Supplementaryfile2.xlsx Supplementary file 2: Strategic redesign of the central metabolism in Pseudomonas putida boosts NADPH generation and product formation 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. 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-6821618","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472640011,"identity":"3ccdc3f0-d589-4189-9a40-590c7731d4d3","order_by":0,"name":"Richard van 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