BioModelsMartha Ligia Ascencio GalvanNon-curatedconstraint-based model0.1Manuscript Acceptedhttps://www.ebi.ac.uk/biomodels/MODEL2502270001falseBioModelsCOMBINE archiveModelsAscencio2025 GEM Cupriavidus necator iCNH2025A2026MODEL2502270001Martha L. Ascencio-GalvánMartha L. Ascencio-Galván, Víctor A. López-Agudelo, Andreas Dräger, Julio C. Caicedo, David Gómez-Ríos, Rodrigo Andler, Howard Ramírez-Malulenull, Background: The growing demand for biodegradable polymers has positioned polyhydroxybutyrate (PHB) as a sustainable alternative to petrochemical plastics. Microbial synthesis from renewable, low-cost substrates such as cassava starch-derived dextrose offers a promising route, but feeding strategies and metabolic understanding remain critical. This study aimed to evaluate the effect of fed-batch pulse feeding on PHB production by Cupriavidus necator DSM 428 and to integrate experimental results with flux balance analysis (FBA). Results: Three pulse-feeding strategies were assessed. Two pulses containing carbon and nitrogen at 24 and 48 h (C/N ratio 73) led to 36.87% (w/w) PHB accumulation. A single dextrose-only pulse at 48 h resulted in 25.12% (w/w), A single dextrose-only pulse at 48 h led to 25.12% (w/w), while delaying this pulse to 60 h increased PHB accumulation to 50.40% (w/w). Feeding at the onset of stationary phase enhanced biomass growth but reduced PHB storage, whereas late feeding favored polymer accumulation, likely due to nitrogen limitation. PHB was characterized by XRD, FTIR, SEM, EDS, TGA, and DSC, confirming its expected structural and thermal properties. FBA with iCNH2025A showed strong correlation with experimental data, accurately predicting growth and carbon flux redirection toward PHB synthesis. Conclusion: The integration of fed-batch pulse feeding with FBA provides new insights into the metabolic and process factors driving PHB accumulation from cassava-based substrates. The results highlight the importance of feeding timing for optimizing yields and demonstrate the value of metabolic modeling in guiding sustainable bioprocess design.. null, null. School of Chemical Engineering, Universidad del Valle, Cali 760042, Colombia; martha.ascencio@correounivalle.edu.co (M.L.A.-G.); david.andres.gomez@correounivalle.edu.co (D.G.-R.) Institute of Clinical Molecular Biology, Kiel University and University Medical Center Schleswig-Holstein, 24105 Kiel, Germany; v.lopez-agudelo@ikmb.uni-kiel.de (V.A.L.A) Data Analytics and Bioinformatics, Institute of Computer Science, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany; andreas.draeger@informatik.uni-halle.de (A.D) Tribology, Polymers, Powder Metallurgy and Solid Waste Transformations Research Group, Universidad del Valle, Cali 760042, Colombia; julio.cesar.caicedo@correounivalle.edu.co (J.C.C) Escuela de Ingeniería en Biotecnología, Centro de Biotecnología de los Recursos Naturales (Cenbio), Universidad Católica del Maule, Talca 3460000, Chile; randler@ucm.cl (R.A) * Correspondence: howard.ramirez@correounivalle.edu.co (H.R-M)martha.ascencio@correounivalle.edu.cofalseAscencio2025 - GEM Cupriavidus necator iCNH2025AOur genome-scale metabolic model, iCNH2025A, differs from previously published models of Cupriavidus necator in several important aspects. First, it represents a larger and more extensively curated reconstruction, including 2,737 reactions, 1,803 metabolites, and 1,059 annotated genes, with improved mass and charge balancing, reaction directionality, and gene–protein–reaction associations. Second, the model underwent systematic thermodynamic curation, including the identification and removal of thermodynamically infeasible cycles, which improves the reliability of flux simulations. Third, iCNH2025A incorporates expanded pathways related to PHA biosynthesis, including both PHB and PHBV production routes, whereas earlier models primarily focused on PHB synthesis from sugars. In addition, this model was validated against experimental data obtained in this study, including growth rates, intracellular flux distributions, and PHB production under different cultivation strategies, demonstrating strong agreement with experimental observations.2026-02-122026-02-122025-02-27MODEL2502270001MODEL2502270001