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Enzyme-capacity constraint flux balance analysis (ecFBA) and flux-sum analysis To investigate metabolic states under various conditions, we utilized the iCHO2291 model, a comprehensive reconstruction of the CHO cell metabolic network 33 , adapted by incorporating experimentally measured uptake and secretion rates as boundary constraints for each condition, with exchange flux bounds for unmeasured or unexpected metabolites set to zero to prevent artificial nutrient uptake, while adjusting the bounds for glucose uptake, lactate secretion, and the biomass reaction to 10% of their measured rates to accommodate potential experimental errors, a range determined to effectively simulate physiological behavior

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