Validation of predictive MFA for metabolic engineering. (A) To validate our framework as a powerful tool to assign targets for metabolic engineering, the complete set of 13 experiments was purposefully split into calibration and validation subsets in two manners. In strategy 1, experiments 6, 7, 8, 9 and 13, which have been rationally designed to manipulate the cellular energetic state, were left aside for validation. In strategy 2, the top three producers (experiments 1, 6 and 7) were chosen as validation cultures to avoid data interpolation. The number of latent variables used to build the calibration models in each case was 1 and 3, respectively. Productivity is expressed in 103 infectious particles × (106 cells × h)-1. (B) Metabolic decomposition for each validation strategy, showing the common selection of TCA cycle/respiration as important pathways for viral replication. Also for validation strategy 1, the catabolism of phenylalanine, methionine and histidine had α values higher than 1, as well as other fluxes with lighter correlations with the target (other: catabolism of maltose, proline and tyrosine, formation of alanine).