Bacillus subtilis genome-scale model (GEM) reconstruction was stimulated by the recent sequencing and consequent re-annotations. The updated gene-enzyme-reaction data were collected from databases to reconstruct B. subtilis reaction network BsRN-2016 containing 1144 genes linked to 1955 reactions and 1103 metabolites. Thermodynamic analysis was conducted to identify reversibility and directionality of the reactions. By elimination of unconnected-reactions from BsRN-2016, reconstruction process of the first third-generation GEM iBsu1 144 employing 1083 reactions linked to 719 genes was completed. The stoichiometric flux-balance based model was solved using time-profiles of serine alkaline protease fermentation-data at three different oxygen-transfer conditions creating perturbations on the intracellular reaction-network. Testing iBsu1 144 dignity with three different objective functions indicated superior robustness of the GEM. Comparison of iBsu1 144 results with a second-generation GEM was demonstrated. Insights obtained from flux distributions were used to determine metabolic engineering sites. Asparagine, isoleucine, threonine, and aspartic acid were determined as the primary rate-limiting amino acids to be considered as corresponding metabolic engineering sites in SAP synthesis in B. subtilis. Flux variability analysis carried out for the optimum condition reveals that 288 reactions are active and linked to 317 genes, so called substantial genes. The blocked 735 reactions linked to 533 genes, formed a platform for guided, gene deletions in B. subtilis to generate simplified host strains. (C) 2017 Elsevier B.V. All rights reserved.