Microbial cell factories are widely used for the production of high-value chemicals. However, maximizing production titers is made difficult by the complicated regulatory mechanisms of these cell platforms. Here,k(cat)values were incorporated to construct anEscherichia colienzyme-constrained model. The resulting ec_iML1515 model showed that the protein demand and protein synthesis rate were the key factors affecting lysine production. By optimizing the expression of the 20 top-demanded proteins, lysine titers reached 95.7 +/- 0.7 g/L, with a 0.45 g/g glucose yield. Moreover, adjusting NH(4)(+)and dissolved oxygen levels to regulate the synthesis rate of energy metabolism-related proteins caused lysine titers and glucose yields to increase to 193.6 +/- 1.8 g/L and 0.74 g/g, respectively. The ec_iML1515 model provides insight into how enzymes required for the biosynthesis of certain products are distributed between and within metabolic pathways. This information can be used to accurately predict and rationally design lysine production.