Kinetic‐model‐based pathway optimization with application to reverse glycolysis in mammalian cells

Over the last two decades, model‐based metabolic pathway optimization tools have been developed for the design of microorganisms to produce desired metabolites. However, few have considered more complex cellular systems such as mammalian cells, which requires the use of nonlinear kinetic models to capture the effects of concentration changes and cross‐regulatory interactions. In this study, we develop a new two‐stage pathway optimization framework based on kinetic models that incorporate detailed kinetics and regulation information. In Stage 1, a set of optimization problems are solved to identify and rank the enzymes that contribute the most to achieving the metabolic objective. Stage 2 then determines the optimal enzyme interventions for specified desired numbers of enzyme adjustments. It also incorporates multi‐scenario optimization, which allows the simultaneous consideration of multiple physiological conditions. We apply the proposed framework to find enzyme adjustments that enable a reverse glucose flow in cultured mammalian cells, thereby eliminating the need for glucose feed in the late culture stage and enhancing process robustness. The computational results demonstrate the efficacy of the proposed approach; it not only captures the important regulations and key enzymes for reverse glycolysis but also identifies differences and commonalities in the metabolic requirements for different carbon sources. This study introduces a new kinetic‐model‐based optimization framework for pathway optimization in metabolic engineering.t The proposed two‐stage framework is applied to find optimal enzyme adjustments in energy metabolism that enable a reverse glucose flow in cultured mammalian cells. The computational results demonstrate the efficacy of the proposed approach; it captures the important regulations and key enzymes for reverse glycolysis and identifies differences and commonalities in the metabolic requirements for different carbon sources.