A Metabolic Valve of Glycolytic Flux for the Design of Optimal Cell Factory

As an end metabolite of glycolysis in almost all organism, pyruvate is a critical intermediate from sugar uptake (i.e., glycolytic flux) to product formation and imbalance between glycolysis and product synthesis may accumulating pyruvate or reducing the productivity. Specifically, excess supply of pyruvate is inevitably secreted from the cell causing the significant reduction of yield whereas lack of pyruvate limits the product formation rate as the rate-limiting step. Therefore, balanced maximization of the pathway efficiency between glycolysis and product-forming pathway is required to construct the microbial cell factory with the maximum yield and productivity. Here, we propose synthetic UTR design of ptsG (encoding glucose transporter) is effectively controllable overall glycolytic flux to balance with the capacity of product formation pathway as a metabolic valve. Using the metabolic valve, optimal glycolytic flux was explored to maximize both yield and productivity of cell factory depending on the various efficiency of product-forming pathways, such as n-butanol, butyric acid, and 2,3-butanediol as model system. In result, tuning down of glycolytic flux was significantly reduced the leaking of pyruvate as major byproduct in n-butanol system, which increased 20% in yield while maximizing the parental productivity (84% of the theoretical maximum yield). Conversely, amplification of glycolytic flux transferred to the 7% and 12.5% higher productivity of butyric acid and 2, 3-butanediol, respectively, with the negligible yield change to the parental strain (around 83% and 60% of theoretical maximum, respectively). Our results demonstrated that fine control of the activity of PTS sugar transfer system allows a universal metabolic valve to accomplish balanced maximum metabolic performance for the maximum yield and productivity and consequently provides a breakthrough for designing the optimal microbial cell factory in the bio-based chemical industry.