A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate.

► Tuning of competing gene flux is an effective strategy for pathway optimization.
► Static strategies result in issues such as reduced growth or enzyme expression.
► Valves, dynamic strategies, improve pathway yield without these issues.
► Glucose valve built to redirect glucose away from glycolysis into a model pathway.
► Glucose valves enable the creation of efficient pathways that use glucose directly.