A molecular rheostat maintains ATP levels to drive a synthetic biochemistry system

An enzymatic rheostat controls ATP levels by regulating flux through two pathways depending on free phosphate levels. Incorporation of this rheostat overcomes ATPase contamination and improves isobutanol production from glucose in a cell-free system. Synthetic biochemistry seeks to engineer complex metabolic pathways for chemical conversions outside the constraints of the cell. Establishment of effective and flexible cell-free systems requires the development of simple systems to replace the intricate regulatory mechanisms that exist in cells for maintaining high-energy cofactor balance. Here we describe a simple rheostat that regulates ATP levels by controlling the flow down either an ATP-generating or non-ATP-generating pathway according to the free-phosphate concentration. We implemented this concept for the production of isobutanol from glucose. The rheostat maintains adequate ATP concentrations even in the presence of ATPase contamination. The final system including the rheostat produced 24.1 ± 1.8 g/L of isobutanol from glucose in 91% theoretical yield with an initial productivity of 1.3 g/L/h. The molecular rheostat concept can be used in the design of continuously operating, self-sustaining synthetic biochemistry systems.