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

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Figure 1: Pathway designs for converting glucose to isobutanol.
Figure 2: Isobutanol production using the stoichiometric pathway.
Figure 3: Reaction modeling of isobutanol production by a system with or without the molecular rheostat in the presence of contaminating ATPase activity.
Figure 4: Final production of isobutanol.


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The authors thank members of the Bowie lab for helpful comments. This work was supported by DOE grants DE-FC02-02ER63421 and DE-AR0000556 to J.U.B.

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P.H.O., T.P.K. and J.U.B. contributed to the system design. P.H.O., T.P.K., L.I. and J.U.B. contributed to the design of experiments and data analysis. P.O., L.I. and T.K. performed the experiments. P.H.O., T.P.K. and J.U.B. wrote the paper.

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Correspondence to James U Bowie.

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Competing interests

The authors have formed a company, Invizyne Technologies, that will seek to exploit cell-free technologies.

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Opgenorth, P., Korman, T., Iancu, L. et al. A molecular rheostat maintains ATP levels to drive a synthetic biochemistry system. Nat Chem Biol 13, 938–942 (2017).

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