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

Nature Chemical Biology volume 13, pages 938942 (2017) | Download Citation

Abstract

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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Acknowledgements

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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Affiliations

  1. Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA.

    • Paul H Opgenorth
    • , Tyler P Korman
    • , Liviu Iancu
    •  & James U Bowie

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Contributions

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.

Competing interests

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

Corresponding author

Correspondence to James U Bowie.

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DOI

https://doi.org/10.1038/nchembio.2418

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