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

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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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.

References

  1. Cameron, D.E., Bashor, C.J. & Collins, J.J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).

    Article  CAS  Google Scholar 

  2. Kwok, R. Five hard truths for synthetic biology. Nature 463, 288–290 (2010).

    Article  CAS  Google Scholar 

  3. Zhang, Y.-H.P. Simpler is better: high-yield and potential low-cost biofuels production through cell-free synthetic pathway biotransformation (SyPaB). ACS Catal. 1, 998–1009 (2011).

    Article  CAS  Google Scholar 

  4. Zhang, Y.-H.P. Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: challenges and opportunities. Biotechnol. Bioeng. 105, 663–677 (2010).

    CAS  PubMed  Google Scholar 

  5. Schultheisz, H.L., Szymczyna, B.R., Scott, L.G. & Williamson, J.R. Pathway engineered enzymatic de novo purine nucleotide synthesis. ACS Chem. Biol. 3, 499–511 (2008).

    Article  CAS  Google Scholar 

  6. Schultheisz, H.L., Szymczyna, B.R., Scott, L.G. & Williamson, J.R. Enzymatic de novo pyrimidine nucleotide synthesis. J. Am. Chem. Soc. 133, 297–304 (2011).

    Article  CAS  Google Scholar 

  7. Rollin, J.A., Tam, T.K. & Zhang, Y.-H.P. New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chem. 15, 1708–1719 (2013).

    Article  CAS  Google Scholar 

  8. Krutsakorn, B. et al. In vitro production of n-butanol from glucose. Metab. Eng. 20, 84–91 (2013).

    Article  CAS  Google Scholar 

  9. Hodgman, C.E. & Jewett, M.C. Cell-free synthetic biology: thinking outside the cell. Metab. Eng. 14, 261–269 (2012).

    Article  CAS  Google Scholar 

  10. Zhu, Z., Kin Tam, T., Sun, F., You, C. & Percival Zhang, Y.-H. A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nat. Commun. 5, 3026 (2014).

    Article  Google Scholar 

  11. Zhang, Y.-H.P., Evans, B.R., Mielenz, J.R., Hopkins, R.C. & Adams, M.W.W. High-yield hydrogen production from starch and water by a synthetic enzymatic pathway. PLoS One 2, e456 (2007).

    Article  Google Scholar 

  12. Ye, X. et al. Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. ChemSusChem 2, 149–152 (2009).

    Article  CAS  Google Scholar 

  13. Welch, P. & Scopes, R.K. Studies on cell-free metabolism: ethanol production by a yeast glycolytic system reconstituted from purified enzymes. J. Biotechnol. 2, 257–273 (1985).

    Article  CAS  Google Scholar 

  14. Korman, T.P. et al. A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates. Protein Sci. 23, 576–585 (2014).

    Article  CAS  Google Scholar 

  15. Guterl, J.-K. et al. Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5, 2165–2172 (2012).

    Article  CAS  Google Scholar 

  16. Opgenorth, P.H., Korman, T.P. & Bowie, J.U. A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat. Chem. Biol. 12, 393–395 (2016).

    Article  CAS  Google Scholar 

  17. Opgenorth, P.H., Korman, T.P. & Bowie, J.U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 5, 4113 (2014).

    Article  CAS  Google Scholar 

  18. Atsumi, S. et al. Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol. Syst. Biol. 6, 449 (2010).

    Article  Google Scholar 

  19. Atsumi, S. et al. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl. Microbiol. Biotechnol. 85, 651–657 (2010).

    Article  CAS  Google Scholar 

  20. Li, X., Shen, C.R. & Liao, J.C. Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942. Photosynth. Res. 120, 301–310 (2014).

    Article  CAS  Google Scholar 

  21. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008).

    Article  CAS  Google Scholar 

  22. Valverde, F., Losada, M. & Serrano, A. Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene. FEBS Lett. 449, 153–158 (1999).

    Article  CAS  Google Scholar 

  23. Van Schaftingen, E. et al. Metabolite proofreading, a neglected aspect of intermediary metabolism. J. Inherit. Metab. Dis. 36, 427–434 (2013).

    Article  Google Scholar 

  24. Didierjean, C. et al. Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate. J. Biol. Chem. 278, 12968–12976 (2003).

    Article  CAS  Google Scholar 

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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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Authors

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.

Corresponding author

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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Supplementary Results, Supplementary Tables 1–7, Supplementary Figures 1–4 and Supplementary Note (PDF 1061 kb)

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

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