A synthetic biochemistry molecular purge valve module that maintains redox balance

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Abstract

The greatest potential environmental benefit of metabolic engineering would be the production of high-volume commodity chemicals, such as biofuels. Yet, the high yields required for the economic viability of low-value chemicals is particularly hard to achieve in microbes owing to the myriad competing biochemical pathways. An alternative approach, which we call synthetic biochemistry, is to eliminate the organism by constructing biochemical pathways in vitro. Viable synthetic biochemistry, however, will require simple methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge valve module for maintaining NADP+/NADPH balance. We test the purge valve in the production of polyhydroxybutyryl bioplastic and isoprene—pathways where cofactor generation and utilization are unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily transportable. The molecular purge valve provides a step towards developing continuously operating, sustainable synthetic biochemistry systems.

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Figure 1: A synthetic biochemistry purge valve system for the production of PHB.
Figure 2: Design of the PDHNADPH enzyme.
Figure 3: Production of PHB using an optimized system.
Figure 4: Time course of pyruvate to PHB optimization reaction using sub-optimal ratios of PDHNADPH and PDHNADH.
Figure 5: The purge valve system is robust.
Figure 6: Employing the purge valve for the production of isoprene.

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Acknowledgements

We thank members of the Bowie lab for critical reading of this manuscript. The work was supported by the DOE grant DE-FC02-02ER63421 to J.U.B.

Author information

P.H.O., T.P.K. and J.U.B. designed the experiments, analysed experimental results and wrote the maunscript. P.H.O. and T.P.K. conducted the experiments.

Correspondence to James U. Bowie.

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The authors declare no competing financial interests.

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Supplementary Figures 1-2, Supplementary Tables 1-4 and Supplementary References (PDF 423 kb)

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