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Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli

Abstract

Carbapenems, a family of β-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.

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Fig. 1: The Car biosynthesis pathway and LC–MS detection of synthetic intermediates and products.
Fig. 2: Metabolic engineering of the Car biosynthesis pathway in E. coli.
Fig. 3: Car production causes lysis and limits achievable titers.
Fig. 4: Inhibition of fatty acid synthesis increases antibiotic tolerance and Car pathway flux.

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Acknowledgements

We thank D.E. Cameron and J.J. Collins (Massachusetts Institute of Technology) for the gift of strains for designing tunable protein degradation. We are grateful to V. Libis (Rockefeller University) for the gift of the plasmid pCFR. We thank F. Wu for valuable scientific discussions throughout the project and B. Beaumont for helpful comments with the manuscript. This work was supported by startup funds provided to G.B. from TU Delft and the Department of Bionanoscience, as well as the Netherlands Organization for Scientific Research (NWO/OCW), as part of the Frontiers of Nanoscience program. P.-L.H. was supported by grant NWO–CW 711.014.006 from the Council for Chemical Sciences of the NWO.

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G.B. and H.S. designed the experiments. H.S. performed the experiments and analyzed the results. S.G. constructed strains and performed experiments for studying the function of CarE. H.T.M. performed preliminary experiments to study FAS inhibition. P.-L.H. conducted EPR spectroscopy and analyzed related data. H.S. and N.v.d.B. developed LC–MS methods, with early assistance from M.J.N. G.B. supervised the research. H.S. and G.B. wrote the manuscript.

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Correspondence to Gregory Bokinsky.

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G.B. and H.S. have filed a patent application covering metabolic engineering of carbapenem pathways.

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Shomar, H., Gontier, S., van den Broek, N.J.F. et al. Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli. Nat Chem Biol 14, 794–800 (2018). https://doi.org/10.1038/s41589-018-0084-6

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