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Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli

Nature Chemical Biology volume 14, pages 794–800 (2018)Cite this article

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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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References

  1. Gillespie, D. E. et al. Isolation of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA. Appl. Environ. Microbiol. 68, 4301–4306 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rutledge, P. J. & Challis, G. L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat. Rev. Microbiol. 13, 509–523 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Brady, S. F., Simmons, L., Kim, J. H. & Schmidt, E. W. Metagenomic approaches to natural products from free-living and symbiotic organisms. Nat. Prod. Rep. 26, 1488–1503 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Olano, C., Lombó, F., Méndez, C. & Salas, J. A. Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab. Eng. 10, 281–292 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Katz, L. & Khosla, C. Antibiotic production from the ground up. Nat. Biotechnol. 25, 428–429 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Demain, A. L. From natural products discovery to commercialization: a success story. J. Ind. Microbiol. Biotechnol. 33, 486–495 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Medema, M. H., Breitling, R., Bovenberg, R. & Takano, E. Exploiting plug-and-play synthetic biology for drug discovery and production in microorganisms. Nat. Rev. Microbiol. 9, 131–137 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Awan, A. R. et al. Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast. Nat. Commun. 8, 15202 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Freeman, M. F., Vagstad, A. L. & Piel, J. Polytheonamide biosynthesis showcasing the metabolic potential of sponge-associated uncultivated ‘Entotheonella’ bacteria. Curr. Opin. Chem. Biol. 31, 8–14 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Hamed, R. B., Henry, L., Claridge, T. D. W. & Schofield, C. J. Stereoselective production of dimethyl-substituted carbapenams via engineered carbapenem biosynthesis enzymes. ACS Catal. 7, 1279–1285 (2017).

    Article  CAS  Google Scholar 

  12. Zhang, H., Wang, Y., Wu, J., Skalina, K. & Pfeifer, B. A. Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host. Chem. Biol. 17, 1232–1240 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. McKenna, M. Antibiotic resistance: the last resort. Nature 499, 394–396 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Papp-Wallace, K. M., Endimiani, A., Taracila, M. A. & Bonomo, R. A. Carbapenems: past, present, and future. Antimicrob. Agents Chemother. 55, 4943–4960 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wei, W. J., Yang, H. F., Ye, Y. & Li, J. B. Bin. New Delhi metallo-beta-lactamase-mediated carbapenem resistance: Origin, diagnosis, treatment and public health concern. Chin. Med. J. (Engl.) 128, 1969–1976 (2015).

    Article  Google Scholar 

  16. Kahan, J. S. et al. Thienamycin, a new beta-lactam antibiotic. I. Discovery, taxonomy, isolation and physical properties. J. Antibiot. (Tokyo) 32, 1–12 (1979).

    Article  CAS  Google Scholar 

  17. Hamed, R. B. et al. The enzymes of β-lactam biosynthesis. Nat. Prod. Rep. 30, 21–107 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Coulthurst, S. J., Barnard, A. M. L. & Salmond, G. P. C. Regulation and biosynthesis of carbapenem antibiotics in bacteria. Nat. Rev. Microbiol. 3, 295–306 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. McGowan, S. J. et al. Analysis of bacterial carbapenem antibiotic production genes reveals a novel beta-lactam biosynthesis pathway. Mol. Microbiol. 22, 415–426 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Pfaendler, H. R., Gosteli, J., Woodward, R. B. & Rihs, G. Structure, reactivity, and biological activity of strained bicyclic β-lactams. J. Am. Chem. Soc. 103, 4526–4531 (1981).

    Article  CAS  Google Scholar 

  21. McGowan, S. J. et al. Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel β-lactam resistance mechanism. Mol. Microbiol. 26, 545–556 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. van Vugt-Lussenburg, B. M., van der Weel, L., Hagen, W. R. & Hagedoorn, P. L. Biochemical similarities and differences between the catalytic [4Fe-4S] cluster containing fumarases FumA and FumB from Escherichia coli. PLoS One 8, e55549 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. McHugh, J. P. et al. Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J. Biol. Chem. 278, 29478–29486 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Stiefel, E. I. & George, G. N. in Bioinorganic Chemistry (eds. Bertini, I., Gray, H.B., Lippard, S.J. & Valentine, J.S.) Ch. 7 (University Science Books, Mill Valley, CA, 1994).

  25. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  26. Gerratana, B., Stapon, A. & Townsend, C. A. Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora. Biochemistry 42, 7836–7847 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Stapon, A., Li, R. & Townsend, C. A. Carbapenem biosynthesis: confirmation of stereochemical assignments and the role of CarC in the ring stereoinversion process from L-proline. J. Am. Chem. Soc. 125, 8486–8493 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Chang, W. C. et al. Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics. Science 343, 1140–1144 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pérez-Arellano, I., Carmona-Álvarez, F., Gallego, J. & Cervera, J. Molecular mechanisms modulating glutamate kinase activity. Identification of the proline feedback inhibitor binding site. J. Mol. Biol. 404, 890–901 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Korch, S. B. & Hill, T. M. Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation. J. Bacteriol. 188, 3826–3836 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bokinsky, G. et al. HipA-triggered growth arrest and β-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis. J. Bacteriol. 195, 3173–3182 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Heath, R. J., Jackowski, S. & Rock, C. O. Guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in Escherichia coli is relieved by overexpression of glycerol-3-phosphate acyltransferase (plsB). J. Biol. Chem. 269, 26584–26590 (1994).

    CAS  PubMed  Google Scholar 

  33. Rodionov, D. G. & Ishiguro, E. E. Dependence of peptidoglycan metabolism on phospholipid synthesis during growth of Escherichia coli. Microbiology 142, 2871–2877 (1996).

    Article  CAS  PubMed  Google Scholar 

  34. Omura, S. The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis. Bacteriol. Rev. 40, 681–697 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Rogers, J. K. & Church, G. M. Genetically encoded sensors enable real-time observation of metabolite production. Proc. Natl Acad. Sci. USA 113, 2388–2393 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yang, Y., Lin, Y., Li, L., Linhardt, R. J. & Yan, Y. Regulating malonyl-CoA metabolism via synthetic antisense RNAs for enhanced biosynthesis of natural products. Metab. Eng. 29, 217–226 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Fehér, T., Libis, V., Carbonell, P. & Faulon, J.-L. A sense of balance: experimental investigation and modeling of a malonyl-CoA sensor in Escherichia coli. Front. Bioeng. Biotechnol. 3, 46 (2015).

    PubMed  PubMed Central  Google Scholar 

  38. Cameron, D. E. & Collins, J. J. Tunable protein degradation in bacteria. Nat. Biotechnol. 32, 1276–1281 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rosi, D. et al. Mutants of Streptomyces cattleya producing N-acetyl and deshydroxy carbapenems related to thienamycin. J. Antibiot. (Tokyo) 34, 341–343 (1981).

    Article  CAS  Google Scholar 

  40. Wilson, K. E., Kempf, A. J., Liesch, J. M. & Arison, B. H. Northienamycin and 8-epi-thienamycin, new carbapenems from Streptomyces cattleya. J. Antibiot. (Tokyo) 36, 1109–1117 (1983).

    Article  CAS  Google Scholar 

  41. Wagman, G. H. & Cooper, R. Natural Products Isolation: Separation Methods for Antimicrobials, Antivirals and Enzyme Inhibitors (Elsevier Science, New York, 1988).

  42. Bateson, J. H., Baxter, A. J. G., Roberts, P. M., Smale, T. C. & South-Gate, R. Olivanic acid analogues. Part 1. Total synthesis of the 7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate system and some related β-lactams. J. Chem. Soc. Perkin Trans. I 1981, 3242–3249 (1981).

    Article  Google Scholar 

  43. Zabala, D., Braña, A. F., Flórez, A. B., Salas, J. A. & Méndez, C. Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus. Metab. Eng. 20, 187–197 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Gómez, C. et al. Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus. J. Bacteriol. 193, 4214–4223 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, M. M., Wang, Y., Ang, E. L. & Zhao, H. Engineering microbial hosts for production of bacterial natural products. Nat. Prod. Rep. 33, 963–987 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Marous, D. R. et al. Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proc. Natl Acad. Sci. USA 112, 10354–10358 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kirby, J. et al. Engineering a functional 1-deoxy-d-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae. Metab. Eng. 38, 494–503 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tuomanen, E. Phenotypic tolerance: the search for β-lactam antibiotics that kill nongrowing bacteria. Rev. Infect. Dis. 8 (Suppl. 3), S279–S291 (1986).

  49. Briand, L. et al. A self-inducible heterologous protein expression system in Escherichia coli. Sci. Rep. 6, 33037 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Neidhardt, F. C., Bloch, P. L. & Smith, D. F. Culture medium for enterobacteria. J. Bacteriol. 119, 736–747 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Lee, T. S. et al. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J. Biol. Eng. 5, 12 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hagen, W. R. Biomolecular EPR Spectroscopy. CRC Press. (CRC Press, Boca Raton, 2008).

    Book  Google Scholar 

  54. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  PubMed  Google Scholar 

  55. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Parker, W. L. et al. SQ 27,860, a simple carbapenem produced by species of Serratia and Erwinia. J. Antibiot. (Tokyo) 35, 653–660 (1982).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands

    Helena Shomar, Niels J. F. van den Broek, Héctor Tejeda Mora, Marek J. Noga & Gregory Bokinsky

  2. Centre for Research and Interdisciplinarity (CRI), Paris Descartes University, Paris, France

    Sophie Gontier

  3. Department of Biotechnology, Delft University of Technology, Delft, the Netherlands

    Peter-Leon Hagedoorn

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  1. Helena Shomar
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Contributions

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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