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Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Nature Chemical Biology volume 14, pages 142147 (2018) | Download Citation

Abstract

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

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Acknowledgements

We are grateful to the Biotechnology and Biological Sciences Research Council of the UK for a strategic LoLa Award to M.J.W., D.N.W., P.V. and W.-F.X. (BB/M002969/1). D.N.W. holds a Royal Society Wolfson Research Merit Award. We thank the Wolfson Bioimaging Facility and BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (L01386X), for access to confocal and electron microscopes; K. Howland for assistance with GC–MS analysis; R. Sessions and I. Uddin for preparing images used in Supplementary Figure 1; L. Harrington and P. Schwille for advice on the MinD system; and the entire BMC-SAGE LoLa group for helpful discussions.

Author information

Affiliations

  1. Industrial Biotechnology Centre, School of Biosciences, University of Kent, Canterbury, UK.

    • Matthew J Lee
    • , Ian R Brown
    • , Wei-Feng Xue
    •  & Martin J Warren
  2. School of Biochemistry, University of Bristol, Medical Sciences Building, University Walk, Bristol, UK.

    • Judith Mantell
    • , Lorna Hodgson
    • , Paul Verkade
    •  & Derek N Woolfson
  3. Wolfson Bioimaging Facility, Medical Sciences Building, University Walk, Bristol, UK.

    • Judith Mantell
    • , Dominic Alibhai
    •  & Paul Verkade
  4. School of Chemistry, University of Bristol, Cantock's Close, Bristol, UK.

    • Jordan M Fletcher
    •  & Derek N Woolfson
  5. Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London, UK.

    • Stefanie Frank
  6. BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol, UK.

    • Paul Verkade
    •  & Derek N Woolfson

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Contributions

M.J.L. made constructs, prepared samples for TEM and confocal analysis, imaged samples by TEM, purified nanotubes and analyzed them by TEM and AFM and conducted the ethanol production experiments and analyses. J.M. undertook tomography and 3D reconstructions. L.H. undertook CLEM sample preparation and imaging. D.A. undertook confocal imaging. I.R.B. sectioned samples for TEM analysis. W.-F.X. assisted with AFM and statistical analysis. M.J.L., J.M., L.H., J.M.F., S.F., P.V., D.N.W. and M.J.W. designed the experiments. All authors contributed to the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Derek N Woolfson or Martin J Warren.

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DOI

https://doi.org/10.1038/nchembio.2535

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