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Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

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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Figure 1: Transmission electron micrographs and analysis of PduA*-based constructs and filaments in E. coli.
Figure 2: Localization of fluorescent proteins to a bacterial cytoscaffold.
Figure 3: Ethanol production in vivo.
Figure 4: Targeting the bacterial cytoscaffold to the inner membrane of E. coli.

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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

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Authors

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.

Corresponding authors

Correspondence to Derek N Woolfson or Martin J Warren.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4, Supplementary Figures 1–14 (PDF 2536 kb)

41589_2018_BFnchembio2535_MOESM2_ESM.pdf

Reporting Summary (PDF 129 kb)

41589_2018_BFnchembio2535_MOESM3_ESM.mov

Tomography of CC-di-B-PduA filaments and automated microtubule tracing (MOV 21397 kb)

41589_2018_BFnchembio2535_MOESM4_ESM.mov

Refined tracing model on a small area of the tomogram shown in movie S1 (MOV 17382 kb)

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Lee, M., Mantell, J., Hodgson, L. et al. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm. Nat Chem Biol 14, 142–147 (2018). https://doi.org/10.1038/nchembio.2535

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