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Evolution of enzyme catalysts caged in biomimetic gel-shell beads

Nature Chemistry volume 6, pages 791–796 (2014)Cite this article

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Abstract

Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these man-made compartments can be identified readily by fluorescence-activated sorting at rates >107 GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components.

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Figure 1: Directed evolution in biomimetic GSBs.
Figure 2: Model selections and directed evolution of active PTE in GSBs.
Figure 3: Triester hydrolysis by PTE and its evolved variants.

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Acknowledgements

This research was funded by the Engineering and Physical Sciences Research Council, by European Union Marie-Curie fellowships (to M.F. and M.F.M.) and by fellowships from the Schering Foundation, the Cambridge Overseas Trust and Trinity Hall (to Y.S.). F.H. is a European Research Council Starting Investigator. We thank N. Miller for help with FACS and several colleagues for helpful discussions and comments on the manuscript. We thank RainDance Technologies (Lexington, USA) for a sample of the EA-surfactant.

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  1. Martin Fischlechner

    Present address: Present address: Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK,

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK

    Martin Fischlechner, Yolanda Schaerli, Mark F. Mohamed & Florian Hollfelder

  2. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Martin Fischlechner, Yolanda Schaerli, Santosh Patil & Chris Abell

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  1. Martin Fischlechner
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Contributions

M.F. developed the concept and designed, conducted and analysed the experiments. Y.S. developed the concept and designed, conducted and analysed preliminary experiments. M.F., Y.S., M.F.M. and F.H. wrote the manuscript. M.M. and S.P. synthesized enzyme substrates. F.H. and C.A. directed the research.

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Correspondence to Florian Hollfelder.

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Fischlechner, M., Schaerli, Y., Mohamed, M. et al. Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nature Chem 6, 791–796 (2014). https://doi.org/10.1038/nchem.1996

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