Catalysis Science & Engineering, Short talk
CE-024

Development of a microfluidics-based assay for the evolution of artificial metalloenzymes using a cell surface display strategy

J. Vallapurackal1, A. Stucki2, P. Dittrich2*, T. Ward1*
1Department of Chemistry, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich

Incorporation of a transition metal catalyst within a protein scaffold enables the formation of artificial metalloenzymes (ArMs) with non-natural reactivities.[1]The catalytic activity and selectivity of such systems can be optimized by either chemical optimization of the metal cofactor or genetic evolution of the host protein. Integration of the biotinylated ruthenium complex[CpRu(Biot-Quinoline)(H2O)][2]into the binding pocket of streptavidin (Sav) affords an artificial deallylase capable of uncaging a protected coumarin yielding a fluorescent readout.[3]
Herein, we present a microfluidics-based high-throughput assay[4]to evolve the catalytic activity of an artificial deallylase. Single E. colicells with surface-displayed Sav are encapsulated in droplets together with the protected coumarin substrate and the biotinylated cofactor. The catalytically active mutants can be sorted by fluorescence-assisted droplet sorting (FADS) and the identified hits can be subjected to a further round of optimization. This method can be applied to evolve various ArMs in vivoand enable the screening of large libraries in an iterative and straightforward manner.

[1] Fabian Schwizer, Yasunori Okamoto, Tillmann Heinisch, Yifan Gu, Michela M. Pellizzoni, Vincent Lebrun, Raphael Reuter, Valentin Köhler, Jared C. Lewis, Thomas R. Ward, Chem. Rev., 2018, 118, 142–231.
[2] T. Völker, F. Dempwolff, P. L. Graumann, E. Meggers, Angew. Chem,. Int. Ed., 2014, 53, 10536–10540.
[3] T. Heinisch, F. Schwizer, B. Garebedian, E. Csibra, M. Jeschek, J. Vallapurackal, V. B. Pinheiro,   P.Marlière, S. Panke, T. R. Ward, Chem. Sci., 2018, 9, 5383–5388.
[4] R. Obexer, A. Godina, X. Garrabou, P. R. Mittl, A. D. Griffiths, D. Hilvert, Nat. Chem., 2017, 9, 50–56.