Herein, we present a project combining microfluidics-based single cell encapsulation and artificial metalloenzymes (ArMs) for the high throughput directed evolution of the latter. The catalytic activity and selectivity of ArMs can be optimized by either chemical optimization of the metal cofactor or genetic evolution of the host protein. Single E. coli cells with surface-displayed Sav are encapsulated in double emulsion droplets together with a caged coumarin substrate and a biotinylated cofactor. The artificial deallylase catalyzes the uncaging of the protected coumarin yielding a fluorescent product, and the catalytically active mutants can be sorted by fluorescence activated cell sorting (FACS). The identified hits can be subjected to further rounds of optimization. This method can be applied to evolve various ArMs in vivo and enables the screening of large libraries in an iterative and straightforward manner.

We present a high-throughput screening assay to perform directed evolution on single E. coli encapsulated in double emulsion droplets. The compartmentalization in droplets allowed us to screen a library of over 5’000 mutants within a week, significantly reducing the work load of six months necessary for traditional screening approaches in 96-well plates.

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