Artificial metalloenzymes result from combining a catalytically competent organometallic moiety with a host protein. The resulting hybrid catalyst combine attractive features of both chemo- and biocatalysts. In recent years, the Ward group has exploited the biotin-streptavidin towards the creation of artificial metalloenzymes for hydrogenation, allylic alkylation, sulfoxidation, alcohol oxidation, dihydroxylation, transfer-hydrogenation and olefin metathesis. The latter two systems were shown to be particularly stable towards E. coli cellular extracts. Within this funding period, it is proposed to exploit this finding towards the implementation of directed evolution protocols for the optimization of the performance of artificial metalloenzymes. Four complementary and intedisciplinary sub-projects will be investigated: i) exploiting streptavidin expressed in the periplasm; ii) cascade reactions with artificial metalloenzymes; iii) optimization of artificial transfer-hydrogenase for the production of high-added value amines and aminoacids and iv) directed evolution of artificial metathesases. i) In order to circumvent the inhibition of the biotinylated precious metal catalyst by glutathione (present in milimolar amounts in the cytoplasm), it is proposed to target streptavidin to the periplasm. This will allow us to sidestep the lengthy purification of streptavidin prior to catalysis, eventually allowing the implementation of directed evolution protocols. ii) We have shown that artificial metalloenzymes are compatible with a variety of biocatalysts. Combining artificial metalloenyzmes with natural enzymes will lead to complex reaction cascades that can be used a) as a high-throughput colorimetric assay or b) to complement metabolic pathways. iii) The above developments will be exploited towards the preparation of a) high-added value amines via the enantioselective imine reduction and b) leucine by relying on a selection strategy based on E. coli leucine auxotrophs. iv) Thanks to the inertness of artificial metathesases based on the biotin-streptavidin technology, the performance of these will be optmized using crude E. coli cell extracts. For this purpose, we will rely screening a fluorophore–quencher substrate which, upon ring closing metathesis releases the quencher, thus becoming fluorescent. Ultimately, we aim at developing artificial metalloenzymes that outperform classical organometallic catalysts. In a biomimetic spirit and thanks to Darwinian protocols, we anticipate that the presence of an optimized second coordination sphere provided by the protein environment will allow to achieve this ambitious goal.