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Directed Evolution of Artificial Metalloproteins and Metalloenzymes

Research Project
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01.10.2018
 - 30.09.2022

Artificial metalloenzymes have emerged as a prolific field of research in the past decade. Such hybrid biosystems result from the incorporation of a metallocofactor within a protein scaffold to endow the macromolecule with novel catalytic properties. Thanks to the progress achieved in recombinant protein expression and evolution, artificial metalloproteins offer an attractive means to complement or substitute natural enzymes. Building on the group's expertize in the design and optimization of artificial metalloenzymes, this proposal sets out develop artificial monooxygenases based on the biotin-streptavidin technology. This work complements our work on C-H activation relying on Rh-based artificial metalloenzymes (Hyster et. al., Science 2012, 338, 500). The field of bioinorganic chemistry has developed a handful of nitrogen-based small coordination compounds containing earth-abundant metals that, in combination with hydrogen peroxide, have been used for the oxidation of hydrocarbons. Building on this, we will prepare a series of iron- and manganese complexes bearing biotinylated tetradentate aminopyridine ligands. Their activity will be screened in the presence of streptavidin. A chemo-genetic optimization strategy will be initially applied to improve the performance of the artificial monooxygenases. For this purpose, a series of model alkanes and alkyl ethers with increasing C-H bond dissociation energies will be screened using a 96-well plate format. Next, it is proposed to implement an E. coli surface display of streptavidin to significantly increase the throughput of the directed evolution effort. Here, we will rely on a fluorescence-activated droplet sorting high-throughput screen using coumarin methyl-ether as pro-fluorescent substrate. With the aim of exploiting artificial monooxygenases for the late-stage functionalization of complex molecules, we will subsequently evolve the regioselectivity of the most promising biohybrids. We anticipate that such systems may offer attractive alternatives to both natural enzymes and small molecule catalysts for the (late-stage) functionalization of inert C-H bonds.

Funding

Directed Evolution of Artificial Metalloproteins and Metalloenzymes

SNF Projekt (GrantsTool), 10.2018-09.2022 (48)
PI : Ward, Thomas R..

Members (1)

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Thomas R. Ward

Principal Investigator