21ENGBIO: In Cell Assembly of Artificial Imine Reductases for Whole-Cell Catalysis

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The project aims at the assembly of artificial enzymes that consist of a synthetic catalyst that is attached via an anchor group to a protein scaffold inside living bacterial cells. By combining the reactivity of the synthetic catalyst with the selectivity and biocompatibility of the protein, the resulting artificial metalloenzymes aim to perform efficient catalytic reactions within the cellular environment of engineered cells. To achieve the uptake of the catalysts by bacterial cells we propose to take advantage of active bacterial iron-transport pathways. Using bacterial proteins that are involved in the transport of essential iron is advantageous since it they are produced by the bacterial cell and secreted into the periplasm, a suitable compartment for catalysis to take place. There the proteins are ready for the capture of the anchored catalyst that is actively transported through the outer membrane disguised as an iron-containing 'Trojan horse'. Small substrates can enter through porins and the products formed can either be released or react further. Consequently, this approach could eventually utilise the metabolism of the bacterial cell for the production of valuable target molecules in living cells.Following catalysis, the release of the iridium-based catalyst from the protein scaffold, its recovery and re-use will be investigated.