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

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.

This proposal targets the assembly of artificial imine reductases that consist of synthetic catalysts attached via an anchor group to cognate protein scaffolds in the periplasm of living E. coli cells. By combining highly-reactive iridium-based transfer hydrogenation catalysts with the selectivity and biocompatibility of periplasmic binding proteins, the resulting artificial imine reductases may perform efficient transfer hydrogenation reactions within the cellular environment of engineered cells.

To achieve active transport of the iridium catalysts into bacterial cells we propose to take advantage of bacterial iron-uptake pathways that are mediated by siderophores. Using bacterial iron-siderophore binding proteins as scaffolds is advantageous since they are produced by the bacterial cell and exported into the periplasm, a suitable compartment for catalysis to take place. There they are ready for the capture of siderophore-anchored catalysts that are actively transported through the outer membrane. In this way, the metabolism of the bacterial cell can be exploited to support chemical transformations, such as the production of valuable enantiopure amines, in vivo. Small substrates can enter through porins and the products can either be released or react further as part of reaction cascades. Following catalysis, the reduction-triggered release of the iridium-based catalyst from the protein scaffold, its recovery and re-use will be investigated.

The ultimate aim will be to integrate artificial imine reductases and other artificial metalloenzymes into engineered biochemical pathways for application in whole-cell biocatalysis.