A Cholesterol Analogue for Cell‐Surface Enzyme Display

The cell membrane is a prime target for the introduction of novel cellular functionalities, as it is a complex system with many routes for surface modification. Several chemical coating and genetic engineering methods have thus been developed for this purpose. Here, a distinct way to enable enzyme‐binding onto the surface of bacterial cells is explored using biomimetic lipids that integrate within the cell membrane. E. coli cells were equipped with a cholesterol‐based artificial lipid containing a nitrilotriacetic acid (NTA) group which, when loaded with Ni 2+ ions, selectively binds His‐tagged enzymes through affinity interactions. This interaction is stable and selective for tagged proteins including green fluorescent protein, enabling their direct one‐step purification and immobilisation from cell lysates. Furthermore, the process is biocompatible and preserves both intracellular and cell‐surface enzymatic activity. This strategy further enables binding of benzaldehyde lyase or amine transaminase enzymes to the surface of bacterial cells for recyclable single‐step enzymatic reactions. Importantly, it allowed the creation of a single‐cell system for the two‐step cascade reaction from benzyl alcohol to (R) ‐benzoin using both intracellular and surface‐immobilised enzymes. This provides a solid proof of concept for the streamlined development of cascade reaction systems in a single cell through non‐genetic cell surface enzyme immobilisation.