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 Ni²⁺ 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.