Artificial metalloenzyme assembly in cellular compartments for enhanced catalysis

Artificial metalloenzymes (ArMs) integrated within whole cells have emerged as promising catalysts; however, their sensitivity to metal centers remains a systematic challenge, resulting in diminished activity and turnover. Here we address this issue by inducing in cellulo liquid-liquid phase separation through a self-labeling fusion protein, HaloTag-SNAPTag. This strategy creates membraneless, isolated liquid condensates within Escherichia coli as protective compartments for the assembly of ArMs using the same fusion protein. The approach allows for high ArM loading and stabilization by localizing the ArMs within the phase-separated regions. Consequently, the performance of ArM-based whole-cell catalysts is improved, with a demonstrated turnover per cell of up to 7.1 × 10⁹ for the olefin metathesis reaction. Furthermore, we apply this to an engineered E. coli system in live mice, where host bacterial cells confine the metal catalytic species, and in a mouse colorectal cancer model, where ArM-containing whole-cell catalysts mediate concurrent reactions to activate prodrugs.