Creation of Artificial Subcellular Organelles Using Compartmentalized Escherichia coli Bodies for Artificial Metalloenzyme-Mediated Abiotic Catalysis in Eukaryotic Cells

Artificial metalloenzymes (ArMs) stand out as excellent tools mediating intracellular abiotic transformations due to their multifaceted advantages, including their adaptability through directed evolution and availability as whole-cell catalysts. However, the applications of ArMs as exogenous agents in eukaryotic systems remain challenging due to issues with protein purification and delivery, metalloenzyme stability, and complex catalyst preparation. In this article, we present a method inspired by nature's endosymbiotic process, enabling the direct use of ArMs residing within the bacterial cells that express them as whole-cell-based catalytic platforms in eukaryotic cells. This approach utilizes HaloTag-SNAPTag fusion protein as the ArM scaffold, which undergoes liquid-liquid phase separation to form sanctuaries in Escherichia coli for different ArMs created from the same fusion protein. Such compartmentalized E. coli are then sterilized and granted cell permeability with polymer decoration so that they may enter eukaryotic cells and work as artificial subcellular organelles, mediating abiotic transformations using those well-protected ArMs residing within. We further demonstrate the potential of this strategy in therapeutic applications in proof-of-concept demonstrations, by showing that these encapsulated ArMs can be viable options for intracellular bacterial pathogen elimination and cancer therapy through prodrug activation in live cells and animals. Likely, this strategy will suggest a different pathway for expanding ArM applications in chemical biology and biomedicine.