Deploying Artificial Metalloenzymes in Complex Environments: Strategies and Applications

Artificial metalloenzymes (ArMs) are engineered metalloproteins with modified metal coordination spheres or abiotic metal cofactors, and they are granted the ability to catalyze reactions rarely or never observed in natural biological processes. Due to their customizable, wide‐spectrum catalytic abilities, ArMs are believed to have high potential in multiple fields, such as synthetic biology, chemical biology, and biomedical research. However, a prerequisite for ArMs to serve well in their roles is the robustness that allows them to function in complex biological environments. This presents multiple challenges, including the difficulties in assembling ArMs in complex environments, metal cofactor instability, and the interference from cellular components. Strategies are established to stabilize ArMs and adapt them for operation in three distinct scenarios: (i) in cell lysates, which provide convenient conditions for ArM screening and function; (ii) as the core functionality of whole‐cell catalysts, in which ArMs can work on or in the cells; and (iii) in live animals, for which the use of ArMs is being explored for chemical biology studies and therapeutics. This review highlights the design principles, breakthroughs, and opportunities for advancing ArMs as robust tools in these contexts, hoping to bring inspirations for scientists who are interested in this area.