Artificial Metalloenzymes in Artificial Sanctuaries Through Liquid–Liquid Phase Separation

Artificial metalloenzymes (ArMs) hold great promise for expanding the toolbox of non-natural transformations usable in living systems, such as cells, plants, and animals. However, their practical application remains challenging, primarily due to their unsatisfactory stability and inefficient intracellular assembly. We recently reported a new strategy, called artificial metalloenzymes in artificial sanctuaries (ArMAS) through liquid-liquid phase separation (LLPS), to enhance the performance of ArMs in cells by placing them in more friendly artificial microenvironments. Here, this protocol describes the detailed method for using this ArMAS-LLPS strategy, a robust way to create artificial compartments using an ArM protein scaffold through LLPS and construct ArMs within using self-labeling cofactor anchoring reactions. In detail, in <i>Escherichia coli</i>, membraneless protein condensates are formed by expressing a self-labeling fusion protein, HaloTag-SNAPTag (HS) and act as intracellular sanctuaries. Simultaneously, the HS scaffolds enable site-specific, bioorthogonal conjugation with synthetic metal cofactors, facilitating efficient ArM formation within the LLPS domains. This strategy can significantly enhance the intracellular catalytic activity and stability of the named HS-based ArMs, allowing whole-cell catalysis to be performed to enable abiotic transformations both in vitro and in vivo. The protocol provides a proof-of-concept approach for researchers aiming to develop stable ArM-based whole-cell catalytic systems for synthetic biology and therapeutic applications. Key features • Describes a robust and reproducible protocol for constructing artificial metalloenzymes (ArMs) in living <i>E. coli</i> cells using protein-driven liquid-liquid phase separation (LLPS). • Demonstrates how intracellular LLPS regions can serve as protective catalytic microenvironments, significantly improving ArM stability and catalytic turnover. • Applicable to various abiotic catalytic transformations, including olefin metathesis.