De Novo Design and Evolution of an Artificial Metathase for Cytoplasmic Olefin Metathesis

Artificial metalloenzymes (ArMs) present a promising avenue for abiotic catalysis within living systems. However, their in vivo application is currently limited by critical challenges, particularly in selecting suitable protein scaffolds capable of binding abiotic cofactors and maintaining catalytic activity in complex media. Herein, we address these limitations by introducing an artificial metathase—an artificial metalloenzyme designed for ring-closing metathesis (RCM)— for whole cell biocatalysis. Our approach integrates a tailored metal cofactor into a hyper-stable, de novo designed protein. By combining computational design with genetic optimization, a binding affinity (K_D ≤ 0.2 μM) between the protein scaffold and cofactor is achieved through supramolecular anchoring. Directed evolution of the artificial metathase yielded variants exhibiting excellent catalytic performance (TON ≥ 1000) and biocompatibility. This work represents a pronounced leap in the de novo design and in vivo engineering of artificial metalloenzymes, paving the way for abiological catalysis in living systems.