De novo design and evolution of an artificial metathase for cytoplasmic olefin metathesis

Abstract Artificial metalloenzymes 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. Here we address these limitations by introducing an artificial metathase—an artificial metalloenzyme designed for ring-closing metathesis—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 (turnover number ≥1,000) and biocompatibility. This work represents a pronounced leap in the de novo design and in cellulo engineering of artificial metalloenzymes, paving the way for abiological catalysis in living systems.