Continuous Evolution of Calmodulin for High‐Purity Separation of Rare Earth Elements

ABSTRACT The grand challenge of separating critical rare earth elements (REEs) stems from their similar physicochemical properties, requiring complex process flow‐sheets to achieve high‐purity products. Protein‐based strategies offer selective and sustainable alternatives to conventional separation processes. While native proteins have been identified for REE processing, protein engineering to enhance the separation of REE mixtures remains constrained by low‐throughput methods that limit the exploration of amino acids beyond the metal coordination sphere. Here, we developed a selection circuit based on a lanthanide‐mediated protein‐protein interaction for phage‐assisted continuous evolution (PACE). This system rapidly selected an evolving calmodulin‐derived peptide library, yielding a dominant sequence within days. Molecular dynamics simulations of the evolved protein suggest a restructured hydrogen‐bond network, which enhances second‐shell ion coordination and protein packing. These changes improved binding affinity and thermal stability, enabling single‐stage, chelator‐free, high‐purity separations of individual REEs. This high‐throughput approach can be readily extended to evolve other critical metal‐binding proteins.