Towards Directed Evolution of Rare Earth Element Binding Peptides Using Genetically Encoded Sensitizing Amino Acids

As demand for the rare earth elements (REEs) that power new and emerging technologies increases rapidly, there is a growing need for next-generation separations systems that can provide more selective isolation of these critical, but difficult-to-purify materials. Due to their evolvability and compatibility with existing separations platforms, polypeptides represent an ideal and environmentally-friendly chassis with which to meet this challenge; however, there exist no high-throughput approaches to selecting polypeptides with improved affinity and selectivity towards the various REEs, chiefly due to the difficulty of detecting a selectable phenotype. Here, we present work towards overcoming this obstacle by developing a bacterial display platform for the direct sensing of REE binding in living bacterial cells. Our approach exploits the antenna effect, which allows a sufficiently proximal fluorophore to sensitize a bound europium ion and stimulate emission through resonant energy transfer, and makes use of genetic code expansion to insert a fluorescent amino acid directly into the polypeptide backbone in cellulo. We find that targeting a model lanthanide-binding peptide to the periplasm enables the accumulation of sufficient concentrations of this binder in an easily accessible subcellular localization such that robust signal can be detected. Through optimization of encoding, sensitization, and handling, we further improve our signal up to >20-fold over control cells lacking binders and demonstrate facile differentiation and characterization of several variant peptides. Our work paves the way for high-throughput synthetic evolution of REE binding polypeptides with expanded functionality.