Rare earth elements (REEs) consist of the lanthanides, scandium, and yttrium. Their superior magnetic properties are critical in the transition to a green economy. However, separating REEs from one another and contaminants like iron and calcium is challenging due to their similar physical and chemical properties, requiring energy-intensive and chemical-intensive processes. Protein-based separation methods have been explored at the lab scale, utilizing lanthanide-binding and calcium-binding proteins. Nonetheless, the effectiveness of these separations has been limited by the capabilities of the identified wild-type proteins. Engineering peptides and proteins to bind REEs, such as lanthanide-binding tags (LBTs), is a slow and labor-intensive process, as testing protein libraries for binding affinity typically occurs with pure proteins in vitro. In this work, we demonstrate the evolution of REE binding affinity in-vivo using phage-assisted continuous evolution (PACE). Phage replication was linked to the protein-protein interaction between lanthanide-bound calmodulin and the peptide C20W. We modulated the selection stringency by varying the dilution rate and the concentration of REEs in the bioreactor. After 200 hours of continuous evolution, a mutant calmodulin emerged as the dominant phage species in the reactor. In-vitro analysis of the evolved mutant shows a tenfold increase in affinity for REEs compared to the wild type, as measured by tyrosine-Tb FRET, isothermal titration calorimetry (ITC), and circular dichroism (CD). The developed circuit can further improve affinity and selectivity through positive and negative selection circuits. Finally, the developed mutant was immobilized to successfully demonstrate single-stage high-purity separation of a mixture rare earth elements.
This research was developed with funding from the Defense Advanced Research Projects Agency (DARPA). The views, opinions and/or findings expressed are those of the author and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. Approved for Public Release, Distribution Unlimited.
