Lanthanide protein biosensors with a single ion-binding site

Rising demand for rare earth elements, including lanthanides (Lns), has intensified environmental pressures and supply-chain vulnerabilities, motivating the development of bio-based methods for their extraction and separation. However, the lack of high-throughput assays for analysing the selectivity of lanthanide-binding proteins remains a key bottleneck in engineering bio-based Ln-extraction systems. Here, we report the development of high-throughput assays based on Ln-responsive protein biosensors. These {beta}-lactamase-based biosensors contain receptors with a single Ln-binding site derived from either lanmodulin or the AI-designed protein RF2. We established multiplexed colourimetric assays that quantify biosensor activity and selectivity in vitro and in the periplasm of E. coli. We further demonstrate that E. coli cells expressing these biosensors exhibit Ln-dependent survival in the presence of {beta}-lactam antibiotics. These platforms enable large-scale testing of Ln biosensors and Ln-binding proteins.