Let it sense! Biosensor creation using computational and structural approaches

Gaining insight into the physiology of individual microbes and their interactions within intricate networks necessitates a profound understanding of dynamic metabolic processes. Deciphering and quantifying these processes hinges on key factors, including pathways of metabolite transport, nutrient exchange, and signaling. High temporal and spatial resolution of metabolic pathways can be achieved through the use of genetically encoded biosensors in combination with advanced imaging techniques.

Here, we establish a pipeline for biosensor development based on rational design, leveraging state-of-the-art molecular techniques integrated with computational structural biology to create novel biosensors for various metabolites, such as ATP, ferrous iron, and sucrose. This pipeline currently employs two distinct biosensor designs: the FRET-based and Matryoshka biosensor cassettes. FRET-based biosensors are relatively straightforward and largely empirical in their design, in contrast to the Matryoshka biosensors, which require a more sophisticated design process. In Matryoshka biosensors, fluorescent proteins must be embedded within the recognition protein in a way that enables them to report metabolite binding through changes in fluorescence intensity. Additionally, ratiometric Matryoshka biosensors not only track metabolite binding but also monitor the biosensor"s expression levels within cells, facilitating direct comparisons across experiments and various bacterial growth phases. Our design pipeline has significantly accelerated the development of biosensors, achieving high success rates, whereas previous empirical design approaches were often time-consuming and heavily reliant on trial and error approaches.

Here we will highlight the crucial steps in the design and show how computional structural biology can help to change from emperical to rational sensor design. As an example, we developed a novel biosensor for ferrous iron, validated both in vitro and in vivo, which senses within the micromolar range, that is based on the DtxR recognition protein.