A versatile method for designing biosensors via regulatory domains of allosteric enzymes.

Genetically encoded fluorescent biosensors (GEFBs) are invaluable tools for spatiotemporal metabolite monitoring in cellular metabolism, yet their development for many key metabolites is hampered by a lack of specific biorecognition elements. Here, we report a versatile strategy to engineer metabolite-responsive GEFBs by leveraging the allosteric properties of regulatory domains from allosteric enzymes. Using regulatory domains from chorismate mutase, 2-acetolactate synthase, and D-citramalate synthase as biorecognition elements, we construct three biosensors for specific L-phenylalanine, L-valine, and L-isoleucine detection. We further demonstrate that multi-ligand-binding regulatory domains can be exploited to derive diverse specific biosensors, and apply this strategy to develop two S-adenosyl-L-methionine biosensors and an S-methyl-5'-thioadenosine biosensor. We also showcase the utility of these biosensors for real-time, in situ tracking of target metabolites in living cells, as well as bioprocess monitoring and clinical diagnostics. Overall, this study establishes a flexible strategy that provides insights to construct GEFBs targeting other metabolites.