Engineering of a biosensor for intracellular aspartate

ABSTRACT Aspartate is a limiting metabolite in proliferating cells with its production closely linked to glutamine and mitochondrial metabolism. To date, measuring aspartate concentrations in live cells was deemed impossible. We present iAspSnFR, a genetically-encoded biosensor for intracellular aspartate, engineered by displaying and screening biosensor libraries in HEK293 cells. In live cells, iAspSnFR exhibits a dynamic range of 130% fluorescence change and detects reduced aspartate levels upon glutamine deprivation or glutaminase inhibition. Furthermore, iAspSnFR tracks aspartate uptake by excitatory amino acid transporters, or of asparagine after co-expression of an asparaginase. Importantly, iAspSnFR reports aspartate depletions upon electron transport chain inhibition, and therefore it can serve as a proxy for mitochondrial respiration. Consequently, iAspSnFR can dissect the major cellular pathways of aspartate production, offering immediate applications, particularly in cancer biology, such as screening small molecules targeting aspartate and glutamine metabolism.