High-performance genetically-encoded green and red fluorescent biosensors for pyruvate

Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., Δ F / F ₀ > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change (Δ F / F ₀ ∼ 20 to >40) and a range of affinities (10s of μM to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.