Abstract

The ratio between the cytosolic concentrations of monocarboxylates lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches to detect these metabolites do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a FRET-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable to be used as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules like acetate and β-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed a robust stability over time and found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.