A genetically encoded biosensor for in vitro and in vivo detection of NADP

NADP+, the oxidized form of nicotinamide adenine dinucleotide phosphate, plays an essential role as a coenzyme in cellular electron transfer reactions. The concentration of NADP+ in cytoplasm or organelles is dynamic due to its conversion to many important derivatives. To track the NADP+ concentration in single living cells, we developed a genetically encoded NADP+ biosensor by inserting a reporter element, ketopantoate reductase (KPR), between the Förster resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This recombinant sensor showed a NADP+ concentration-dependent decrease in the fluorescence ratio in vitro assay. In order to optimize this biosensor, we performed peptide-length optimization and site-directed mutagenesis in the binding pocket of KPR guided by predictions from computational protein redesign. This modified biosensor showed a 70% Δratio increase compared to the wild type and was found to be highly specific to NADP+, with a detection limit of 1μM. The sensor also reported NADP+ real-time cellular dynamics in Escherichia coli (E. coli) after the addition of its precursor, nicotinic acid (NA). Altogether, these results demonstrate the feasibility of the biosensor for visualizing NADP+ both in vitro and in vivo. •We generated a novel fluorescent sensor NADPsor for NADP detection.•The sensor NADPsor was optimized by the computational protein redesign.•This NADPsor sensor exhibited great sensitivity, selectivity, and kinetics.•NADP dynamics in Escherichia coli cells were reported in real time with this sensor.