Generation of Circularly Permuted Fluorescent-Protein-Based Indicators for In Vitro and In Vivo Detection of Citrate

<div><p>Indicators for citrate, particularly those applicable to its <i>in vivo</i> detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate <i>in vitro</i> at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the <i>in vivo</i> applicability of CF98 to estimation of the intracellular citrate concentration in <i>Escherichia coli</i> co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate <i>in vitro</i> and a non-invasive tool for real-time estimation of intracellular concentrations of the compound <i>in vivo</i>.</p>