Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations

Abstract

Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

Figure 1. Fluorescence emission spectrum of CitA with and without 500 µM citrate.

The FRET sensor CFP-CitA-Venus was purified and the fluorescence emission recorded at an excitation of 433 nm in a fluorescence plate reader. The dotted line represents the spectrum of the protein in buffer, the dashed line the protein after addition of 500 µM citrate.

Figure 2. Binding curve of citrate to the FRET nanosensor CIT8μ.

The FRET sensor CFP-truncated CitA-Venus was purified and the fluorescence emission of CFP and Venus recorded at an excitation of 433 nm in a fluorescence plate reader. The emission ratio 530/488 nm was determined at different citrate (A) and isocitrate (B) concentrations. Data points are averages of three independent protein extractions. Error bars indicate standard deviations. Data were fitted to a single site binding curve (black line) as described in the method section.

Figure 3. Response of citrate concentration in E. coli upon addition of different carbon substrates after starvation.

The response was monitored by citrate nanosensors of different affinities. Time zero indicates the addition of glucose, acetate or citrate; this interrupted the measurement for approximately 20 s. White dots represent the buffer controls. FI: fluorescence intensity.

Figure 4. Citrate concentration changes in E. coli in response to carbon substrates after 24 hours of starvation.

Addition of substrates is indicated as time point t = 0. Manual addition interrupted the measurement for approximately 10 s. Black dots: glucose, white dots: acetate. Time points are averaged over three independent experiments.