title: 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.
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Ewald
authors.forename: Sabrina
authors.initials: S
authors.lastname: Reich
authors.forename: Stephan
authors.initials: S
authors.lastname: Baumann
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
authors.forename: Nicola
authors.initials: N
authors.lastname: Zamboni
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000235:genetics; D036103:Nanotechnology / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics; D012492:Salts / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
chemical_list: C485182:Cyan Fluorescent Protein; D017931:DNA Primers; D029968:Escherichia coli Proteins; D009711:Nucleotides; D012492:Salts; D049452:Green Fluorescent Proteins; D019343:Citric Acid; D002244:Carbon; D011494:Protein Kinases; C460095:CitA protein, E coli