title: Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells.
abstract: BACKGROUND
  Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer (FRET) based genetically encoded nanosensors represent a promising tool for measuring the metabolite levels and corresponding rate changes in live cells. Here, we report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of L-lysine is of major interest in industrial biotechnology.
  
  
  RESULTS
  The lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor. The LAO was sandwiched between GFP variants i.e. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Affinity, pH stability, specificity and metal ions effects was scrutinized for the in vitro characterization of this nanosensor, named as FLIPK. The FLIPK is specific to lysine and found to be stable with the pH within the physiological range. The calculated affinity (K d ) of FLIPK was 97 µM. For physiological applications, mutants with different binding affinities were also generated and investigated in vitro. The developed nanosensor efficiently monitored the intracellular level of lysine in bacterial as well as yeast cell.
  
  
  CONCLUSIONS
  The developed novel lysine fluorescence resonance energy transfer sensors can be used for in vivo monitoring of lysine levels in prokaryotes as well as eukaryotes. The potential of these sensors is that they can be used as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular lysine during fermentation.
authors.affiliation: Department of Botany, Faculty of Science, Hamdard University, New Delhi, India.
authors.forename: Seema
authors.initials: S
authors.lastname: Ameen
authors.affiliation: Department of Botany, Faculty of Science, Hamdard University, New Delhi, India.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ahmad
authors.affiliation: Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Mohsin
authors.affiliation: Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, India.
authors.forename: M Irfan
authors.initials: MI
authors.lastname: Qureshi
authors.affiliation: Department of Botany & Microbiology, Science College, King Saud University, P.O. Box 2455, Riyadh, Saudi Arabia.|Department of Botany & Microbiology, Faculty of Science, Alexandria University, P.O. Box 21511, Alexandria, Egypt.
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Ibrahim
authors.affiliation: Department of Biotechnology, Faculty of Science, Hamdard University, New Delhi, India.
authors.forename: Malik Z
authors.initials: MZ
authors.lastname: Abdin
authors.affiliation: Department of Botany, Faculty of Science, Hamdard University, New Delhi, India. aahmad.bo@amu.ac.in.|Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India. aahmad.bo@amu.ac.in.
authors.forename: Altaf
authors.identifier: http://orcid.org/0000-0003-4099-7338
authors.initials: A
authors.lastname: Ahmad
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D012485:Salmonella typhi / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D008239:Lysine
keywords: Fluorescent protein; Fluorescent resonance energy transfer (FRET); Genetically encoded nanosensor; Lysine; Periplasmic binding protein