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Quantitative monitoring of 2-oxoglutarate in Escherichia coli cells by a fluorescence resonance energy transfer-based biosensor

  • Applied microbial and cell physiology
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Abstract

2-Oxoglutarate (2OG) is a metabolite from the highly conserved Krebs cycle and not only plays a critical role in metabolism but also acts as a signaling molecule in a variety of organisms. Environmental inorganic nitrogen is reduced to ammonium by microorganisms, whose metabolic pathways involve the conversion of 2OG to glutamate and glutamine. Tracking of 2OG in real time would be useful for studies on cell metabolism and signal transduction. Here, we developed a genetically encoded 2OG biosensor based on fluorescent resonance energy transfer by inserting the functional 2OG-binding domain GAF of the NifA protein between the fluorescence resonance energy transfer (FRET) pair YFP/CFP. The dynamic range of the sensors is 100 μM to 10 mM, which appeared identical to the physiological range observed in E. coli. We optimized the peptide lengths of the binding domain to obtain a sensor with a maximal ratio change of 0.95 upon 2OG binding and demonstrated the feasibility of this sensor for the visualization of metabolites both in vitro and in vivo.

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Acknowledgments

This study was supported by the China NSF (21276079), SRFDP (no. 20120074110009), the Key Grant Project (no. 313019) of the Chinese Ministry of Education, and the Fundamental Research Funds for the Central Universities.

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  1. Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Chang Zhang, Zi-Han Wei & Bang-Ce Ye

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  1. Chang Zhang
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Correspondence to Bang-Ce Ye.

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Zhang, C., Wei, ZH. & Ye, BC. Quantitative monitoring of 2-oxoglutarate in Escherichia coli cells by a fluorescence resonance energy transfer-based biosensor. Appl Microbiol Biotechnol 97, 8307–8316 (2013). https://doi.org/10.1007/s00253-013-5121-5

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  • DOI: https://doi.org/10.1007/s00253-013-5121-5

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