A single fluorescent protein-based sensor for in vivo 2-oxogluatarate detection in cell

Area of Science:

• Metabolic Engineering
• Cellular Biology
• Biochemistry

Background:

• 2-Oxoglutarate (2OG) is a critical metabolite linking carbon and nitrogen metabolism.
• 2OG plays signaling roles in prokaryotic nitrogen metabolism.
• Altered tricarboxylic acid (TCA) cycle metabolism, including 2OG, is observed in tumor cells (Warburg effect).

Purpose of the Study:

• To develop a novel sensor for visualizing and monitoring 2-oxoglutarate (2OG) metabolism in single living cells.
• To create a tool for real-time tracking of cellular 2OG dynamics.

Main Methods:

• Engineered a novel sensor by fusing the 2OG-binding GAF domain of NifA protein with YFP.
• Validated sensor specificity and fluorescence response to 2OG.
• Tested sensor performance in *E. coli* under varying nutritional conditions.

Main Results:

• The developed sensor exhibits high specificity for 2OG.
• Fluorescence intensity increases with 2OG concentration, showing a 1.5-fold signal change.
• The sensor demonstrates fast kinetics and a dynamic response range of 100 µM-100 mM.
• In vivo and in vitro dissociation of 2OG from the sensor was confirmed.
• Real-time cellular 2OG dynamics in *E. coli* were reported, revealing differences in accumulation and consumption rates.

Conclusions:

• The novel sensor enables real-time visualization of 2-oxoglutarate (2OG) metabolism in single living cells.
• This tool provides insights into cellular metabolic responses to nutritional challenges.
• The sensor facilitates the study of 2OG dynamics in various biological contexts, including prokaryotes and potentially cancer cells.