Engineering Riboswitch as In Vivo Biosensor for ATP

Cellular ATP levels are a critical indicator of metabolic state and energy balance in bacteria. In this study, we employed an engineered ATP riboswitch to function as an in vivo biosensor for monitoring intracellular ATP dynamics in Escherichia coli. The riboswitch was designed to regulate the translation of a green fluorescent protein (GFP) reporter in response to ATP binding. To validate the system, intracellular ATP concentrations were modulated using two complementary strategies: permeabilization-mediated ATP supplementation and chemical uncoupling with carbonyl cyanide m-chlorophenyl hydrazone (CCCP). When ATP was added to the cells at concentrations of 5, 10, and 20 mM, GFP expression increased by 38.44% , 840.83%, and 2230.88% respectively, compared to the control group. In contrast, treatment with CCCP at concentrations of 5, 50, 500, and 5000 µM resulted in decreased GFP expression by 26.19% , 21.99%, 20.32%, and 45.10% respectively. These results indicate that higher ATP levels enhance riboswitch activity, while ATP depletion leads to a reduction in riboswitch-mediated gene expression. Overall, the ATP riboswitch demonstrated a sensitive and dynamic response to intracellular energy changes in living bacteria. This system highlights the potential of riboswitch-based biosensors for applications in metabolic engineering, including real-time monitoring of cellular energy flux and optimization of biosynthetic pathways.