Tyler, J.; Amrapali Vishwanath, A.; Menon, T.; Duarah, T.; Adhikari, R.; Koberstein, J. N.; Feliciano, D.; Espinosa-Medina, I.; Colon-Ramos, D.; Tebo, A. G.

Fructose-1,6-bisphosphate (FBP) is the product of the first committed step of glycolysis, and its concentration is tightly correlated with glycolytic flux. Glycolytic activity varies across tissues and cell types: some tissues, such as the brain, dynamically regulate glycolysis in response to demand, while others, such as the liver have characterized spatial heterogeneity. Here, we report HYlight2, an improved sensor for FBP developed through random whole-gene mutagenesis in E. coli lysate. After four rounds of screening, we isolated HYlight2, which retains its binding affinity while displaying a {Delta}R/R ~9 in vitro, a three-fold improvement in mammalian cells, and a two-fold improvement in detecting glycolytic responses during stimulated neuronal activity. We further demonstrate its use in vivo to detect altered glycolytic activity in C. elegans neurons, zebrafish pancreatic islets, and mouse liver.