Single-cell ATP monitoring in Escherichia coli engineered for polyhydroxybutyrate production using the fluorescent...

Polyhydroxyalkanoates (PHAs) are polyesters accumulated by various microorganisms as intracellular inclusions. In this study, Escherichia coli was engineered to co-express PHA biosynthetic genes and the ratiometric fluorescent biosensor QUEEN. QUEEN is a genetically encoded fusion protein comprising green fluorescent protein and the ε subunit of ATP synthase, which functions as an ATP-binding domain. Its fluorescence intensity ratio at two wavelengths reflects intracellular ATP levels. In this study, we aimed to monitor in situ ATP levels during poly(3-hydroxybutyrate) [P(3HB)] production in E. coli. The metabolic pathway was designed to initiate and enhance P(3HB) synthesis through precursor (3HB) supplementation, while PHA biosynthetic genes were constitutively expressed. Consequently, P(3HB) production was successfully induced in recombinant cells harboring the QUEEN gene, with 78 % of Nile Blue-stained cells exhibiting PHA inclusions under microscopy. QUEEN remained functionally expressed under both P(3HB)-producing and non-producing conditions. Single-cell fluorescence measurements using QUEEN revealed no significant difference in ATP levels between the two conditions. These results suggested that E. coli possesses homeostatic functions in energy metabolism during PHA production.