An optical BOD biosensor based on intracellular ATP measurements in genetically modified Saccharomyces cerevisiae.

Abstract

A biosensor for biochemical oxygen demand (BOD) was developed based on intracellular 5'-adenosine triphosphate (ATP) measurements in Saccharomyces cerevisiae. Intracellular ATP was measured using an engineered protein named ATeam, comprising a bacterial F₀F₁-ATP synthase ε subunit sandwiched between cyan fluorescent protein and mVenus, a modified yellow fluorescent protein. Because the binding of ATP to ATeam induces changes in the fluorescence spectra owing to Fӧrster resonance energy transfer, S. cerevisiae expressing ATeam is expected to show spectral changes owing to the intracellular ATP produced by the metabolism of the BOD sample. A glycogen phosphorylase knockout S. cerevisiae strain expressing ATeam was prepared, and the fluorescence spectra of the strain were analyzed. Changes in the fluorescence spectra of glucose in the medium were observed, which exhibited a linear relationship with the glucose concentration (0-100 mg/L, R² = 0.970). Responses to lactose, fructose, sucrose, Glu, Asp, His, and Gly were evaluated and compared with typical BOD measurements. The results of this comparison suggest that a BOD biosensor based on intracellular ATP can be used for BOD measurements. A BOD standard solution comprising glucose and glutamic acid (GGA) was calibrated across a concentration range of 0 to 100 mg/L. Finally, simulated real samples were prepared using real pond water and GGA was measured. The correlation between the BOD value evaluated using intracellular ATP and that evaluated using the 5-day BOD test showed a linear relationship with R² = 0.944.