Discriminating changes in intracellular NADH/NAD⁺ levels due to anoxicity and H₂ supply in R. eutropha cells using...

Area of Science:

• Microbiology and Biotechnology
• Biochemical Engineering
• Metabolic Engineering

Background:

• Ralstonia eutropha is a versatile bacterium capable of both aerobic and anaerobic growth, with adaptable metabolic pathways.
• The soluble hydrogenase (SH) in R. eutropha facilitates H2 oxidation, linking it to NAD+ reduction to NADH, essential for cofactor regeneration.
• Understanding the interplay between SH activity and the cellular NADH/NAD+ pool is critical for optimizing biotechnological processes.

Purpose of the Study:

• To evaluate the fluorescent biosensor Frex for measuring cytoplasmic NADH levels in R. eutropha under varying gas conditions.
• To assess Frex's ability to differentiate SH-mediated redox changes from general anaerobiosis effects.
• To explore the utility of Frex for real-time monitoring of the NADH/NAD+ redox state in living bacteria.

Main Methods:

• Application of the Frex fluorescent biosensor to quantify cytoplasmic NADH concentrations in R. eutropha.
• Exposure of bacterial cultures to different gas supplies (including H2) to induce specific metabolic responses.
• Comparative analysis of Frex performance against the Peredox reporter, focusing on NADH affinity and sensor saturation.

Main Results:

• Frex successfully distinguished SH-mediated NADH changes from general respiratory effects.
• A robust fluorescence response was observed upon H2 supply, enabling monitoring of rapid NADH fluctuations.
• Frex demonstrated lower NADH affinity than Peredox, preventing sensor saturation at typical bacterial NADH levels.

Conclusions:

• The Frex biosensor is a suitable tool for monitoring cytoplasmic NADH dynamics in R. eutropha.
• Frex allows for real-time tracking of the NADH/NAD+ redox state, providing insights into SH activity.
• The findings support the optimization of fluorescent NADH sensors for applications in microbial biotechnology.