Redox-guided dynamic regulation for enhanced bioproduction using engineered NADH/NAD⁺ biosensors

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Efficient microbial production relies on balancing carbon flux and intracellular redox state (NADH/NAD+ ratio).
• NADH/NAD+ imbalance frequently limits the performance of synthetic pathways dependent on these cofactors.

Purpose of the Study:

• To engineer a transcription factor Rex-based biosensor for sensing and guiding microbial redox states.
• To optimize microbial production of value-added chemicals by dynamically regulating metabolic pathways based on intracellular redox status.

Main Methods:

• Engineered a Staphylococcus aureus Rex transcription factor-based biosensor for NADH/NAD+ sensing in Escherichia coli.
• Systematically tuned intracellular redox states using NADH-consuming and generating enzymes for biosensor characterization.
• Employed rational mutagenesis to create a Rex variant (Rexm) with an expanded operational range.
• Integrated the biosensor into a dynamic regulatory circuit to control glycolysis and enhance mevalonate biosynthesis.

Main Results:

• The optimized biosensor demonstrated a 4.37-fold dynamic range for decreased NADH/NAD+ ratios.
• The Rexm variant showed a 3.14-fold response to increased NADH/NAD+ ratios.
• The dynamic regulatory circuit increased mevalonate titer by 28% (to 6.23 g/L) by balancing NADH supply and carbon flux.

Conclusions:

• Engineered Rex-based biosensors offer a tunable platform for intracellular redox sensing.
• Dynamic regulation guided by these biosensors enhances microbial production of chemicals.
• This approach provides a robust strategy for optimizing metabolic pathways and improving yields.