A Regulatory NADH/NAD+ Redox Biosensor for Bacteria

ACS Synth Biol. 2019 Feb 15;8(2):264-273. doi: 10.1021/acssynbio.8b00485. Epub 2019 Jan 23.


NADH and NAD+ cofactors drive hundreds of biochemical reactions, and their ratio is a key metabolic marker of cellular state. Traditional assays to measure the NADH/NAD+ ratio is laborious, prone to inaccuracies, and not suitable for high-throughput screening. We report a genetically encoded ratiometric biosensor for NADH/NAD+ based on redox-responsive bacterial transcription factor Rex that overcomes these limitations. We engineered a Rex-regulated E. coli promoter with improved biosensor characteristics by tuning the affinity of Rex and the operator site. Since NADH is oxidized during aerobic respiration, we used the biosensor-reporter to investigate the effect of removing respiratory chain enzymes on NADH/NAD+ ratio during aerobiosis. We found that the NADH/NAD+ signal increased in five of the nine mutants by over 3-fold compared to wildtype, including an NADH dehydrogenase double mutant with 6-fold elevation. We also found that among several common carbon sources, E. coli grown on acetate exhibited higher NADH/NAD+ compared to E. coli grown on glucose. As a proof-of-concept for high-throughput redox screening, we were able to enrich high NADH mutants present at 1 in 10 000 among wildtype cells by biosensor-guided pooled screen. Thus, our Rex biosensor-reporter enables facile, noninvasive, high-throughput redox measurement to understand and engineer redox metabolism.

Keywords: NADH/NAD+; bacterial physiology; high-throughput screening; redox biosensor; respiratory chain.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / genetics
  • Bacteria / metabolism
  • Bacterial Proteins / metabolism
  • Biosensing Techniques / methods*
  • NAD / metabolism*
  • Oxidation-Reduction
  • Promoter Regions, Genetic / genetics


  • Bacterial Proteins
  • NAD