The mechanism for regulating ethanol fermentation by redox levels in Thermoanaerobacter ethanolicus

Anaerobes can obtain the entire cell's ATP by glycolysis and remove resulting reducing power by fermentation. There is a delicate balance in redox status to obtain a maximal growth of these cells, and the conditions to change redox fluxes can induce kinds of changes in metabolism. The fundamental knowledge on sensing redox status and coupling redox signals with fermentation pathways is essential for the metabolic engineering to control redox fluxes at the molecular level. A redox sensing protein (RSP) was isolated by DNA affinity chromatography, and corresponding gene was mined from genomic sequences of Thermoanaerobacter spp. The RSP shares up to 41% identity with the regulatory proteins which sense NADH and control the expression of NADH dehydrogenase in aerobic microorganisms. The operator sites for RSP were located in all the operons for ethanol fermentation rather than in that of NADH dehydrogenase. The typical operator was identified as a palindromic sequence, -ATTGTTANNNNNNTAACAAT-. NADH caused a transition of RSP from an α-helix rich to β-sheet rich conformation. In an in vitro transcription system of T. ethanolicus, RSP repressed the transcription of an alcohol dehydrogenase, whereas the repression was reversed by adding NADH. Base substitutes in the repeats of the palindrome reduced the affinity between RSP and the operator, and thus delicate regulation could be achieved. This study reveals for the first time a repressor/operator system that couples a redox signal with a fermentation pathway, and the results presented here provide valuable insights for the design of metabolic engineering.