Questing the extremes of the cellular NAD(H) level using an Escherichia coli NAD+ auxotrophic mutant

NAD(H) is an essential cofactor in cell activity participating in over 300 redox reactions in vivo. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ is tightly controlled with the biosynthesis regulation mechanism. Here, we developed a directed manipulation strategy to determine the extreme NAD(H) levels in Escherichia coli cells by providing exogenous NAD+ using the NAD(H) transporter NTT4, and blocking the NAD+ biosynthesis pathways. Firstly, we validated the function of NTT4 expressed in an E. coli mutant lacking the de novo NAD+ biosynthesis pathway. We then constructed the NAD+ auxotrophic mutant YJE003 by disrupting the essential NAD+ biosynthesis gene nadE in cells with an NTT4 expression background. The minimal NAD+ level was determined in M9 medium by proliferating YJE003 cells that were fed with exogenous NAD+. The maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044- and 9.6-fold amounts of those of the wide-type cells, respectively. Finally, the potential application of this strategy in biotechnology was briefly discussed.