Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

Abstract

ABSTRACT NAD (NAD + ) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD + level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD + auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD + de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD + . We then constructed the NAD + -auxotrophic mutant YJE003 by disrupting the essential gene nadE , which is responsible for the last step of NAD + biosynthesis in cells harboring the ntt4 gene. The minimal NAD + level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD + , while 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× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.