Open AccessDetermining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant
Author(s) -
Yongjin J. Zhou,
Lei Wang,
Fan Yang,
Xinping Lin,
Sufang Zhang,
Zongbao K. Zhao
Publication year - 2011
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00630-11
Subject(s) - nad+ kinase , auxotrophy , mutant , cofactor , escherichia coli , biochemistry , biology , intracellular , glycerol 3 phosphate dehydrogenase , biosynthesis , microbiology and biotechnology , gene , enzyme
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 inEscherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed thentt4 gene encoding the NAD(H) transporter in theE. coli mutant YJE001, which had a deletion of thenadC 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 genenadE , which is responsible for the last step of NAD+ biosynthesis in cells harboring thentt4 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.