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Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+ -Auxotrophic Mutant.
- Source :
-
Applied & Environmental Microbiology . Sep2011, Vol. 77 Issue 17, p6133-6140. 8p. - Publication Year :
- 2011
-
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 0.044x and 9.6x those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00992240
- Volume :
- 77
- Issue :
- 17
- Database :
- Academic Search Index
- Journal :
- Applied & Environmental Microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 66742194
- Full Text :
- https://doi.org/10.1128/AEM.00630-11