RRC ID 12325
Author Zhou Y, Wang L, Yang F, Lin X, Zhang S, Zhao ZK.
Title Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant.
Journal Appl. Environ. Microbiol.
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.
Volume 77(17)
Pages 6133-40
Published 2011-9
DOI 10.1128/AEM.00630-11
PII AEM.00630-11
PMID 21742902
PMC PMC3165392
MeSH Culture Media / chemistry Cytoplasm / chemistry Escherichia coli / chemistry Escherichia coli / genetics Escherichia coli / metabolism* Gene Deletion Gene Expression Membrane Transport Proteins / genetics Membrane Transport Proteins / metabolism Metabolic Networks and Pathways / genetics* Mutation* NAD / metabolism* Recombinant Proteins / genetics Recombinant Proteins / metabolism
IF 3.633
Times Cited 17
Prokaryotes E. coli JW0106-KC