title: Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant.
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.
authors: Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.
authors: Yongjin
authors: Y
authors: Zhou
authors: Lei
authors: L
authors: Wang
authors: Fan
authors: F
authors: Yang
authors: Xinping
authors: X
authors: Lin
authors: Sufang
authors: S
authors: Zhang
authors: Zongbao K
authors: ZK
authors: Zhao
mesh_terms: D003470:Culture Media / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D009243:NAD