論文 - 詳細
| RRC ID | 12325 |
|---|---|
| 著者 | Zhou Y, Wang L, Yang F, Lin X, Zhang S, Zhao ZK. |
| タイトル | Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant. |
| ジャーナル | 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. |
| 巻・号 | 77(17) |
| ページ | 6133-40 |
| 公開日 | 2011-9-1 |
| 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 | 4.016 |
| 引用数 | 41 |
| WOS 分野 | BIOTECHNOLOGY & APPLIED MICROBIOLOGY MICROBIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 4 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 原核生物(大腸菌) | JW0106-KC |