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Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD(+)-Auxotrophic Mutant
Zhou, Yongjin1,3; Wang, Lei1,3; Yang, Fan1; Lin, Xinping1,3; Zhang, Sufang1; Zhao, Zongbao K.1,2
刊名APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2011-09-01
DOI10.1128/AEM.00630-11
77期:17页:6133-6140
收录类别SCI
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
类目[WOS]Biotechnology & Applied Microbiology ; Microbiology
研究领域[WOS]Biotechnology & Applied Microbiology ; Microbiology
关键词[WOS]SACCHAROMYCES-CEREVISIAE ; NADH AVAILABILITY ; SEQUENCE-ANALYSIS ; GLYCOLYTIC FLUX ; NAD(P)(H) POOL ; METABOLISM ; ENHANCEMENT ; TRANSPORTER ; EXPRESSION ; MEMBRANE
英文摘要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 x and 9.6 x those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.
语种英语
WOS记录号WOS:000294205700036
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文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/142672
专题中国科学院大连化学物理研究所
作者单位1.Chinese Acad Sci, Dalian Inst Chem Phys, Dalian 116023, Peoples R China
2.Dalian Natl Lab Clean Energy, Dalian 116023, Peoples R China
3.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
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Zhou, Yongjin,Wang, Lei,Yang, Fan,et al. Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD(+)-Auxotrophic Mutant[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2011,77(17):6133-6140.
APA Zhou, Yongjin,Wang, Lei,Yang, Fan,Lin, Xinping,Zhang, Sufang,&Zhao, Zongbao K..(2011).Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD(+)-Auxotrophic Mutant.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,77(17),6133-6140.
MLA Zhou, Yongjin,et al."Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD(+)-Auxotrophic Mutant".APPLIED AND ENVIRONMENTAL MICROBIOLOGY 77.17(2011):6133-6140.
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