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学科主题: 生物化学
题名: Questing the extremes of the cellular NAD(H) level using an Escherichia coli NAD+ auxotrophic mutant
作者: Zhou YJ(周雍进) ;  Wang L(王磊) ;  Yang F(杨帆) ;  Lin XP(林心萍) ;  Zhang SF(张素芳) ;  Zhao ZB(赵宗保)
会议文集: Gordon Research Conferences on Bioorganic Chemistry
会议名称: Gordon Research Conferences on Bioorganic Chemistry
会议日期: 2011-6-12
出版日期: 2011
会议地点: 安度华
通讯作者: 赵宗保
出版者: 待补充
出版地: 待补充
合作性质: 墙报
部门归属: 1816
主办者: The Gordon Research Conferences Organization
摘要: NAD(H) is an essential cofactor in cell activity participating in over 300 redox reactions in vivo. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ is tightly controlled with the biosynthesis regulation mechanism. Here, we developed a directed manipulation strategy to determine the extreme NAD(H) levels in Escherichia coli cells by providing exogenous NAD+ using the NAD(H) transporter NTT4, and blocking the NAD+ biosynthesis pathways. Firstly, we validated the function of NTT4 expressed in an E. coli mutant lacking the de novo NAD+ biosynthesis pathway. We then constructed the NAD+ auxotrophic mutant YJE003 by disrupting the essential NAD+ biosynthesis gene nadE in cells with an NTT4 expression background. The minimal NAD+ level was determined in M9 medium by proliferating YJE003 cells that were fed with exogenous NAD+. 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-fold amounts of those of the wide-type cells, respectively. Finally, the potential application of this strategy in biotechnology was briefly discussed.
英文摘要: NAD(H) is an essential cofactor in cell activity participating in over 300 redox reactions in vivo. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ is tightly controlled with the biosynthesis regulation mechanism. Here, we developed a directed manipulation strategy to determine the extreme NAD(H) levels in Escherichia coli cells by providing exogenous NAD+ using the NAD(H) transporter NTT4, and blocking the NAD+ biosynthesis pathways. Firstly, we validated the function of NTT4 expressed in an E. coli mutant lacking the de novo NAD+ biosynthesis pathway. We then constructed the NAD+ auxotrophic mutant YJE003 by disrupting the essential NAD+ biosynthesis gene nadE in cells with an NTT4 expression background. The minimal NAD+ level was determined in M9 medium by proliferating YJE003 cells that were fed with exogenous NAD+. 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-fold amounts of those of the wide-type cells, respectively. Finally, the potential application of this strategy in biotechnology was briefly discussed.
语种: 英语
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/116100
Appears in Collections:中国科学院大连化学物理研究所_会议论文

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Recommended Citation:
Zhou YJ,Wang L,Yang F,et al. Questing the extremes of the cellular NAD(H) level using an Escherichia coli NAD+ auxotrophic mutant[C]. 见:Gordon Research Conferences on Bioorganic Chemistry. 安度华. 2011-6-12.
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