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题名: Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis
作者: Wang, Xueying1, 2;  Zhou, Yongjin J.1;  Wang, Lei1;  Liu, Wujun1;  Liu, Yuxue1, 2;  Peng, Chang1, 2;  Zhao, Zongbao K.1, 3
关键词: cofactor engineering ;  NAD biosynthesis ;  protein engineering
刊名: APPLIED AND ENVIRONMENTAL MICROBIOLOGY
发表日期: 2017-07-01
DOI: 10.1128/AEM.00692-17
卷: 83
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
英文摘要: NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering.
关键词[WOS]: FLUORESCENT PROTEIN ;  RF CLONING ;  KEY ENZYME ;  EXPRESSION ;  ASSAY ;  IDENTIFICATION ;  TARGETS ;  GENE
语种: 英语
WOS记录号: WOS:000403495700023
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/152103
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Chinese Acad Sci, Dalian Inst Chem Phys, Div Biotechnol, Dalian, Peoples R China
2.Univ Chinese Acad Sci, Beijing, Peoples R China
3.Chinese Acad Sci, Dalian Inst Chem Phys, State Key Lab Catalysis, Dalian, Peoples R China

Recommended Citation:
Wang, Xueying,Zhou, Yongjin J.,Wang, Lei,et al. Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2017,83.
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