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Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis
Wang, Xueying1,2; Zhou, Yongjin J.1; Wang, Lei1; Liu, Wujun1; Liu, Yuxue1,2; Peng, Chang1,2; Zhao, Zongbao K.1,3
KeywordCofactor Engineering Nad Biosynthesis Protein Engineering
Source PublicationAPPLIED AND ENVIRONMENTAL MICROBIOLOGY
2017-07-01
DOI10.1128/AEM.00692-17
Volume83
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
WOS SubjectBiotechnology & Applied Microbiology ; Microbiology
WOS Research AreaBiotechnology & Applied Microbiology ; Microbiology
WOS KeywordFLUORESCENT PROTEIN ; RF CLONING ; KEY ENZYME ; EXPRESSION ; ASSAY ; IDENTIFICATION ; TARGETS ; GENE
AbstractNAD 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.
Language英语
WOS IDWOS:000403495700023
Citation statistics
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/152103
Collection中国科学院大连化学物理研究所
Affiliation1.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
GB/T 7714
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.
APA Wang, Xueying.,Zhou, Yongjin J..,Wang, Lei.,Liu, Wujun.,Liu, Yuxue.,...&Zhao, Zongbao K..(2017).Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,83.
MLA Wang, Xueying,et al."Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis".APPLIED AND ENVIRONMENTAL MICROBIOLOGY 83(2017).
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