Wang Xueying, Zhou Yongjin J, Wang Lei, Liu Wujun, Liu Yuxue, Peng Chang, Zhao Zongbao K
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, People's Republic of China.
University of Chinese Academy of Sciences, Beijing, People's Republic of China.
Appl Environ Microbiol. 2017 Jun 16;83(13). doi: 10.1128/AEM.00692-17. Print 2017 Jul 1.
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 , NAD 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 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. Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.
烟酰胺腺嘌呤二核苷酸（NAD）及其还原形式烟酰胺腺嘌呤二核苷酸磷酸（NADH）作为重要的氧化还原辅助因子发挥作用，在决定细胞代谢特征方面具有主要作用。NAD可以通过脱酰胺途径和酰胺化途径合成，其中关键反应分别涉及烟酸单核苷酸（NaMN）和烟酰胺单核苷酸（NMN）的腺苷酸化。在[具体生物名称未给出]中，NAD生物合成依赖于蛋白质NadD催化NaMN腺苷酸化生成烟酸腺嘌呤二核苷酸（NaAD），随后由NAD合酶催化酰胺化。在本研究中，我们对NadD进行工程改造，使其更倾向于NMN，以提高酰胺化途径的活性。我们设计了NadD突变体文库，通过苹果酸酶偶联比色法进行筛选，并鉴定出两个变体，11B4（Y84V/Y118D）和16D8（A86W/Y118N），它们对NMN具有高度偏好性。在存在NMN的情况下，这两个变体都能够使BW25113衍生的NAD营养缺陷型菌株YJE003的细胞存活，该菌株脱酰胺途径的最后一步被阻断，而16D8表达菌株可以在没有外源NMN的情况下生长，并且在稳定期积累的细胞内NAD（H）水平高于BW25113。这些突变体建立了完全活跃的酰胺化NAD生物合成途径，并为通过生物催化和代谢工程来操纵NAD代谢提供了新机会。烟酸单核苷酸（NaMN）的腺苷酸化和烟酰胺单核苷酸（NMN）的腺苷酸化分别是NAD生物合成脱酰胺途径和酰胺化途径中的关键步骤。在大多数生物体中，经典的NAD生物合成遵循脱酰胺途径。在这里，我们对NaMN腺苷酸转移酶进行工程改造，使其更倾向于NMN，并在一个脱酰胺途径最后一步被阻断的NAD营养缺陷型菌株中表达突变酶。工程改造后的菌株能够在M9培养基中存活，这表明实现了一条用于NAD生物合成的功能性酰胺化途径。这些结果丰富了我们对NAD生物合成的理解，对于通过代谢工程操纵NAD稳态具有重要价值。
