烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)是维生素B族的衍生物,天然存在于果蔬和肉类中,广泛应用于食品、保健品和退行性疾病治疗等领域。烟酰胺磷酸核糖基转移酶(nicotinamide phosphoribosyltransferase,NAMPT)是NMN补救合成途径中的限速酶,当前研究最佳的NAMPT为来源于弧菌噬菌体的VpNadV,存在活性较差、底物利用率低等缺陷,其晶体结构尚未完全解析。该研究旨在基于NMN荧光反应体系构建了一种高通量筛选系统,结合酶工程改造获得高活性的VpNadV突变体,并应用于大肠杆菌中NMN的高效合成。通过高通量筛选与酶工程改造,获得了一种高酶活性的突变体VpNadVL139V+R382G,其酶活性为野生型的1.46倍。经发酵条件优化后,NMN在摇瓶水平的最高产量达到3.68 g/L,放大培养至5 L发酵罐后,NMN的产量最高达到24.4 g/L。该研究首先构建了可用于筛选NAMPT突变体的高通量筛选系统,并简要解析VpNadV的催化机理,最后通过发酵进一步验证了突变体合成NMN的正向效果,为NMN与烟酰胺类其他衍生物的高效合成提供了新的思路。
Nicotinamide mononucleotide (NMN) is a derivative of the vitamin B group, which naturally exists in fruits, vegetables, and meat.It has been widely used in the field of food, health care products, and degenerative diseases treatment.Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the NMN salvage synthesis pathway.The best NAMPT in current research is VpNadV derived from Vibrio bacteriophage, which has several defects, such as poor activity and low substrate utilization.In addition, its crystal structure has not been fully resolved.Based on the NMN fluorescence reaction system, this study aimed to construct a high-throughput screening system, and then to screen highly active VpNadV mutants combined with enzyme engineering, finally to apply for efficient synthesis of NMN in Escherichia coli.Through the high-throughput screening and enzyme engineering, a mutant with high enzymatic activity, VpNadVL139V+R382G, was selected, which enzyme activity was 1.46-fold than that of the wild type.Based on the mutant, 3.68 g/L NMN was achieved in shake flasks, and 24.4 g/L NMN was obtained after scaling up in a 5 L fermenter.In this study, a high-throughput screening system for Nicotinamide phosphoribosyltransferase (NAMPT) mutants was constructed, and then the catalytic mechanism of VpNadV was briefly analyzed, and finally the positive effect of NMN synthesis by mutants was further verified by fermentation, providing ideas for the efficient production of NMN and nicotinamide derivatives.
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