β-烟酰胺单核苷酸(β-nicotinamide mononucleotide,NMN)作为人体内重要辅酶烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)的直接前体受到了广泛关注,目前工业上主要采用化学法或化学-酶催化法进行合成,发酵法仍未取得突破。为获得一株高产NMN的工程菌株,对大肠杆菌进行了代谢途径改造。首先在大肠杆菌中异源表达了来自松噬几丁质菌(Chitinophaga pinensis)的烟酰胺磷酸核糖转移酶,构建了NMN的补救合成途径;为增强前体5-磷酸核糖-1-焦磷酸(5-phosphoribose 1-pyrophosphate,PRPP)的供应,过表达了从葡萄糖出发的多个途径酶,促进了葡萄糖向磷酸戊糖途径的流动;同时,为有利于NMN积累,通过敲除NMN下游代谢分解途径基因pncC、ushA以及调控NAD+衍生物基因nadR,所获得的重组菌产量摇瓶水平达到60 mg/L,是初始产量的4.95倍;进一步在5 L罐上进行发酵试验,产量最高达到390.1 mg/L。该菌株用于NMN的发酵生产具有良好的潜力。
β-nicotinamide mononucleotide (NMN) as the direct precursor of the important coenzyme NAD+ in the human body has received widespread attention, and the current industry mainly uses chemical method or chemical-enzyme catalytic method for synthesis, and the fermentation method has not yet made a breakthrough. In order to obtain an engineered strain with high NMN yield, the metabolic pathway of Escherichia coli was modified. Firstly, nicotinamide phosphoribosyl transferase from Chitinophaga pinensis was expressed heterologous in E. coli, and a remedial synthesis pathway for NMN was constructed. In order to enhance the supply of the precursor 5-phosphate ribose-1-pyrophosphate (PRPP), multiple pathway enzymes originating from glucose were expressed, which promoted the flow of glucose to the pentose phosphate pathway; Further, in order to facilitate the accumulation of NMN, by knocking out the NMN downstream metabolic catalysis pathway genes pncC and ushA and regulating the NAD+ derivative gene nadR, the yield of recombinant strain obtained reached 60 mg/L, notably, which was 4.95 times over the initial level, and the fermentation was further performed on a 5 L fermenter, and the yield reached 390.1 mg/L. The strain exhibits significant potential for fermentation production of NMN.
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