基于双菌耦合发酵策略的烟酰胺单核苷酸合成

A Dual-Bacterial Coupled Fermentation Strategy for Nicotinamide Mononucleotide Synthesis

本研究构建了分别含有烟酰胺核苷激酶(nicotinamideribosidekinase,NRK)和多聚磷酸酶(polyphosphatekinase,PPK)的双菌耦合发酵体系,实现了基于PPK的ATP再生系统在烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)生产中的应用。首先分别构建表达NRK1和NRK2的工程菌株,筛选得到高活性的大肠杆菌(Escherichia coli)BL21(DE3)-pET28a-NRK1,NMN产量5.17 g/L,产率77.4%;然后对NRK1的诱导表达条件进行优化,发现低温16℃、异丙基-β-D-硫代半乳糖吡喃糖苷0.7 mmol/L、接种量3%、诱导时长22 h更利于蛋白的可溶性表达;进一步对E. coli BL21(DE3)-pET28a-NRK1合成NMN的最优体系进行探索,发现在菌体质量浓度100 g/L、温度18℃、时间12 h、ATP与烟酰胺核糖(nicotinamide riboside,NR)浓度比1∶1.5时,NMN产量最高为5.73 g/L,产率85.78%;最后,通过对E. coli BL21(DE3)pET28a-PPK和E. coli BL21(DE3)-pET28a-NRK1耦合发酵系统进行优化,得到最优体系为ATP与NR浓度比1∶3.5、菌体质量浓度比1∶2、发酵时间16 h,NMN产量达11.81 g/L。本研究所建立的高密度双菌耦合发酵产NMN工艺为高效、低成本的大规模发酵生产NMN开辟了新途径。

In this study,a dual-bacterial coupled fermentation system containing nicotinamide nucleoside kinase(NRK)and polyphosphatase(PPK)was constructed,and the application of PPK-based ATP regeneration system in NMN production was achieved.First,engineering strains expressing NRK1 and NRK2 were constructed,and the highly active Escherichia coli BL21(DE3)-pET28a-NRK1 was selected,with NMN yield and productivity of 5.17 g/L and 77.4%,respectively.Then,the induced expression conditions of NRK1 were optimized,and a low temperature of 16℃,an isopropyl-β-D-thiogalactopyranoside(IPTG)concentration of 0.7 mmol/L,an inoculation amount of 3%and an induction duration of 22 h were found to be optimal the soluble expression of NRK1 protein.The optimal synthesis conditions of NMN by E.coli BL21(DE3)-pET28a-NRK1 were explored.It was found that after 12 h culture at 18℃at an initial cell concentration of 100 g/L and a ratio of ATP to NR of 1:1.5,the highest yield of NMN of 5.73 g/L was obtained with a productivity of 85.78%.Finally,the optimal conditions that provided maximal NMN production(11.81 g/L)by coupled fermentation with E.coli BL21(DE3)pET28a-PPK and E.coli BL21(DE3)-pET28a-NRK1 were determined as 1:3.5,1:2 and 16 h for ATP to NR ratio,initial cell concentration and fermentation time,respectively.The high-density dual-bacterial coupled fermentation strategy established in this study opens up a new pathway for high-efficiency,low-cost and large-scale production of NMN.