A Dual-Bacterial Coupled Fermentation Strategy for Nicotinamide Mononucleotide Synthesis
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-NRKl 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-NRKl 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.
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