FOOD SCIENCE ›› 2023, Vol. 44 ›› Issue (24): 201-210.doi: 10.7506/spkx1002-6630-20230321-203

• Bioengineering • Previous Articles     Next Articles

A Dual-Bacterial Coupled Fermentation Strategy for Nicotinamide Mononucleotide Synthesis

SUN Ting, ZHANG Hongtao, YANG Feng, CHAI Wengang, XUE Haoyang, TAN Shuyin   

  1. (1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; 2. Department of Inorganic Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China; 3. Glycosciences Laboratory, Faculty of Medicine, Imperial College London, London HA13UJ, UK; 4. School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China)
  • Online:2023-12-25 Published:2024-01-02

Abstract: 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.

Key words: nicotinamide mononucleotide; nicotinamide nucleoside kinase; polyphosphate kinase; ATP regeneration; coupled fermentation

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