Efficient expression of NADH pyrophosphatase promotes the synthesis of NMNH by biotransformation

Abstract:

[Objective] As an important coenzyme in human body, coenzyme I (nicotinamide adenine dinucleotide, NAD+) plays an important role in maintaining cell growth, differentiation, and energy metabolism and protecting cells. Reduced nicotinamide mononucleotide (NMNH), an effective NAD+ enhancer, can efficiently elevate the levels of NAD+ in tissues. NADH pyrophosphatase can transform reduced nicotinamide adenine dinucleotide (NADH) into NMNH to promote the regeneration of NAD+. The purpose of this study is to construct a NADH pyrophosphatase expression system in Bacillus subtilis and realize the synthesis of NMNH by biotransformation. [Methods] NADH pyrophosphatase was successfully expressed in B. subtilis WB600 by vector screening, and promoter engineering was employed to improve the enzyme activity. Furthermore, the industrial application potential of the recombinant enzyme was further investigated by medium optimization and amplified fermentation in a 5 L fermenter. On this basis, the whole cell catalytic system was used for biotransformation to synthesize NMNH. [Results] The initial activity of NADH pyrophosphatase and the yield of NMNH were 1.70 U/mL and 135 mg/L, respectively. After promoter engineering, the enzyme activity was improved by 41%. In addition, the enzyme activity was increased to 5.02 U/mL after optimization of the culture medium and amplified fermentation in a 5 L fermenter, which was 1.09 times higher than that in a shake flask. On this basis, the whole-cell catalytic system was used for biotransformation, and the yield of NMNH reached 1.20 g/L, which was 7.75 times higher than the initial yield. [Conclusion] We built an efficient expression system of NADH pyrophosphatase in B. subtilis and realized the efficient transformation from NADH to NMNH by whole-cell catalysis, providing a new idea for the biosynthesis of NMNH.

Key words:reduced nicotinamide mononucleotide;promoter engineering;fermentation optimization;whole-cell catalysis;Bacillus subtilis