基于NADH焦磷酸酶高效表达的NMNH生物转化合成

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

摘要:

辅酶I (nicotinamide adenine dinucleotide, NAD⁺)作为人体内重要的辅酶，在维持细胞生长、分化和能量代谢以及细胞保护方面起着重要作用。还原型烟酰胺单核苷酸(reduced nicotinamide mononucleotide, NMNH)是一种有效的NAD+增强剂，可以快速、高效地提高组织中NAD⁺水平。NADH焦磷酸酶可将还原型辅酶I (reduced nicotinamide adenine dinucleotide, NADH)转化为NMNH以促进NAD⁺的再生。【目的】在枯草芽孢杆菌中构建NADH焦磷酸酶表达体系并实现NMNH的生物转化合成。【方法】通过载体筛选成功在枯草芽孢杆菌WB600中实现NADH焦磷酸酶的胞内表达，结合启动子工程提升其酶活，同时通过培养基优化及5 L发酵罐放大发酵策略进一步考察重组酶的工业应用潜力。在此基础上采用全细胞催化体系进行NMNH的生物转化。【结果】NADH焦磷酸酶的初始表达酶活为1.70 U/mL，NMNH产量为135 mg/L。通过启动子工程化改造，将酶活提升了41%；此外，培养基优化及5 L发酵罐放大发酵策略将酶活进一步提升至5.02 U/mL，较摇瓶水平提升1.09倍；在此基础上采用全细胞催化体系进行NMNH生物转化，获得NMNH产量为1.20 g/L，较初始产量提高了7.88倍。【结论】本研究开发了NADH焦磷酸酶在枯草芽孢杆菌中的高效表达体系，并采用全细胞催化方式实现了NADH到NMNH的高效转化，为NMNH的生物合成提供了新思路。

关键词:还原型烟酰胺单核苷酸;启动子工程;发酵优化;全细胞催化;枯草芽孢杆菌

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