烟酰胺核糖激酶在大肠杆菌中的高效异源表达、酶学特性分析和发酵工艺探究  

Efficient heterologous expression,enzymatic property analysis,and fermentation process investigation of nicotinamide riboside kinase in Escherichia coli

作　　者：毛歆安 龚劲松 苏畅 李恒 徐国强[2] 许正宏[2,3] 史劲松 MAO Xinan;GONG Jinsong;SU Chang;LI Heng;XU Guoqiang;XU Zhenghong;SHI Jinsong(School of Life Science and Health Engineering,Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;College of Biomass Science and Engineering,Sichuan University,Chengdu 610065,China)

机构地区：[1]江南大学生命科学与健康工程学院,糖化学与生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [3]四川大学轻工科学与工程学院,四川成都610065

出　　处：《食品与发酵工业》2025年第6期50-57,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2023YFA0914500);国家自然基金面上项目(32171261)。

摘　　要：烟酰胺核糖激酶(nicotinamide riboside kinase,Nrk)催化烟酰胺核糖磷酸化生成β-烟酰胺单核苷(β-nicotinamide mononucleotide,β-NMN),是目前生物酶法合成β-NMN的重要途径。该研究通过异源表达体系筛选,实现Nrk在大肠杆菌中的可溶表达,初始酶活力为2.14 U/mL。通过核糖体结合位点(ribosome binding site,RBS)序列和启动子优化,将翻译起始速率为35000 a.u.的RBS序列与双启动子P alsR-T7组合,构建的重组菌酶活力可达5.56 U/mL,是初始水平的2.6倍。Nrk酶学性质研究表明,该酶的最适反应条件为50℃,pH 7.0,10 mmol/L Mg^(2+)。最后,对产Nrk重组菌进行培养条件优化,并在5 L发酵罐中进行罐上产酶放大工艺研究,最终使得酶活力达到72.33 U/mL,是摇瓶发酵水平的13倍。该研究显著提升了Nrk在大肠杆菌中的异源表达水平,为β-NMN的生物酶法合成奠定了基础。Nicotinamide riboside kinase(Nrk)catalyzes the phosphorylation of nicotinamide riboside to generateβ-nicotinamide mononucleotide(β-NMN),serving as a crucial route for the enzymatic synthesis ofβ-NMN.An efficient heterologous expression system was used in this research for the expression of Nrk in Escherichia coli with an initial enzyme activity of 2.14 U/mL.Through ribosome binding site(RBS)and promoter optimization,this study combined an RBS sequence with a translation initiation rate of 35000 a.u.with the dual promoter P alsR-T7,and the recombinant strain demonstrated an enzyme activity of 5.56 U/mL,2.6 times the initial level.The enzymatic properties of Nrk revealed optimal reaction conditions at 50℃,pH 7.0,and 10 mmol/L Mg^(2+).Finally,cultivation conditions for the Nrk-producing recombinant strain were optimized and the fermentation was scaled up to a 5 L bioreactor,achieving an enzyme activity of 72.33 U/mL,13 times the shake flask fermentation level.This research significantly improved the heterologous expression of Nrk in Escherichia coli,laying the foundation for the enzymatic synthesis ofβ-NMN.

关 键 词：烟酰胺核糖激酶 β-烟酰胺单核苷酸 核糖体结合位点 启动子工程 异源表达 

分 类 号：TQ925[轻工技术与工程—发酵工程]