关键酶基因转录水平对重组大肠杆菌合成NAD;的影响

Effects of transcription levels of key enzyme genes on NAD;production in recombinant Escherichia coli

【目的】烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide)是生物体内一种重要的辅因子,其细胞内含量对于NAD;依赖型氧化还原反应和有关生化合成代谢具有重要意义。为强化辅因子合成,本文通过不同诱导条件下关键酶基因转录水平与产物合成水平的相关性分析,利用增加正向关键酶基因拷贝数策略提高胞内氧化型辅酶NAD^(+)的浓度。【方法】以实验室前期构建的大肠杆菌NA016为出发菌株,分别从诱导温度、诱导剂浓度、诱导时机三个方面进行了诱导条件的优化,并利用实时荧光定量PCR(RT-qPCR)技术对代谢改造中的有关过表达基因进行了转录水平分析,确定了NAD^(+);量与这些过表达基因转录水平之间的相关性,增加对NAD;合成代谢具有正向作用的基因拷贝数以进一步提高胞内NAD^(+)水平。【结果】通过诱导条件优化实验确定菌株NA016的最适诱导温度,在诱导时机为OD600达到0.6时加入0.8mmol/L的IPTG可使胞内NAD^(+)含量提高35.37%;转录水平方面分析发现基因nadE和pncB的表达对NAD^(+)的合成具有正向调节作用。进一步增加菌株NA016中关键酶基因nadE和pncB的拷贝数可使辅酶含量再次提高22.46%,最高可达41.66μmol/g DCW。【结论】优化诱导条件和增加关键酶基因拷贝数可以提高氧化型辅酶NAD^(+)的含量,这为促进大肠杆菌胞内NAD^(+)合成的研究提供借鉴意义。

[Objective] Nicotinamide adenine dinucleotide(NAD;) is an important cofactor in organisms, and its intracellular concentration plays an important role in NAD;-dependent redox reaction and related biochemical synthesis. In order to strengthen the synthesis of cofactors, we optimized the induction conditions and increased the copies of key enzyme genes to elevate the intracellular NAD;concentration. [Methods] First, the induction temperature, inducer concentration, and induction time were optimized for the starting strain NA016 of Escherichia coli. Meanwhile, the transcriptional levels of overexpressed genes in metabolic modification were determined by real-time fluorescence quantitative PCR. Subsequently, the correlations between NAD;content and the transcription levels of these overexpressed genes were explored. Finally, the copies of the genes positively regulating NAD;production were up-regulated to improve the intracellular NAD^(+) concentration. [Results] At the optimized induction conditions, the NAD;concentration in NA016 increased by 35.37%. The genes nadE and pncB positively regulated the production of NAD^(+), and up-regulating the copies of these two genes in NA016 increased the NAD^(+) concentration by 22.46% to 41.66 μmol/g DCW. [Conclusion] Optimizing the induction conditions and up-regulating the copy number of key enzyme genes can increase the NAD^(+) concentration. These research findings provide reference for the study of NAD^(+) synthesis.