大肠杆菌NAD^+合成关键酶的克隆表达及发酵优化

Cloning and expression of key enzymes for NAD^+ synthesis and optimization of fermentation in Escherichia coli

【目的】烟酰胺腺嘌呤二核苷酸(NAD^+)在细胞基因表达、氧化还原反应、能量代谢以及调控细胞生命周期中具有重要的作用,其细胞内含量是能量效率的关键因素。强化辅因子合成策略,获得高产NAD^+菌株,对于NAD^+依赖型氧化还原反应的速率和调节相关生化合成途径的代谢流具有重要意义。【方法】首先通过内源性调节,对代谢途径中的关键酶基因进行强化,过量表达和共表达NAD^+合成途径中的关键酶基因pncB、nadD和nadE;其次,通过外源调节增加NAD^+前体物,优化诱导条件提高发酵过程中关键酶的表达量,增加NAD^+的合成量;最后在单因素优化试验的基础上,以NAD^+含量为响应值,采用Box-Bohnken试验设计方法,研究3个显著性影响因素相互作用对NAD^+积累量的影响,确定最佳的优化条件。【结果】根据关键酶基因强化策略,构建了7株重组菌,其中重组菌E.coli BL21/p ET-21a-nad E-pncB胞内NAD^+含量相比初始菌株E.coli BL21/pET-21a提高了405.2%。通过对该菌株诱导条件和NAD^+合成前体的优化,使用Design Expert 8.0分析实验数据,得出该重组菌株的最佳发酵条件为:诱导温度控制在15–20 oC,OD_(600)为0.6–0.8时添加IPTG 0.63 mmol/L、烟酸15.8 mg/L、诱导时长控制在24 h。NAD^+含量在最优条件下实验验证值可达43.16μmol/g DCW,与优化前相比提高了123.6%,与初始菌株相比提高了1029.8%。【结论】在大肠杆菌中共表达关键酶基因pncB和nadE,胞内NAD^+合成量明显增加,前体物以及诱导条件的外源调节使NAD^+积累量达到最佳优化值。实现了提高NAD^+含量的目标,胞内辅因子浓度的增加为提高生物催化效率奠定了可行性基础。

[Objective] Nicotinamide adenine dinucleotide （NAD^＋） plays a crucial role in controlling metabolism network. Improving its intracellular concentration or getting a high-NAD^＋-yield strain is of great significance for NAD^＋-dependent redox reaction rate. [Methods] First, we used endogenous regulation means to enhance the key genes of NAD^＋ synthesis pathway, such as over-expressing and co-expressing the key enzyme genes pncB, nadD and nadE. Second, we optimized NAD^＋ precursors supplement and fermentation conditions to increase NAD^＋ synthesis. Finally, we used Box-Bohnken method for optimal synthesis condition by 3 significant factors＇ interaction based on single-factor experiments and the response value of NAD^＋ content. [Results] According to different expression strategies, we constructed seven recombinant strains. Besides, the intracellular NAD^＋ content of the recombinant strain E. coli BL21/pET-21 a-nadE-pncB was 405.2% higher than that of the original strain. Moreover, after optimization of induction conditions and NAD^＋ precursor concentration by Design Expert 8.0, NAD^＋ content reached 43.16 μmol/g DCW, 123.6% higher than that before optimization and 1029.8% higher than the original strain. [Conclusion] Co-expression the key enzyme genes pncB and nadE are essential to improve NAD^＋ synthesis. The recombinant strain with high NAD^＋ provides the feasibility to improve biocatalytic efficiency.