Physiological Mechanism for Enhanced Cyclic Adenosine Monophosphate Biosynthesis by Sodium Nitrate in Arthrobacter sp. CCTCC M2013431
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摘要: 目的：阐明硝酸盐促进环磷酸腺苷发酵合成的生理机制。方法：首先通过摇瓶实验确定硝酸钠最适添加条件，并在发酵罐上进行添加硝酸钠的cAMP发酵实验，然后对发酵过程动力学数据、硝酸盐代谢、还原力水平、关键酶活性、氨基酸水平和能量代谢进行分析。结果：确定在发酵24 h添加3 g/L-broth硝酸钠为最适条件，cAMP发酵产量和得率分别达到5.02 g/L和0.097 g/g，比对照批次分别提高了22.7%和29.8%，发酵性能得到明显提升。硝酸盐利用过程消耗了大量NADPH，缓解了对6-磷酸葡萄糖脱氢酶的抑制作用，同时糖酵解途径受到抑制而磷酸戊糖途径和三羧酸循环中关键酶活性明显提高，更多碳流分配到产物合成途径。此外，胞内前体氨基酸水平、NADH/NAD+和ATP/AMP均得到明显提高，为cAMP发酵合成提供了物质和能量基础。结论：硝酸盐利用过程消耗了大量的还原力，改变了代谢流分配情况，提高了胞内氨基酸水平和ATP合成，进而促进cAMP的发酵合成。硝酸盐作用机制的阐明，为提高核苷酸类发酵产品的生产水平提供了参考。Abstract: Objectives: The aim of this study is to elucidate the physiological mechanism for enhanced cyclic adenosine monophosphate biosynthesis by nitrate in Arthrobacter sp. CCTCC M2013431. Methods: Firstly,optimal sodium nitrate adding condition was determined by fermentations conducted in shaking flask and cAMP fermentation with optimal condition was carried out in a 7 L fermentation tank. Then,fermentation kinetic parameters,nitrate utilization,reducing force level,key enzymes activities,intracellular amino acids levels and energy metabolism were analyzed. Results: Due to the addition of 3 g/L-broth sodium nitrate into fermentation broth at 24 h,cAMP concentration and yield on glucose achieved 5.02 g/L and 0.097 g/g,with the improvements of 22.7% and 29.8%,respectively,when compared with those of control. Activities of glucose-6-phosphate dehydrogenase were improved greatly by lower NADPH levels caused by plentiful consumption during nitrate reduction process,meanwhile,the metabolic intensity of glycolysis pathway was weakened while activities of key enzymes in pentose phosphate pathway and tricarboxylic acid cycle were significantly increased. More carbon flux was directed into pentose phosphate pathway and purine synthesis pathway for cAMP production. In addition,intracellular precursor amino acids levels,NADH/NAD+ and ATP/AMP ratios were also enhanced greatly by nitrate which supply sufficient energy for cAMP synthesis. Conclusion: Nitrate utilization consumed redundant reducing force which changed the distribution of metabolic flux among different pathways,and improved intracellular amino acids levels and ATP synthesis,then cAMP fermentation synthesis was promoted greatly. The physiological mechanism of nitrate for enhanced cAMP biosynthesis provided reference materials for nucleotide fermentation production.
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Keywords:
- sodium nitrate /
- NADPH /
- amino acids /
- energy metabolism /
- cAMP
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