基于能量代谢优化的丁二酸生物合成强化  

Efficient production of succinic acid by optimizing an energy-conserving pathway in Escherichia coli

作　　者：白冰[1,2] 杨茂华[1,2] 刘谊兰[1,2] 邢建民[1,2] 

机构地区：[1]生化工程国家重点实验室 [2]中国科学院过程工程研究所,北京100190 [2]中国科学院大学,北京100049

出　　处：《中国科学：化学》2015年第5期564-570,共7页SCIENTIA SINICA Chimica

基　　金：国家高技术研究发展计划(2012AA101807,2011AA02A203,2012AA022301)资助

摘　　要：磷酸烯醇式丙酮酸(PEP)的羧化是大肠杆菌合成丁二酸的关键反应.由于PEP羧化激酶(PCK)催化过程伴随三磷酸腺苷(ATP)的产生,故更有利于菌体生长和丁二酸生成,但是PCK对于底物HCO3-亲和性较弱,也存在着催化效率低的问题.针对该问题,本研究构建了产丁二酸工程菌BS101(W3110△ldh A△pfl B::Kan/p Trc His A-130ZPCK),探讨酶的表达水平、HCO3-的供应方式及浓度对PCK催化效率的影响,优化能量代谢途径,提高菌株积累丁二酸的能力.结果表明,PCK在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0 mmol/L和Na HCO3浓度15 g/L条件下催化效率最高,丁二酸产量和摩尔产率达到5.68 g/L和1.15 mol/mol,较原来分别提高20%和28%,同时几乎没有副产物的积累.同时考察胞内ATP水平发现,在最优条件下胞内ATP的含量是未经优化时的2倍.这种基于优化能量代谢的策略为强化丁二酸的生物合成提供新的思路.Phosphoenolpyruvate （PEP） carboxylation is a crucial reaction for succinate synthesis in E. coli. PCK （phosphoenolpyruvate carboxykinase）-catalyzed reaction for PEP carboxylation is associated with the generation of ATP, which favors cell growth and succinate production. However, the catalytic efficiency of PCK is relatively low due to poor affinity for bicarbonate. To solve this problem, engineered strain BS101 （W3110△ldh△lxpflB：： Kan/pTrcHis-13OZPCK） was constructed in present study and effects of expression level of PCK, supply patterns and concentration of HCO3 , on catalytic efficiency of PCK were investigated for optimizing this energy-conserving pathwayand improving succinate production. The results showed that PCK could exhibit the highest catalytic ability under 0 mmol/L IPTG and 15 g/L NaHCO3. Under optimized conditions, the final succinate concentration and molar yield reached to 5.68 g/L and 1.15 mol/mo], respectively, which increased by 20% and 28% compared with those of control group. Meanwhile, the intracellular ATP concentration in optimized conditions above was nearly 2 fold higher than before. This strategy based on optimization of energetic pathway provides a promising direction for succinate biosynthesis.

关 键 词：丁二酸 大肠杆菌 磷酸烯醇式丙酮 酸羧化激酶 表达水平 HCO 

分 类 号：TQ225.144[化学工程—有机化工]