产琥珀酸大肠杆菌工程菌株的构建

Construction of engineered Escherichia coli for succinate producition

大肠杆菌在厌氧条件及不额外添加电子受体的条件下可利用葡萄糖进行混合酸发酵,主要产物为甲酸、乙酸和乙醇,丁二酸及乳酸含量较低。为减少副产物的生成,使更多的代谢流流向丁二酸,本研究拟失活厌氧条件下甲酸、乙酸及乳酸主要生成途径相关的酶(乳酸脱氢酶和丙酮酸甲酸裂解酶)。借助Red重组系统和位点特异性重组技术,无痕敲除了野生大肠杆菌W3110染色体上编码2个酶的基因pfl和ldh,构建了1株pfl和ldh双突变重组菌JM130(7△pfl△ldh),并引入PYC前后对比实验表明:JM1307(pTrc99a-pyc)较出发菌株解除了生长抑制,在8g/L初糖条件下,发酵48h,菌体密度可提高至OD6002.5,产物主要为丁二酸(2.8g/L),乙酸含量很低,无甲酸、乳酸生成。

Under anaerobic conditions and in the absence of exogenous electron acceptors, Escherichia coli ferments glucose to a mixture of products consisting of acetate, formate and ethanol, as well as a small amounts of lactate and succinate. In order to reduce the co-products lactate and formate, a Idh pt/double mutant has been constructed based on the RED recombination system and the site-specific recombinant technology. The fermentation results indicated that deficient in both of the two enzymes showed significant growth defects because of pyruvate accumulation which would negatively affect the phosphoenolpyruvate-dependent sugar transport. Since E. coil lacks the anaplerotic enzyme pyruvate carboxylase, pyruvate accumulation and succinate formation in this strain could be altered by directing pyruvate to the succinate branch with this enzyme. Hence, a recombinant plasmid pTrc99a- pyc was constructed and transformed to JMI307. Results showed： with 8 g/L initial glucose, the cell density of JM1307 （pTrc99a-pyc） enhanced to OD600 2.5, and the main product was succinate （2.8 g/L） with little acetic acid, and no formic acid and lactic acid were produced.