乳糖诱导丙酮酸羧化酶基因在大肠杆菌中的表达及对丁二酸产量的影响

Expression of heterogenous pyruvate carboxylase in Escherichia coli with lactose as inducer and its effect on succinate production

摘要:

大肠杆菌DC1515是敲除葡萄糖磷酸转移酶(ptsG)、乳酸脱氢酶(ldhA)、丙酮酸甲酸裂解酶(pflA)基因的菌株, 具有发酵生产丁二酸的潜力。为进一步提高菌株DC1515的丁二酸生产能力, 将枯草芽孢杆菌丙酮酸羧化酶(pyc)基因转入其中。用乳糖代替IPTG诱导pyc表达, 确定了最佳乳糖加入时间、乳糖浓度及诱导温度。在此基础上, 考察了补加乳糖对丁二酸产量的影响。结果表明: 由于ptsG基因缺失, 当培养基中葡萄糖浓度达到15 g/L时, 乳糖诱导作用并不受葡萄糖抑制。优化诱导条件后, pyc过表达菌株的丁二酸产量达15.17 g/L, 为对照菌株的1.78倍。间歇补加乳糖2次至浓度为1 g/L, 丁二酸产量可进一步增至17.54 g/L。研究结果为以葡萄糖为底物生产丁二酸的过程中乳糖诱导外源基因在大肠杆菌中的表达奠定了基础。乳糖诱导降低了成本, 有利于实现丁二酸发酵生产的工业化。

Abstract:

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.