过量表达Bacillus subtilis磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响.docVIP

过量表达Bacillus subtilis磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响 于丽 姜岷* 马江锋 岳方方 刘树文 (南京工业大学生物与制药工程学院 材料化学工程国家重点实验室 江苏 南京 210009) 摘 要: 在大肠杆菌厌氧混合酸发酵途径中, 磷酸烯醇式丙酮酸羧化酶(PPC)和磷酸烯醇式丙酮酸羧化激酶(PCK)皆可催化由磷酸烯醇式丙酮酸(PEP)到草酰乙酸(OAA)的反应。鉴于经由PCK催化的反应伴有ATP的生成, 理论上更有利于菌体生长和产酸, 本研究以大肠杆菌W3110 (△pfl, △ldh)为出发菌株, 利用λ-Red同源重组系统构建了其ppc缺陷菌株并在此基础上过量表达了Bacillus subtilis pck基因。初步的厌氧发酵实验表明: 过量表达pck可在一定程度上恢复初始菌株厌氧代谢葡萄糖的能力。其中又以ppc缺陷株更为明显, 其耗糖能力和产酸能力分别为对照菌株的4.2和15.3倍。 关键词: 磷酸烯醇式丙酮酸羧化激酶, 磷酸烯醇式丙酮酸羧化酶, 大肠杆菌, 琥珀酸 Effect of Overexpression of Bacillus subtilis Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli YU Li JIANG Min* MA Jiang-Feng YUE Fang-Fang LIU Shu-Wen (State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, Jiangsu 210009, China) Abstract: Both of the phosphoenolpyruvate carboxylase (PPC) and phosphoenolpyruvate carboxykinase (PCK) could catalyze the reaction from phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA) in the pathways of anaerobic mixed acid fermentation for E. coli. In addition, the reaction catalyzed by PCK generates ATP, which is more beneficial to the growth of the strain and the succinic acid production theoretically. In this study, we constructed a ppc defective strain using λ-Red homologous recombination system with the E. coli W3110 (△pfl, △ldh) as the parent strain. Based on that, Bacillus subtilis pck was overexpressed. The preliminary anaerobic fermentation experiments showed that both strains partially recovered the ability to consume glucose through the overexpression of pck. Besides, the ppc defective strain showed the most excellent performance, the rate of glucose consumption and succinate production were 4.2 and 15.3 folds as much as those of the parent strain, respectively. Keywords: Phosphoenolpyruvate carboxykinase, Phosphoenolpyruvate carboxylase, E. coli, Succinic acid 琥珀酸(又称丁二酸), 被广泛应用于医药、农药、染料、香料、油漆、食品和塑料等行业, 同时, 作为优秀的C4平台化合物, 可用