微生物學(xué)通報(bào) MAR 20,2010,37(3):325～330
過量表達(dá)Bacillus subtilis磷酸烯醇式丙酮酸羧化激酶對大腸桿菌產(chǎn)琥珀酸的影響
于麗 姜岷* 馬江鋒 岳方方 劉樹文
(南京工業(yè)大學(xué)生物與制藥工程學(xué)院 材料化學(xué)工程國家重點(diǎn)實(shí)驗(yàn)室 江蘇 南京 210009)
摘 要: 在大腸桿菌厭氧混合酸發(fā)酵途徑中, 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)的反應(yīng),。鑒于經(jīng)由PCK催化的反應(yīng)伴有ATP的生成, 理論上更有利于菌體生長和產(chǎn)酸, 本研究以大腸桿菌W3110 (△pfl, △ldh)為出發(fā)菌株, 利用λ-Red同源重組系統(tǒng)構(gòu)建了其ppc缺陷菌株并在此基礎(chǔ)上過量表達(dá)了Bacillus subtilis pck基因,。初步的厭氧發(fā)酵實(shí)驗(yàn)表明: 過量表達(dá)pck可在一定程度上恢復(fù)初始菌株厭氧代謝葡萄糖的能力,。其中又以ppc缺陷株更為明顯, 其耗糖能力和產(chǎn)酸能力分別為對照菌株的4.2和15.3倍。
關(guān)鍵詞: 磷酸烯醇式丙酮酸羧化激酶, 磷酸烯醇式丙酮酸羧化酶, 大腸桿菌, 琥珀酸
Effect of Overexpression of Bacillus subtilis Phosphoenolpy-ruvate 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 path-ways 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 ex-cellent 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
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