Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements
Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements
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DOI：
10.1016/j.femsle.2004.04.003
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发表时间：
2004-06-01
影响因子：
2.1
通讯作者：
Shimizu, K
中科院分区：
文献类型：
--
作者：
Peng, LF;Arauzo-Bravo, MJ;Shimizu, K
采用气相色谱-质谱联用（GC-MS）和二维核磁共振（2D NMR）技术，利用c -13标记实验获得的代谢通量分布，结合酶活性测定和细胞内代谢物浓度测定，对ppc突变体大肠杆菌的生理和中心代谢进行了研究。与野生型相比，它的ppc突变体排出的醋酸盐很少，产生的二氧化碳也更少，但代价是生长速度较慢，葡萄糖摄取率较低。因此，在突变体pp中观察到葡萄糖的生物量产量的提高。酶活性测量显示，异柠檬酸裂解酶活性在ppc突变体中增加了3倍以上。部分TCA循环酶如柠檬酸合成酶、乌头酸酶和苹果酸脱氢酶也上调，而糖酵解酶和戊糖磷酸途径酶下调。因此，在ppc突变体中，糖酵解和戊糖磷酸途径的细胞内中间体积累，而乙酰辅酶A和草酰乙酸浓度下降。细胞内代谢通量分析发现，ppc的缺失导致了乙醛酸盐分流的出现，18.9%的碳通量通过乙醛酸盐分流。然而，在ppc突变体中，戊糖磷酸途径的通量显著降低。(C) 2004年欧洲微生物学会联合会。Elsevier B.V.版权所有。
The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by C-13-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the pp, mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway sitinificantly decreased in the ppc mutant. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.