Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements
Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements
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DOI：
10.1007/s00253-004-1627-1
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发表时间：
2004-07-01
影响因子：
5
通讯作者：
Shimizu, K
中科院分区：
文献类型：
--
作者：
Kabir, MM;Shimizu, K
通过对三羧酸循环中异柠檬酸脱氢酶（ICDH）基因（icd）敲除后大肠杆菌细胞生长特性、酶活性和蛋白表达模式的综合研究，探讨了ICDH基因敲除后大肠杆菌中心代谢的变化。icd基因的缺失导致比生长速率和比葡萄糖消耗速率降低。icd突变株比生长速率的降低主要是由于胞内ATP/ADP比值降低和NADPH/NADP（+）比值降低所致。然而，发现icd突变株的二氧化碳释放速率比亲本E.杆菌这可能是由于6-磷酸葡萄糖酸脱氢酶、磷酸烯醇丙酮酸羧激酶和NADP（+）依赖性苹果酸酶的较高活性。乙醛酸途径也被利用，如icd突变体E中异柠檬酸裂解酶和苹果酸合酶活性的显著上调所证明的。杆菌乙醛酸途径的出现导致较低的乙酸生产。在21种表达水平改变的蛋白质中，17种蛋白质通过MALDI-TOF质谱成功鉴定。结果表明，ICDH活性的丧失显著影响呼吸系统和电子传递链，表现为icd突变体E中nuoE、nuoH、cydA和cyoA基因编码的蛋白质显著下调。与亲本相比。
An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP(+) ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP(+)-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.