Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanisms in vivo
Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanisms in vivo
复制标题
DOI：
10.1006/mben.2001.0198
复制
发表时间：
2001-10-01
影响因子：
8.4
通讯作者：
Sahm, H
中科院分区：
文献类型：
--
作者：
Petersen, S;Mack, C;Sahm, H
谷氨酸棒状杆菌在葡萄糖上生长过程中具有高的糖异生磷酸烯醇丙酮酸羧激酶（PEPCk）的体内活性，导致PEP/丙酮酸/草酰乙酸底物循环中的反复羧化反应。本研究研究了产生l -赖氨酸的C. glutamium MH20-22B中PEPCk活性的改变所引起的细胞内通量和代谢物浓度的变化，应用了最近开发的基于C-13标记的策略来测定和定量细胞内通量。通过缺失相应的pck基因来消除PEPCk的活性，导致细胞内草酰乙酸、l -天冬氨酸、a-酮戊二酸、丙酮酸和l -赖氨酸的浓度增加，并使l -赖氨酸的生物合成通量增加60%，而通过pck过表达来增加PEPCk的活性则产生相反的效果。利用酶活性、体内通量和代谢物浓度的联合测量结果，阐明了谷氨酰胺倒转反应的体内调控，并讨论了对氨基酸产生菌株代谢工程的影响。(C) 2001学术出版社。
Corynebacterium glutamicum possesses high in viva activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed C-13 labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate, L-aspartate, a-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in viva fluxes, and metabolite concentrations were exploited to elucidate the in viva regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed. (C) 2001 Academic Press.