Netzer Roman, Krause Malgorzata, Rittmann Doris, Peters-Wendisch Petra G, Eggeling Lothar, Wendisch Volker F, Sahm Hermann
Institute of Biotechnology 1, Research Centre Jülich, 52425 Juelich, Germany.
Arch Microbiol. 2004 Nov;182(5):354-63. doi: 10.1007/s00203-004-0710-4. Epub 2004 Sep 15.
In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.
在许多细菌中，丙酮酸激酶在糖酵解过程中发挥着明确的作用，催化产生ATP的反应。然而，其在利用需要糖异生的碳源生长过程中的作用研究较少。我们分析了谷氨酸棒杆菌中一个明确的丙酮酸激酶基因（pyk）缺失突变体，该突变体不能以核糖作为唯一碳源生长。出乎意料的是，pyk缺失突变体也不能以乙酸盐或柠檬酸盐作为唯一碳源生长，除非在生长培养基中添加少量丙酮酸。分离出了pyk缺失菌株的一个自发抑制突变体，该突变体恢复了在乙酸盐上生长的能力。DNA微阵列实验显示苹果酸酶基因malE的表达增加。通过转录融合分析发现，该突变体中malE上游的点突变导致了碳源依赖性调控的丧失。malE的过表达足以恢复pyk缺失菌株在乙酸盐或柠檬酸盐上的生长。为了补偿丙酮酸激酶的缺失，需要提高苹果酸酶水平来重新引导糖酵解、糖异生和三羧酸循环之间界面处的碳通量，这表明在代谢节点磷酸烯醇丙酮酸处存在代谢通量分支。在谷氨酸棒杆菌利用乙酸盐或柠檬酸盐生长过程中，由草酰乙酸产生的大部分磷酸烯醇丙酮酸在糖异生过程中被代谢，而一小部分则在糖酵解方向上被丙酮酸激酶转化，以维持生物量合成所需的丙酮酸的适当供应。
