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首页> 外文期刊>Journal of bacteriology >Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli
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Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

机译：代谢通量对丙酮酸丙酮酸激酶敲除的响应。

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摘要

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.
机译：通过生物合成定向的分数 13 C标记实验和[U- 13 C 6 ]葡萄糖或氨受限的化学恒温器中的葡萄糖，细胞氨基酸的二维核磁共振（NMR）光谱以及全面的同位素模型。对 E中两种丙酮酸激酶同工酶破坏的一般反应。大肠埃希菌是通过磷酸烯醇丙酮酸（PEP）羧化酶和苹果酸酶的联合反应而重新路由的。磷酸戊糖途径和三羧酸循环中的响应强烈取决于环境条件。另外，在丙酮酸缺乏酶 E的葡萄糖限制的恒化培养物中，以低稀释率鉴定了通过糖异生性PEP羧激酶的高无效循环活性。大肠杆菌，其营业额与特定的葡萄糖摄取率相当。此外，突变培养物中的通量分析表明 E中的葡萄糖摄取。在葡萄糖受限的培养物中，低稀释率不能完全由磷酸转移酶系统催化大肠杆菌。通过统计误差分析并与通过代谢通量比分析从相同NMR数据独立计算的细胞内碳通量比进行比较，从而验证了如此获得的通量估计值的可靠性。

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