13C-Metabolic Flux Analysis for Batch Culture of Escherichia coli and Its pyk and pgi Gene Knockout Mutants Based on Mass Isotopomer Distribution of Intracellular Metabolites
13C-Metabolic Flux Analysis for Batch Culture of Escherichia coli and Its pyk and pgi Gene Knockout Mutants Based on Mass Isotopomer Distribution of Intracellular Metabolites
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DOI：
10.1002/btpr.420
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发表时间：
2010-07-01
影响因子：
2.9
通讯作者：
Shimizu, Kazuyuki
中科院分区：
文献类型：
--
作者：
Toya, Yoshihiro;Ishii, Nobuyoshi;Shimizu, Kazuyuki
由于大多数生物生产过程以分批或补料分批方式进行，因此非常需要相对于时间评估代谢。为此，我们应用C-13-代谢通量分析的非稳态条件下，通过测量细胞内代谢产物的质量同位素分布。我们对野生型大肠杆菌的分批培养物以及Pyk和Pgi突变体进行了分析，获得了通量和代谢物浓度随时间的变化。我们对野生型的研究结果表明，在葡萄糖生长过程中，TCA循环通量趋于增加。葡萄糖耗尽后，细胞控制乙醛酸途径和TCA循环之间的分支比例，这取决于乙酸的可用性。在Pyk突变体中，由于PEP积累引起的倾倒和反馈抑制，糖酵解中间体的浓度随时间急剧变化。然而，通量分布和游离氨基酸浓度变化不大。在Pgi突变体中，生长速率和通量保持恒定，葡萄糖-6-磷酸脱氢酶反应是限速步骤。所测得的通量进行了比较，通过通量平衡分析，使用最大化的生物质产量或ATP生产的预测。我们的研究结果表明，随着时间的推移，生物合成的目标函数变得不那么重要的葡萄糖在野生型，而它仍然非常重要的Pyk突变体。此外，ATP生产是Pgi突变体的主要目标函数。这项研究展示了细胞如何调整其代谢，以应对环境变化和/或分批培养中的遗传扰动。(C)2010年美国化学工程师学会生物技术。程序：26：975-992，2010
Since most bio-production processes are conducted in a batch or fed-batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied C-13-metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild-type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild-type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose-6-phosphate dehydrogenase reaction was the rate-limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild-type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 975-992, 2010