丙酮酸限制甘油连续培养中大肠杆菌适应进化实验的模型分析。

Ronny Feuer, Katrin Gottlieb, Gero Viertel, Johannes Klotz, Steffen Schober, Martin Bossert, Oliver Sawodny, Georg Sprenger, Michael Ederer
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引用次数: 8

摘要

在重要的代谢途径中被遗传阻断并在选择性条件下生长的菌株经历了适应性进化的过程:某些途径可能已被解除管制,因此允许绕过给定的阻断。通过敲除大肠杆菌中丙酮酸激酶和磷酸烯醇丙酮酸羧化酶,实现了内源性甘油合成丙酮酸的阻断。由此产生的突变株能够在含有甘油和乳酸的培养基上生长,作为外源丙酮酸源。利用谷氨酸棒状杆菌丙酮酸羧化酶基因的异源表达对TCA循环进行了修复。用有限的乳酸饲料和过量的甘油饲料控制连续培养的选择条件。200-300代后分离出丙酮酸原生营养突变体。对一个进化菌株的基因组分析表明,重新获得的丙酮酸原生营养的基因型基础并不明显。采用基于约束的代谢模型,通过求解层次线性规划问题,计算给定代谢块周围所有可能的弯路。预计监管网络将负责适应过程。因此,转录因子网络的布尔模型被连接到代谢模型。我们的模型分析仅显示转录控制对底物生物量产量的边际影响,而底物生物量产量是选择过程中的关键变量。在我们的实验中,微阵列分析证实,转录控制可能在规避阻滞的替代途径的解除管制中发挥了次要作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol.

Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol.

Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol.

Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol.

: Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200-300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.

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