Metabolic flux elucidation for large-scale models using 13C labeled isotopes
Metabolic flux elucidation for large-scale models using 13C labeled isotopes
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DOI：
10.1016/j.ymben.2007.05.005
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发表时间：
2007-09-01
影响因子：
8.4
通讯作者：
Maranas, Costas D.
中科院分区：
文献类型：
--
作者：
Suthers, Patrick F.;Burgard, Anthony P.;Maranas, Costas D.
代谢工程中的一个关键考虑因素是确定细胞内代谢物的通量。该确定提供了工程干预之前和/或之后代谢的明确描述。在这里，我们提出了一个计算框架，结合了基于约束的建模框架与同位素标签跟踪大规模。当细胞被喂食具有用13 C标记的某些碳位置的生长底物时，可以基于参与途径的已知生物化学来计算该标记在细胞内代谢物中的分布。大多数标记的研究集中在骨骼代表性的中央代谢，忽略了许多通量的路线，可能有助于观察到的同位素标记模式。相比之下，我们的方法研究了进行同位素标记研究的重要性，使用一个更全面的反应网络，包括350通量和184代谢产物在大肠杆菌中，包括全球代谢平衡的辅因子，如ATP，NADH和NADPH。建议的程序是证明了一个E。这是一种经过基因工程改造的大肠杆菌菌株，可以生产抗疟疾药物青蒿素的前体紫穗槐二烯。细胞在含有20% [U-C-13]葡萄糖的葡萄糖上连续培养;使用GC-MS对从细胞中提取的13种氨基酸进行测量。我们确定通量分布的计算标签模式同意以及与测量暗指网络重建的准确性。此外，我们探讨的鲁棒性的通量计算的变化，在实验MS测量，以及突出的关键实验测量所需的通量测定。最后，我们讨论了减少模型的效果，以及阐明定制的开发计算框架到其他系统。(C)2007爱思唯尔公司All rights reserved.
A key consideration in metabolic engineering is the determination of fluxes of the metabolites within the cell. This determination provides an unambiguous description of metabolism before and/or after engineering interventions. Here, we present a computational framework that combines a constraint-based modeling framework with isotopic label tracing on a large scale. When cells are fed a growth substrate with certain carbon positions labeled with 13 C, the distribution of this label in the intracellular metabolites can be calculated based on the known biochemistry of the participating pathways. Most labeling studies focus on skeletal representations of central metabolism and ignore many flux routes that could contribute to the observed isotopic labeling patterns. In contrast, our approach investigates the importance of carrying out isotopic labeling studies using a more comprehensive reaction network consisting of 350 fluxes and 184 metabolites in Escherichia coli including global metabolite balances on cofactors such as ATP, NADH, and NADPH. The proposed procedure is demonstrated on an E. coli strain engineered to produce amorphadiene, a precursor to the antimalarial drug artemisinin. The cells were grown in continuous culture on glucose containing 20% [U-C-13]glucose; the measurements are made using GC-MS performed on 13 amino acids extracted from the cells. We identify flux distributions for which the calculated labeling patterns agree well with the measurements alluding to the accuracy of the network reconstruction. Furthermore, we explore the robustness of the flux calculations to variability in the experimental MS measurements, as well as highlight the key experimental measurements necessary for flux determination. Finally, we discuss the effect of reducing the model, as well as shed light onto the customization of the developed computational framework to other systems. (C) 2007 Elsevier Inc. All rights reserved.