A transient isotopic labeling methodology for 13C metabolic flux analysis of photo auto trophic microorganisms
A transient isotopic labeling methodology for 13C metabolic flux analysis of photo auto trophic microorganisms
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DOI：
10.1016/j.phytochem.2007.03.042
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发表时间：
2007-08-01
期刊：
影响因子：
3.8
通讯作者：
Morgan, John A.
中科院分区：
文献类型：
--
作者：
Shastri, Avantika A.;Morgan, John A.
代谢流分析越来越被认为是系统生物学的一个组成部分。然而，由于纯光自养系统（以二氧化碳作为唯一碳源生长）中系统范围代谢通量的实验测量技术尚未开发出来，因为此类系统带来了独特的问题。在本文中，我们证明了一种瞬时平衡位置同位素分布的方法是获得纯自养代谢的全系统代谢通量图的唯一途径。概述的瞬态 C-13-MFA 方法能够测量代谢稳态下的通量，同时跟踪代谢中间体的 C-13 标记模式随时间的变化，以响应 C-13 标记输入的阶跃变化。我们使用中心中间体的瞬态同位素标记模式的数学模型来评估光合自养 MFA 的各种实验要求。这包括需要测量细胞内代谢物浓度和作为时间函数的同位素标记测量。我们还讨论了光生物反应器的设计和操作，以便在精确的环境条件下测量通量。瞬态MFA技术可用于测量和比较不同光强、氮源条件下的通量或比较具有各种突变或基因缺失和添加的菌株。 (C) 2007 Elsevier Ltd. 保留所有权利。
Metabolic flux analysis is increasingly recognized as an integral component of systems biology. However, techniques for experimental measurement of system-wide metabolic fluxes in purely photoautotrophic systems (growing on CO2 as the sole carbon source) have not yet been developed due to the unique problems posed by such systems. In this paper, we demonstrate that an approach that balances positional isotopic distributions transiently is the only route to obtaining system-wide metabolic flux maps for purely autotrophic metabolism. The outlined transient C-13-MFA methodology enables measurement of fluxes at a metabolic steady-state, while following changes in C-13-labeling patterns of metabolic intermediates as a function of time, in response to a step-change in C-13-label input. We use mathematical modeling of the transient isotopic labeling patterns of central intermediates to assess various experimental requirements for photoautotrophic MFA. This includes the need for intracellular metabolite concentration measurements and isotopic labeling measurements as a function of time. We also discuss photobioreactor design and operation in order to measure fluxes under precise environmental conditions. The transient MFA technique can be used to measure and compare fluxes under different conditions of light intensity, nitrogen sources or compare strains with various mutations or gene deletions and additions. (C) 2007 Elsevier Ltd. All rights reserved.