Crown Scott B, Long Christopher P, Antoniewicz Maciek R
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark DE 19716, USA.
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark DE 19716, USA.
Metab Eng. 2016 Nov;38:10-18. doi: 10.1016/j.ymben.2016.06.001. Epub 2016 Jun 4.
C-Metabolic flux analysis (C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for C-MFA. The best single tracers were doubly C-labeled glucose tracers, including [1,6-C]glucose, [5,6-C]glucose and [1,2-C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-C]glucose and [1,2-C]glucose. Combined analysis of [1,6-C]glucose and [1,2-C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-C]glucose +20% [U-C]glucose.
碳代谢通量分析（C-MFA）是代谢工程中广泛用于量化细胞内代谢通量的一种方法。由C-MFA确定的通量精度在很大程度上取决于同位素示踪剂的选择和特定的标记测量集。该领域最近的一项进展是使用平行标记实验来提高通量精度和准确性。然而，截至目前，还不存在用于识别平行标记实验最佳示踪剂的系统方法。在本论文中，我们通过引入一种新的评分系统并评估数千种不同的同位素示踪剂方案来解决这个问题。基于这一广泛分析，我们确定了C-MFA的最佳示踪剂。最佳的单一示踪剂是双碳标记的葡萄糖示踪剂，包括[1,6-C]葡萄糖、[5,6-C]葡萄糖和[1,2-C]葡萄糖，无论代谢通量图如何（这里评估了100个随机通量图），它们始终能产生最高的通量精度。此外，我们证明纯葡萄糖示踪剂总体上比葡萄糖示踪剂混合物表现更好。对于平行标记实验，最佳的同位素示踪剂是[1,6-C]葡萄糖和[1,2-C]葡萄糖。与广泛使用的示踪剂混合物80% [1-C]葡萄糖 + 20% [U-C]葡萄糖相比，[1,6-C]葡萄糖和[1,2-C]葡萄糖标记数据的联合分析将通量精度得分提高了近20倍。
