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. 2018 Sep;49:242-247. doi: 10.1016/j.ymben.2018.08.013. Epub 2018 Sep 1.
In this study, we have investigated for the first time the metabolism of E. coli grown on agar using C metabolic flux analysis (C-MFA). To date, all C-MFA studies on microbes have been performed with cells grown in liquid culture. Here, we extend the scope of C-MFA to biological systems where cells are grown in dense microbial colonies. First, we identified new optimal C tracers to quantify fluxes in systems where the acetate yield cannot be easily measured. We determined that three parallel labeling experiments with the tracers [1,2-C]glucose, [1,6-C]glucose, and [4,5,6-C]glucose permit precise estimation of not only intracellular fluxes, but also of the amount of acetate produced from glucose. Parallel labeling experiments were then performed with wild-type E. coli and E. coli ΔackA grown in liquid culture and on agar plates. Initial attempts to fit the labeling data from wild-type E. coli grown on agar did not produce a statistically acceptable fit. To resolve this issue, we employed the recently developed co-culture C-MFA approach, where two E. coli subpopulations were defined in the model that engaged in metabolite cross-feeding. The flux results identified two distinct E. coli cell populations, a dominant cell population (92% of cells) that metabolized glucose via conventional metabolic pathways and secreted a large amount of acetate (~40% of maximum theoretical yield), and a second smaller cell population (8% of cells) that consumed the secreted acetate without any glucose influx. These experimental results are in good agreement with recent theoretical simulations. Importantly, this study provides a solid foundation for future investigations of a wide range of problems involving microbial biofilms that are of great interest in biotechnology, ecology and medicine, where metabolite cross-feeding between cell populations is a core feature of the communities.
在这项研究中，我们首次使用 C 代谢通量分析 (C-MFA) 研究了在琼脂上生长的大肠杆菌的代谢。迄今为止，所有关于微生物的 C-MFA 研究都是在液体培养的细胞中进行的。在这里，我们将 C-MFA 的范围扩展到细胞在密集微生物菌落中生长的生物系统中。首先，我们确定了新的最佳 C 示踪剂，以量化无法轻松测量乙酸盐产率的系统中的通量。我们确定，使用示踪剂 [1,2-C]葡萄糖、[1,6-C]葡萄糖和 [4,5,6-C]葡萄糖进行三个平行标记实验，不仅可以精确估计细胞内通量，还可以估计葡萄糖产生的乙酸盐量。然后在液体培养和琼脂平板上用野生型大肠杆菌和大肠杆菌 ΔackA 进行平行标记实验。最初尝试拟合在琼脂上生长的野生型大肠杆菌的标记数据并没有产生统计学上可接受的拟合。为了解决这个问题，我们采用了最近开发的共培养 C-MFA 方法，其中在模型中定义了两个大肠杆菌亚群，它们通过代谢物交叉喂养进行代谢。通量结果确定了两个不同的大肠杆菌细胞群，一个占主导地位的细胞群（92%的细胞）通过常规代谢途径代谢葡萄糖并分泌大量乙酸盐（~40%的最大理论产量），以及第二个较小的细胞群（8%的细胞）消耗分泌的乙酸盐而没有任何葡萄糖流入。这些实验结果与最近的理论模拟非常吻合。重要的是，这项研究为未来研究涉及微生物生物膜的广泛问题提供了坚实的基础，这些问题在生物技术、生态学和医学中非常感兴趣，其中细胞群体之间的代谢物交叉喂养是群落的核心特征。
