Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
Institute of Biological Chemistry, Academia Sinica, 115 Taipei, Taiwan.
Metab Eng. 2019 Mar;52:273-283. doi: 10.1016/j.ymben.2019.01.004. Epub 2019 Jan 8.
In silico kinetic modeling is an essential tool for rationally designing metabolically engineered organisms based on a system-level understanding of their regulatory mechanisms. However, an estimation of enzyme parameters has been a bottleneck in the computer simulation of metabolic dynamics. In this study, the ensemble-modeling approach was integrated with the transomics data to construct kinetic models. Kinetic metabolic models of a photosynthetic bacterium, Synechocystis sp. PCC 6803, were constructed to identify engineering targets for improving ethanol production based on an understanding of metabolic regulatory systems. A kinetic model ensemble was constructed by randomly sampling parameters, and the best 100 models were selected by comparing predicted metabolic state with a measured dataset, including metabolic flux, metabolite concentrations, and protein abundance data. Metabolic control analysis using the model ensemble revealed that a large pool size of 3-phosphoglycerate could be a metabolic buffer responsible for the stability of the Calvin-Benson cycle, and also identified that phosphoglycerate kinase (PGK) is a promising engineering target to improve a pyruvate supply such as for ethanol production. Overexpression of PGK in the metabolically engineered PCC 6803 strain showed that the specific ethanol production rate and ethanol titers at 48 h were 1.23- and 1.37-fold greater than that of the control strain. PGK is useful for future metabolic engineering since pyruvate is a common precursor for the biosynthesis of various chemicals.
基于对其调控机制的系统理解，计算动力学建模是合理设计代谢工程生物体的重要工具。然而，酶参数的估计一直是代谢动力学计算机模拟的瓶颈。在这项研究中，我们将集合模型方法与转录组学数据集成，构建了动力学模型。我们构建了光合细菌集胞藻 6803 的代谢动力学模型，以在了解代谢调控系统的基础上，确定提高乙醇产量的工程目标。通过随机抽样参数构建了动力学模型集合，并通过将预测的代谢状态与包括代谢通量、代谢物浓度和蛋白质丰度数据在内的测量数据集进行比较，选择了最佳的 100 个模型。使用模型集合进行代谢控制分析表明，3-磷酸甘油酸的大池体积可以作为卡尔文-本森循环稳定性的代谢缓冲液，还确定了磷酸甘油激酶（PGK）是提高丙酮酸供应（如提高乙醇产量）的有前途的工程目标。在代谢工程集胞藻 6803 菌株中过表达 PGK 后，发现 48 小时时特定乙醇生产率和乙醇浓度分别比对照菌株提高了 1.23 倍和 1.37 倍。由于丙酮酸是各种化学物质生物合成的常见前体，PGK 对未来的代谢工程很有用。
