Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Catalonia, Spain.
Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark.
N Biotechnol. 2019 May 25;50:52-59. doi: 10.1016/j.nbt.2019.01.005. Epub 2019 Jan 17.
Metabolic flux analysis based on C-derived constraints has proved to be a powerful method for quantitative physiological characterisation of one of the most extensively used microbial cell factory platforms, Pichia pastoris (syn. Komagataella spp.). Nonetheless, the reduced number of carbon atoms and the symmetry of the glycerol molecule has hampered the comprehensive determination of metabolic fluxes when used as the labelled C-source. Moreover, metabolic models typically used for C-based flux balance analysis may be incomplete or misrepresent the actual metabolic network. To circumvent these limitations, we reduced the genome-scale metabolic model iMT1026-v3.0 into a core model and used it for the iterative fitting of metabolic fluxes to the measured mass isotope distribution of proteinogenic amino acids obtained after fractional C labelling of cells with [1,3-C]-glycerol. This workflow allows reliable estimates to be obtained for in vivo fluxes in P. pastoris cells growing on glycerol as sole carbon source, as well as revising previous assumptions concerning its metabolic operation, such as alternative metabolic branches, calculation of energetic parameters and proposed specific cofactor utilisation.
基于 C 约束的代谢通量分析已被证明是一种强大的方法，可用于对最广泛使用的微生物细胞工厂平台之一——毕赤酵母（同义词：Komagataella 属）进行定量生理特征描述。然而，甘油分子的碳原子数量较少且对称性较高，这阻碍了将其作为标记 C 源时对代谢通量的全面测定。此外，通常用于基于 C 的通量平衡分析的代谢模型可能不完整或不能代表实际的代谢网络。为了规避这些限制，我们将基因组规模的代谢模型 iMT1026-v3.0 简化为核心模型，并将其用于对细胞用 [1,3-C]-甘油进行分数 C 标记后获得的蛋白氨基酸的质量同位素分布进行代谢通量的迭代拟合。该工作流程可用于可靠估计在以甘油为唯一碳源生长的毕赤酵母细胞中的体内通量，同时还可修正以前关于其代谢操作的假设，例如替代代谢分支、能量参数的计算和提出的特定辅因子利用。
