Development of quantitative metabolomics for Pichia pastoris.
Development of quantitative metabolomics for Pichia pastoris.
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DOI：
10.1007/s11306-011-0308-1
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发表时间：
2012-04
期刊：
影响因子：
3.6
通讯作者：
van Gulik, Walter
中科院分区：
文献类型：
--
作者：
Carnicer, Marc;Canelas, Andre B.;ten Pierick, Angela;Zeng, Zhen;van Dam, Jan;Albiol, Joan;Ferrer, Pau;Heijnen, Joseph J.;van Gulik, Walter
准确、可靠和可重复的测量细胞内代谢物水平已成为微生物细胞工厂代谢研究的重要内容。代谢组学研究的第一个关键步骤是建立适当的淬火和洗涤方案，以确保有效地阻止所有代谢活动并去除细胞外代谢物，而不会导致代谢物从细胞中泄漏。以甲醇含量和淬火液温度为关键参数，对五种不同的甲醇冷淬火和过滤分离方法进行了毕赤酵母代谢组学检测。通过质量平衡分析对这些方案进行定量评估，基于所有样品组分的代谢物测量，这些样品组分是全肉汤，淬火和洗涤细胞，培养滤液和淬火和洗涤溶液。最后，利用优化后的方法研究了葡萄糖限化培养中游离氨基酸和中心碳代谢中间体的时间分布。所有淬火工艺均获得了可接受的回收率（bbb90 %）。然而，在- 27°C， 60% v/v甲醇中淬火，在最小化泄漏方面表现略好。我们可以证明在葡萄糖限制下停留五次足以达到稳定的细胞内代谢物池。此外，当比较毕氏酵母和酿酒酵母的代谢组时，在相同的培养条件下，两种酵母的代谢物指纹图谱相似，除了较低的糖酵解，在毕氏酵母中这些代谢物的水平表明酶的能力限制在这部分代谢中。本文的在线版本（doi:10.1007/ s111306 -011-0308-1）包含补充材料，仅供授权用户使用。
Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism. The online version of this article (doi:10.1007/s11306-011-0308-1) contains supplementary material, which is available to authorized users.
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