The development of metabolomic sampling procedures for Pichia pastoris, and baseline metabolome data.
The development of metabolomic sampling procedures for Pichia pastoris, and baseline metabolome data.
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DOI：
10.1371/journal.pone.0016286
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发表时间：
2011-01-21
期刊：
影响因子：
3.7
通讯作者：
Bundy JG
中科院分区：
文献类型：
--
作者：
Tredwell GD;Edwards-Jones B;Leak DJ;Bundy JG
代谢谱越来越多地被用于研究各种各样的生物学问题。由于细胞内代谢物的快速周转，重要的是要有可靠的，可重复的采样和样品处理技术。通过使用非靶向分析技术，如NMR和GC-MS，我们已经进行了全面的定量调查的采样技术，巴斯德毕赤酵母。很明显，使用基于标准冷甲醇方案的溶液淬灭代谢导致巴斯德毕赤酵母细胞的一些代谢物损失。然而，这些水平较低，NMR结果表明，对于75%的已鉴别代谢物，淬灭溶液中代谢物的增加低于其细胞内水平的5%;而GC-MS结果表明水平略高，增加低于其细胞内值的15%。研究的四种淬灭溶液之间存在细微差异，但大体上，它们都给出了类似的结果。使用巴斯德毕赤酵母发酵典型的高细胞密度培养物进行细胞+肉汤的总培养物提取是用于NMR分析的有效取样技术，并提供了细胞内代谢物水平的金标准;然而，培养基中的盐影响GC-MS分析。此外，在淬灭过程中包括额外的洗涤步骤没有益处，因为结果与仅通过单个离心步骤获得的结果基本相同。我们已经确定了主要的高浓度代谢产物中发现的细胞外和细胞内的位置的巴斯德毕赤酵母培养物的NMR光谱和GC-MS。这为我们提供了一个基线代谢组巴斯德毕赤酵母为未来的研究。巴斯德毕赤酵母的代谢组与酿酒酵母的代谢组显著不同，其中最显著的差异是巴斯德毕赤酵母产生高浓度的阿拉伯糖醇。
Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.