Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary ¹³C flux analysis.
Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary ¹³C flux analysis.
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由代谢组学和实例研究研究的Pichia Passtoris中的葡萄糖 - 甲醇共同利用。
DOI：
10.1186/1752-0509-7-17
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发表时间：
2013-02-28
影响因子：
--
通讯作者：
Wahl A
中科院分区：
文献类型：
--
作者：
Jordà J;Suarez C;Carnicer M;ten Pierick A;Heijnen JJ;van Gulik W;Ferrer P;Albiol J;Wahl A
一些研究表明，利用混合碳源代替甲醇作为唯一碳源有利于甲醇营养型酵母巴斯德毕赤酵母的蛋白质生产。特别是，在混合饲料条件下的生长似乎减轻了与蛋白质过量生产和分泌引发的应激反应相关的代谢负担。然而，在混合碳源代谢条件下的代谢组和通量组的详细分析是缺失的。为了获得详细的通量分布的中心碳代谢，包括戊糖磷酸途径在甲醇-葡萄糖条件下，我们已经应用代谢组学和不稳定的13 C通量分析恒化培养。使用GC-MS和LC-MS测量的基于13 C的代谢通量分析允许及时准确地绘制糖酵解、磷酸戊糖和甲醇同化途径的代谢通量。与以前的结果NMR衍生的稳态标记数据（蛋白质氨基酸，METAFoR）更多的通量可以确定更高的准确度。此外，使用热力学代谢网络分析的代谢物测量和代谢通量方向进行了验证。值得注意的是，与参考葡萄糖条件相比，在葡萄糖-甲醇进料下，上糖酵解和戊糖磷酸途径的几种代谢物的浓度增加，表明热力学驱动力发生了变化。相反，与葡萄糖生长的巴斯德毕赤酵母细胞的相应外代谢组相比，所有测量的代谢物的细胞外浓度均较低。不稳定的13 C通量分析导致通量与先前从蛋白质氨基酸的NMR数据集获得的通量相当，但允许几个额外的见解。具体而言，i）将体内代谢通量估计扩展到更大的代谢网络，例如通过包括海藻糖再循环，其占葡萄糖摄取速率的约1.5%; ii）估计糖酵解/异生、TCA循环和戊糖磷酸途径反应的可逆性，揭示从磷酸二羟丙酮/磷酸甘油醛池到葡萄糖-6 P的显著异生通量。这一发现的起源可能是从甲醇同化途径到戊糖磷酸池的碳循环。此外，草酰乙酸与天冬氨酸以及苹果酸的高交换通量表明氨基酸库缓冲和苹果酸/天冬氨酸穿梭的活性; iii）甲醇氧化与利用的比率似乎较低（54 vs 79%同化的甲醇直接氧化为CO2）。总之，对巴斯德毕赤酵母应用基于不稳定13 C的代谢通量分析提供了具有改进能力的实验框架，以探索该酵母的碳和能量代谢的调节，特别是对于甲醇和多碳源代谢的情况。
Several studies have shown that the utilization of mixed carbon feeds instead of methanol as sole carbon source is beneficial for protein production with the methylotrophic yeast Pichia pastoris. In particular, growth under mixed feed conditions appears to alleviate the metabolic burden related to stress responses triggered by protein overproduction and secretion. Yet, detailed analysis of the metabolome and fluxome under mixed carbon source metabolizing conditions are missing. To obtain a detailed flux distribution of central carbon metabolism, including the pentose phosphate pathway under methanol-glucose conditions, we have applied metabolomics and instationary 13C flux analysis in chemostat cultivations. Instationary 13C-based metabolic flux analysis using GC-MS and LC-MS measurements in time allowed for an accurate mapping of metabolic fluxes of glycolysis, pentose phosphate and methanol assimilation pathways. Compared to previous results from NMR-derived stationary state labelling data (proteinogenic amino acids, METAFoR) more fluxes could be determined with higher accuracy. Furthermore, using a thermodynamic metabolic network analysis the metabolite measurements and metabolic flux directions were validated. Notably, the concentration of several metabolites of the upper glycolysis and pentose phosphate pathway increased under glucose-methanol feeding compared to the reference glucose conditions, indicating a shift in the thermodynamic driving forces. Conversely, the extracellular concentrations of all measured metabolites were lower compared with the corresponding exometabolome of glucose-grown P. pastoris cells. The instationary 13C flux analysis resulted in fluxes comparable to previously obtained from NMR datasets of proteinogenic amino acids, but allowed several additional insights. Specifically, i) in vivo metabolic flux estimations were expanded to a larger metabolic network e.g. by including trehalose recycling, which accounted for about 1.5% of the glucose uptake rate; ii) the reversibility of glycolytic/gluconeogenesis, TCA cycle and pentose phosphate pathways reactions was estimated, revealing a significant gluconeogenic flux from the dihydroxyacetone phosphate/glyceraldehydes phosphate pool to glucose-6P. The origin of this finding could be carbon recycling from the methanol assimilatory pathway to the pentose phosphate pool. Additionally, high exchange fluxes of oxaloacetate with aspartate as well as malate indicated amino acid pool buffering and the activity of the malate/Asp shuttle; iii) the ratio of methanol oxidation vs utilization appeared to be lower (54 vs 79% assimilated methanol directly oxidized to CO2). In summary, the application of instationary 13C-based metabolic flux analysis to P. pastoris provides an experimental framework with improved capabilities to explore the regulation of the carbon and energy metabolism of this yeast, particularly for the case of methanol and multicarbon source metabolism.
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影响因子：
7.4
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通讯作者：
van Gulik, Walter