Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived 13C-labelling data
Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived 13C-labelling data
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DOI：
10.1111/j.1567-1364.2006.00180.x
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发表时间：
2007-03-01
影响因子：
3.2
通讯作者：
van Winden, Wouter A.
中科院分区：
文献类型：
--
作者：
Kleijn, Roelco J.;Geertman, Jan-Maarten A.;van Winden, Wouter A.
本研究旨在通过缺失编码丙糖磷酸异构酶(TPI1)、线粒体NADH脱氢酶(NDE1和NDE2)和呼吸链连接的3-磷酸甘油脱氢酶(GUT2)的结构基因，对一株甘油高产的酿酒酵母菌株的碳和氧化还原代谢进行研究。代谢通量的分析采用两种方法：代谢物平衡法和基于C-13标记的代谢通量分析。通过蛋白质来源的氨基酸(核磁共振和气相色谱-MS)直接测量和间接测量细胞内初级代谢物的同位素丰度。由于几个重要代谢节点周围的通量敏感性被证明依赖于所应用的技术，三种C-13量化技术的组合产生了最准确的总体通量模式。当结合在一起时，测量的转化率和C-13标记数据提供了证据，证明同化代谢和戊糖磷酸途径活性的组合转移了甘油形成过程中的一些碳。代谢物平衡表明，这会导致细胞内NADH过量，这表明除了那些被缺失的NADH外，还存在细胞内NADH汇。通过C-13标记数据测量的线粒体膜上四碳二元酸的交换通量支持苹果酸/天冬氨酸或苹果酸/草酰乙酸酯氧化还原穿梭在将这些氧化还原等价物从胞浆转移到线粒体基质中的可能作用。
This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and C-13-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three C-13 quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and C-13-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the C-13-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.