13C-Tracer and Gas Chromatography-Mass Spectrometry Analyses Reveal Metabolic Flux Distribution in the Oleaginous Microalga Chlorella protothecoides1[C][W][OA]
13C-Tracer and Gas Chromatography-Mass Spectrometry Analyses Reveal Metabolic Flux Distribution in the Oleaginous Microalga Chlorella protothecoides1[C][W][OA]
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DOI：
10.1104/pp.110.158956
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发表时间：
2010-08
期刊：
影响因子：
7.4
通讯作者：
Wei Xiong;Lixia Liu;Chao Wu;Chen Yang;Qingyu Wu
中科院分区：
文献类型：
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作者：
Wei Xiong;Lixia Liu;Chao Wu;Chen Yang;Qingyu Wu
绿藻小球藻（Chlorella protothecoides）因其积累中性三酰基甘油（triacylglycerol）而受到广泛关注，通常被认为是生产生物柴油的理想原料。为了更好地了解其代谢，在异养生长的原coides中进行了[U-13C]/[1-13C]葡萄糖示踪实验，以确定代谢网络拓扑结构并估计细胞内通量。气相色谱-质谱分析追踪了蛋白质结合氨基酸的标记模式，揭示了一个由糖酵解、戊糖磷酸途径和三羧酸循环组成的代谢网络，其中有无活性的乙醛酸分流。还获得了磷酸烯醇丙酮酸羧化酶、磷酸烯醇丙酮酸羧化激酶和苹果酸酶活性的证据。结果表明，在氮限制环境下，戊糖磷酸途径糖酵解的相对活性增加，反映了脂质生物合成对NADPH的过量需求。尽管氮限制显著改变了原石斛的生长速率和细胞含油量，但其全球通量分布保持稳定，表现出中心碳代谢的刚性。总之，本研究获得的产油藻类代谢通量分布的定量知识可能对设计理想生物产品的代谢工程策略具有价值。
The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-13C]/[1-13C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts.