School of Life Sciences, Tsinghua University, Beijing, People's Republic of China.
Plant Physiol. 2010 Oct;154(2):1001-11. doi: 10.1104/pp.110.158956. Epub 2010 Aug 18.
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-(13)C]/[1-(13)C]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.
集胞藻（Chlorella protothecoides）因其能够积累中性三酰基甘油而备受关注，通常被认为是生物柴油生产的理想原料。为了更好地了解其代谢途径，我们进行了异养生长集胞藻的[U-(13)C]/[1-(13)C]葡萄糖示踪实验，以确定代谢网络拓扑结构并估算细胞内通量。气相色谱-质谱分析跟踪了蛋白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，其中乙醛酸支路处于非活性状态。还获得了磷酸烯醇丙酮酸羧激酶、磷酸烯醇丙酮酸羧化酶和苹果酸酶活性的证据。结果表明，在氮限制环境下，戊糖磷酸途径相对于糖酵解的相对活性增加，反映了脂质生物合成对过量 NADPH 的需求。尽管氮限制会显著改变细胞的生长速率和细胞内油脂含量，但集胞藻的全局通量分布仍然保持稳定，表现出中心碳代谢的刚性。总之，本研究中获得的关于油脂藻类代谢通量分布的定量知识可能有助于设计理想生物产物代谢工程的策略。
