Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers
Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers
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DOI：
10.1016/j.ymben.2006.10.006
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发表时间：
2007-03-01
影响因子：
8.4
通讯作者：
Vieille, Claire
中科院分区：
文献类型：
--
作者：
McKinlay, James B.;Shachar-Hill, Yair;Vieille, Claire
产琥珀酸放线杆菌是一种很有前途的琥珀酸生产菌。然而，除了产生琥珀酸盐外，它还产生甲酸盐和乙酸盐。为了了解琥珀酸和替代产品的碳通量分布，我们给A.琥珀酸[1-C-13]葡萄糖，并通过气相色谱-质谱法和核磁共振光谱法分析排泄的有机酸、蛋白质氨基酸和糖原单体的同位素异构体。同位素数据，连同葡萄糖消耗和产物形成速率和A。将产琥珀酸菌生物质组成提供给代谢通量模型。氧化戊糖磷酸途径通量提供，最多，20%的细胞生长的估计NADPH的需求。该模型表明，NADPH主要是通过转氢酶和/或依赖于NADP的苹果酸酶将NADH转化为NADPH而产生的。转氢酶活性在A.琥珀酸菌细胞提取物，以及甲酸和丙酮酸脱氢酶，该模型表明这有助于NADH的生产。在细胞提取物中也检测到苹果酸酶活性，与通量分析结果一致。在氨基酸和有机酸的标记模式表明，草酰乙酸和苹果酸被脱羧丙酮酸。这些是第一个体内实验，表明琥珀酸和替代发酵产物之间的流量分配可以发生在A.产琥珀酸菌设计有效的代谢工程策略，以增加A。suceinogenes琥珀酸生产的讨论。(c)2006年爱思唯尔公司All rights reserved.
Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. suceinogenes [1-C-13]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. suceinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. suceinogenes succinate production are discussed. (c) 2006 Elsevier Inc. All rights reserved.