希瓦氏菌Shewanella sp.MR-4在不同末端电子受体条件下的代谢通量分析

Metabolic flux analysis of Shewanella sp.MR-4 with different terminal electron receptors

本研究解析了Shewanella sp.MR-4在厌氧条件下以Fe^(3+)、延胡索酸、硝酸和二甲基亚砜(DMSO)为末端电子受体时代谢通量分布,发现在各种不同末端电子受体条件下细胞内代谢通量分布发生显著改变。与延胡索酸相比,在Fe^(3+)和硝酸为电子受体条件下,乙酸生成速率明显降低,而三羧酸循环代谢通量增加。同时,三羧酸循环回补反应存在显著差异:在延胡索酸为电子受体条件下,磷酸烯醇式丙酮酸羧化酶反应是唯一回补反应,而在Fe^(3+)和硝酸条件下苹果酸酶催化成为唯一回补反应。本研究在代谢通量分析基础上定量解析了还原力NADH合成和消耗,分析了电子流向变化,发现在不同末端电子受体条件下,NADH产生途径和反应发生明显变化。研究结果为希瓦氏菌代谢工程改造提供了一定理论依据。

Shewanella is ubiquitous in natural environments,known to show various unique activities,such as metal reduction and trimethylamine production.The metal reduction capability expands the range of electron acceptors and enables its application in producing current in microbial fuel cells.Previous work primarily focused on the model organism Shewanella oneidensis MR-1,based on which the metabolic models were constructed for three other strains including Shewanella sp.MR-4.Interestingly,the genetic similarity does not always coincide with metabolic phenotype similarity between these two strains,suggesting that potential regulatory effects affecting cellular phenotypes are present in Shewanella sp.MR-4.This study analyzed the metabolic flux distribution of Shewanella sp.MR-4 when Fe^(3+),fumaric acid,nitric acid and dimethyl sulfoxide were used as electron acceptors under anaerobic conditions.The intracellular metabolic flux distribution was significantly changed with different electron acceptors.The formation rate of acetic acid was decreased,while the metabolic flux of the tricarboxylic acid(TCA)cycle was increased with Fe^(3+)and nitric acid as electron acceptors,compared to that with fumaric acid.The anaplerotic reaction in the TCA cycle was also dramatically altered:the phosphoenolpyruvate carboxylase-catalyzed reaction or malic enzymecatalyzed reaction is the only anaplerotic reaction with fumaric acid or Fe^(3+)and nitric acid as electron acceptor,respectively.Based on the metabolic flux map,quantitative analysis was performed to derive the synthesis and consumption rates of reducing agent NADH,and the change of electron transfer.It was found that NADH metabolism varies significantly with the electron acceptors tested.The results provide a theoretical basis for the future metabolic engineering of Shewanella and experimental evidences that can be used for future study of different phenotypes unaccounted for in previous models of Shewanella sp.MR-4 and MR-1.