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A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress.

ABSTRACT: BACKGROUND: The cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed. RESULTS: Flux analysis based on nuclear magnetic resonance (NMR) and mass spectroscopy (MS) measurements and computation provided quantitative results on the metabolic fluxes redistribution of the E. coli central carbon network under paraquat-induced oxidative stress. The metabolic fluxes of the glycolytic pathway were redirected to the pentose phosphate pathway (PP pathway). The production of acetate increased significantly, the fluxes associated with the TCA cycle decreased, and the fluxes in the glyoxylate shunt increased in response to oxidative stress. These global flux changes resulted in an increased ratio of NADPH:NADH and in the accumulation of ?-ketoglutarate. CONCLUSIONS: Metabolic flux analysis provided a quantitative and global picture of responses of the E. coli central carbon metabolic network to oxidative stress. Systematic adjustments of cellular physiological state clearly occurred in response to changes in metabolic fluxes induced by oxidative stress. Quantitative flux analysis therefore could reveal the physiological state of the cell at the systems level and is a useful complement to molecular systems approaches, such as proteomics and transcription analyses.

SUBMITTER: Rui B

PROVIDER: S-EPMC2944137 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress.

Rui Bin B Shen Tie T Zhou Hong H Liu Jianping J Chen Jiusheng J Pan Xiaosong X Liu Haiyan H Wu Jihui J Zheng Haoran H Shi Yunyu Y

BMC systems biology 20100901

<h4>Background</h4>The cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reacti ...[more]

PMID: 20809933

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Project description:Beta (?)-carotene (C40H56; a provitamin) is a particularly important carotenoid for human health. Many studies have focused on engineering Escherichia coli as an efficient heterologous producer of ?-carotene. Moreover, several strains with potential for use in the industrial production of this provitamin have already been constructed via different metabolic engineering strategies. In this study, we aimed to improve the ?-carotene-producing capacity of our previously engineered E. coli strain ZF43?gdhA through further gene deletion and metabolic pathway manipulations. Deletion of the zwf gene increased the resultant strain's ?-carotene production and content by 5.1 and 32.5%, respectively, relative to the values of strain ZF43?gdhA, but decreased the biomass by 26.2%. Deletion of the ptsHIcrr operon further increased the ?-carotene production titer from 122.0 to 197.4 mg/L, but the provitamin content was decreased. Subsequently, comparative transcriptomic analysis was used to explore the dynamic transcriptional responses of the strains to the blockade of the pentose phosphate pathway and inactivation of the phosphotransferase system. Lastly, based on the analyses of comparative transcriptome and reduction cofactor, several strategies to increase the NADPH supply were evaluated for enhancement of the ?-carotene content. The combination of yjgB gene deletion and nadK overexpression led to increased ?-carotene production and content. The best strain, ECW4/p5C-nadK, produced 266.4 mg/L of ?-carotene in flask culture and 2,579.1 mg/L in a 5-L bioreactor. The latter value is the highest reported from production via the methylerythritol phosphate pathway in E. coli. Although the strategies applied is routine in this study, the combinations reported were first implemented, are simple but efficient and will be helpful for the production of many other natural products, especially isoprenoids. Importantly, we demonstrated that the use of the methylerythritol phosphate pathway alone for efficient ?-carotene biosynthesis could be achieved via appropriate modifications of the cell metabolic functions.

Project description:BackgroundSystems biology calls for studying system-level properties of genes and proteins rather than their individual chemical/biological properties, regarding the bio-molecules as system components. By characterizing how critical the components are to the system and classifying them accordingly, we can study the underlying complex mechanisms, facilitating researches in drug target selection, metabolic engineering, complex disease, etc. Up to date, most studies aiming at this goal are confined to the topology-based or flux-analysis approaches. However, proteins have tertiary structures and specific functions, especially in metabolic systems. Thus topological properties such as connectivity, path length, etc., are not good surrogates for protein properties. Also, the manner of individual sensitivity analysis in most flux-analysis approaches cannot reveal the simultaneous impacts on collateral components as well as the overall impact on the system, thus lacking in system-level perspective.ResultsIn the present work, we developed a method to directly assess protein system-level properties based on system dynamics and in silico knockouts, regarding to the conceptual term "criticality". Applying the method to E. coli central carbon metabolic system, we found that multiple enzymes including phosphoglycerate kinase, enolase, transketolase-b, etc., had critical roles in the system in terms of both system states and dynamical stability. In contrast, another set of enzymes including glucose-6-phosphate isomerise, pyruvate kinase, phosphoglucomutase, etc., exerted very little influences when deleted. The finding is consistent with experimental characterization of metabolic essentiality and other studies on E. coli gene essentiality and functions. We also found that enzymes could affect distant metabolites or enzymes even greater than a close neighbour and asymmetry in system-level properties of enzymes catalyzing alternative pathways could give rise to local flux compensation.ConclusionsOur method creates a different angle for evaluating protein criticality to a biological system from the conventional methodologies. Moreover, the method leads to consistent results with experimental references, showing its efficiency in studying protein system-level properties. Besides working on metabolic systems, the application of the method can be extended to other kinds of bio-systems to reveal the constitutive/functional properties of system building blocks.

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A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress.

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A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress.

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