TAILIEUCHUNG - Báo cáo khoa học: Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS

We describehere anovelmethodology for rapiddiagnosis of metabolic changes,which is basedonprobabilistic equations that relate GC-MS-derived mass distributions in proteino-genic aminoacids toin vivoenzyme activities. Thismetabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13 C] and [U-13 C]glucose experiments. | Eur. J. Biochem. 270 880-891 2003 FEBS 2003 doi Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS Eliane Fischer and Uwe Sauer Institute of Biotechnology ETH Zurich Zurich Switzerland We describe here a novel methodology for rapid diagnosis of metabolic changes which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteino-genic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from 1-13C and U-13C glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore several mutants exhibited additional unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently the low in vivo activity of the Entner- Doudoroff pathway in wild-type E. coli increased up to a contribution of 30 to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption. Keywords Entner-Doudoroff pathway flux analysis fluxome METAFoR analysis pentose phosphate pathway. Comprehensive and quantitative understanding of biochemical reaction networks requires not only knowledge about their .

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