ISSN 0003-6838, Applied Biochemistry and Microbiology, 2008, Vol. 44, No. 2, pp. 151–157. © Pleiades Publishing, Inc., 2008. Original Russian Text © A.D. Kivero, E.V. Bocharov, V.G. Doroshenko, A.G. Sobol, M.A. Dubinnyi, A.S. Arseniev, 2008, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 168–175. 151 1 In current basic and applied studies of bacterial metabolism, attention is focused on comprehensive analysis of metabolic pathways rather than expression of particular genes or activity of corresponding pro- teins. In recent years, new biochemical methods have been developed to study metabolism as a whole: enzy- matic activity assays in cells [1, 2], determination of gene expression levels [3], and study of pathways of labeled substrate ( 13 ë, 14 ë, 15 N, 3 H, and 31 P) utilization [4–7]. Three major directions can be recognized in approaches to the determination of quantitative cell metabolism indices by using labels and NMR or chro- matomass spectroscopy: measurement of intermediate pools and modeling metabolism on these grounds [7– 9], in vivo measurement of the level of labeling of var- ious compounds obtained from cells with simultaneous modeling of carbon fluxes and correction of the chosen model [10], and measurement of distribution of labeled atoms in proteinogenic amino acids derived from one glucose molecule as a sole carbon source and determi- nation of the metabolic pathways of these atoms in vitro [5, 11–14]. The first approach allows the study of spe- cific biosynthesis pathways for compounds whose 1 Abbreviations: α-kG, α-ketoglutarate; AcCoA, acetyl coenzyme A; Asp, aspartate; HCS, heteronuclear correlation spectroscopy; Gly, glycine; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6- phosphate; HOCS, heteronuclear one-quantum correlation spec- troscopy; HTCS, heteronuclear total correlation spectroscopy; OA, oxaloacetate; Pyr, pyruvate; PPP, pentose phosphate path- way; R5P, ribose-5-phosphate; Ser, serine; Suc, succinate; PEP, phosphoenol pyruvate; F6P, fructose-6-phosphate; Fum, fuma- rate; TAC, tricarboxylic acid cycle; EDP, Enther–Doudoroff path- way; E4P, erythrose-6-phosphate. pools have been measured. It can be applied only to the description of cells at the stationary phase but provides no information on general pathways of central cell metabolism. The second approach provides informa- tion on pathways involving reactions whose equations were included into the model but does not determine the contributions of various metabolic pathways in cases of identical rearrangements of carbon backbone and, correspondingly, identical distributions of labeled atoms. The third approach allows carbon flux measure- ments only at several nodal points of central metabo- lism and only during intense biomass growth. All of the three approaches are used for construction of industrial producers of biologically active compounds. Combina- tion of elements of all of the three methods can add to the understanding of general cell metabolism. We chose the third method [5], because combination of all of the three methods presents too much technical difficulty. The objectives of the present study are (1) to deter- mine the quantitative distribution of carbon fluxes in glycolysis, pentose phosphate pathway (PPP), and Enther–Doudoroff pathway (EDP) and (2) to investi- gate the effects of major glucose utilization pathways on Escherichia coli physiological indices. MATERIALS AND METHODS Bacterial strains and growth conditions. Experi- ments were carried out with laboratory E. coli strain K12 MG1655 and its derivatives bearing deletions ∆edd-eda, ∆zwf-edd-eda, and ∆pgi + ∆edd-eda. MG1655 is a wild-type E. coli strain, whose chromosomal DNA had been completely sequenced. 2D [ 1 H, 13 C] NMR Study of Carbon Fluxes during Glucose Utilization by Escherichia coli MG1655 A. D. Kivero a , E. V. Bocharov b , V. G. Doroshenko a , A. G. Sobol b , M. A. Dubinnyi b , and A. S. Arseniev b a Ajinomoto-Genetika Research Institute, Moscow, 117545 Russia e-mail: kiveroad@rambler.ru b Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia Received January 24, 2007 Abstract—Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells—glycolysis, pentose phosphate pathway (PPP), and Enther–Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655∆(edd-eda), MG1655∆(zwf, edd-eda), and MG1655∆(pgi, edd- eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation. DOI: 10.1134/S000368380802004X