Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy

We have developed Escherichia coli strains that internalize glucose utilizing the GalP permease instead of the phosphoenolpyruvate:carbohydrate phosphotransferase system. It has been demonstrated that a strain with these modifications (PTS(-)Glc(+)) can direct more carbon flux into the aromatic pathway than the wild-type parental strain (N. Flores et al., 1996, Nat. Biotechnol. 14, 620-623; G. Gosset et al., 1996, J. Ind. Microbiol. 17, 47-52; J. L. Baéz et al., 2001, Biotechnol. Bioeng. 73, 530-535). In this study, we have determined and compared the carbon fluxes of a wild-type strain (JM101), a PTS(-)Glc(-) strain, and two isogenic PTS(-)Glc(+) derivatives named PB12 and PB13 by combining genetic, biochemical, and NMR approaches. It was determined that in these strains a functional glk gene in the chromosome is required for rapid glucose consumption; furthermore, glucokinase-specific activities were higher than in the wild-type strain. (13)C labeling and NMR analysis allowed the determination of differences in vivo which include higher glycolytic fluxes of 93.1 and 89.2% compared with the 76.6% obtained for the wild-type E. coli. In PB12 and PB13 we found a flux through the malic enzymes of 4 and 10%, respectively, compared to zero in the wild-type strain. While flux through the Pck enzyme was absent in PB12 and PB13, in the wild type it was 7.7%. Finally, it was found that in the JM101 and PB12 strains both the oxidative and the nonoxidative branches of the pentose phosphate pathway contributed to ribose 5-phosphate synthesis, whereas in PB13 this pentose was synthesized almost exclusively through the oxidative branch. The determined carbon fluxes correlate with biochemical and genetic characterizations.