title: Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy.
abstract: 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.
authors: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, 62250 Cuernavaca, México.
authors: S
authors: S
authors: Flores
authors: G
authors: G
authors: Gosset
authors: N
authors: N
authors: Flores
authors: A A
authors: AA
authors: de Graaf
authors: F
authors: F
authors: Bolívar
mesh_terms: D001698:Biomedical Engineering; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005941:Glucokinase / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D026901:Membrane Transport Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010427:Pentose Phosphate Pathway; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; C043910:galactose permease; D002244:Carbon; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005941:Glucokinase; D005947:Glucose