Journal
METABOLIC ENGINEERING
Volume 4, Issue 2, Pages 124-137Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/mben.2001.0209
Keywords
NMR; glucose transport; phosphoenolpyruvate; central carbon metabolism; metabolic engineering
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We have developed Escherichia coli strains that internalize glucose utilizing the GaIP 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. Baez et al., 200 1, 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. C-13 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. (C) 2002 Elsevier Science (USA).
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