by Kovaleva E. | Shepelin D. | Baboshin M. | Golubeva L. | Mashko S. | Ajinomoto-Genetika Research
Institute, 117545, Moscow, Russian Federation | Recent affiliation: Novo Nordisk Foundation Center for
Biosustainability, Technical University of Denmark, DK-2800 Lyngby, Denmark
Motivation and aim: 13C metabolic flux analysis (13C-MFA) is complex method for
quantitative estimation of actual carbon metabolic fluxes distribution in vivo.
There are still unsolved questions even about E. coli metabolism. By flux balance analysis
(FBA) of E. coli BL21 with 6-phosphogluconolactonase (pgl) deficient genotype,
investigators assume inactivation of oxidative branch of pentose phosphate pathway (oxPPP)
in this strain. But 13C-MFA of BL21 and MG1655 Δpgl demonstrated only insignificant
decrease of oxPPP flux. We hypothesized that flux through oxPPP is actually reduced, but
not to zero, in pgl mutant.
Methods and Algorithms:
13C-MFA of E. coli strains grown on minimal medium with
20%/80% mixture of [1-13C]- and [U-13C]-glucose. Flux calculation and statistical analysis of
calculated carbon flux distribution was provided by OpenFLUX2 software based on
proteinogenic amino acids and monosaccharides labeling data.
Results: Flux solution with the best accordance with experimental data for Δpgl mutant
corresponds to 18% oxPPP carbon flux of input glucose in contrast with 25% in parent
strain. Flux calculation under assumption that pgl inactivation blocks oxPPP resulted in
statistically unacceptable solutions. We determined that flux through PGL reaction
responded to χ2-criterion on minimal 10% level.
It is known that simultaneous oxPPP and pntAB genes inactivation resulted in drastic
decrease of growth rate caused by NADPH deficiency. Previously it was proposed that the
specific bypass through gluconate can functioning in Δpgl mutant. Simultaneous
inactivation of major gluconate importers reduces growth of Δpgl and ΔpglΔpntAB mutants
on glucose. Gluconate bypass can participate in oxPPP flux keeping in pgl deficient strain.
Conclusions: Proposal of inactive oxPPP in pgl deficient E. coli mutant contradicts to 13CMFA of carbon flux distribution in MG1655Δpgl strain and to growth characteristics of double mutants, with inactivated pntAB and oxPPP genes. According to 13C-MFA the flux
through the oxPPP decreases by no more than 2.5-times in the MG1655Δpgl strain.