title: ExaminingEscherichia coliglycolytic pathways, catabolite repression, and metabolite channeling using Δpfkmutants.
abstract: BACKGROUND
  Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However,Escherichia colido not use their native EDP for glucose metabolism.
  
  
  RESULTS
  Overexpression ofeddandedainE. colito enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressingeddandedain this ΔpfkAmutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via thepfkAdeletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkAmutant failed to improve its growth rates due to metabolic burden. However, the reorganization ofE. coliglycolytic strategies did reduce glucose catabolite repression. The ΔpfkAmutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkAmutant repressed acetate flux toward gluconeogenesis. Moreover,13C-pulse experiments in the ΔpfkAmutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium).
  
  
  CONCLUSIONS
  We engineeredE. colito redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improveE. coliglucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO USA.
authors.forename: Whitney D
authors.initials: WD
authors.lastname: Hollinshead
authors.affiliation: Sandia National Laboratory, Livermore, CA USA ; Joint BioEnergy Institute, Emeryville, CA USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Rodriguez
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA.
authors.forename: Hector Garcia
authors.initials: HG
authors.lastname: Martin
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Sale
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA ; California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA USA ; Department of Bioengineering, University of California, Berkeley, CA USA ; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA USA ; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé, DK2970 Hørsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA USA ; Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA USA.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO USA.
authors.forename: Yinjie J
authors.identifier: 0000-0002-5112-0649
authors.initials: YJ
authors.lastname: Tang
keywords: 13C; Catabolite repression; Channeling; EMP; Metabolic modeling; Synthetic biology; Xylose