Metabolomic and proteomic analyses of a quiescent
Escherichia coli cell factory reveal the mechanisms behind
its production efficiency
Nicholas M. Thomson, Tomokazu Shirai, Marco Chiapello, Akihiko Kondo, Krishna
J. Mukherjee, Easan Sivaniah, David K. Summers and Keiji Numata
Supplementary figures
Figure S1: Individual principal component analyses for each time point of the
metabolites analyzed by LC-MS/MS. Points represent individual fermentations (n =
4) and ellipses represent 95% confidence intervals.
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Figure S2: Individual principal component analyses for each time point of the
metabolites analyzed by GC-MS. Points represent individual fermentations (n = 4)
and ellipses represent 95% confidence intervals.
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Figure S3: Overview of changes in the concentration of metabolites in glycolysis.
Concentrations were measured semi-quantitatively by LC-MS/MS (peak area relative
to an internal control) following addition of 3 mM indole (solid symbols) or 0.3%
ethanol (open symbols) to E. coli W3110 wild-type or hnsΔ93 cultures. Intracellular
lactate is also shown here as it was detected simultaneously and represents an
alternative end-point for glucose metabolism. Concentrations are normalized by the
OD600 at the time at which the sample was taken. Concentrations were normalized by
the OD600 at the time at which the sample was taken, mean-centered and Pareto scaled
to generate relative concentration changes. Error bars represent standard error (n = 4).
3
Figure S4: Overview of changes in the concentration of metabolites in the
pentose phosphate pathway. Concentrations were measured semi-quantitatively by
LC-MS/MS (peak area relative to an internal control) following addition of 3 mM
indole (solid symbols) or 0.3% ethanol (open symbols) to E. coli W3110 wild-type or
hnsΔ93 cultures. For metabolites with no graph shown, the concentration was too low
to detect or the metabolite was not able to be detected by our system. Concentrations
were normalized by the OD600 at the time at which the sample was taken, meancentered and Pareto scaled to generate relative concentration changes. Error bars
represent standard error (n = 4).
4
Figure S5: Overview of changes in the concentration of metabolites in the
tricarboxylic acid (TCA) cycle. Concentrations were measured quantitatively by
GC-MS following addition of 3 mM indole (solid symbols) or 0.3% ethanol (open
symbols) to E. coli W3110 wild-type or hnsΔ93 cultures. For metabolites with no
graph shown, the concentration was too low to detect or the metabolite was not able
to be detected by our system. Concentrations were normalized by the OD600 at the
time at which the sample was taken, mean-centered and Pareto scaled to generate
relative concentration changes. Error bars represent standard error (n = 4).
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Figure S6: Overview of changes in the concentration of cofactors involved in
central carbon metabolism. Concentrations were measured semi-quantitatively by
LC-MS/MS (peak area relative to an internal control) following addition of 3 mM
indole (solid symbols) or 0.3% ethanol (open symbols) to E. coli W3110 wild-type or
hnsΔ93 cultures. NADH was discounted from the analysis because the concentrations
were below the detectable limit. Concentrations were normalized by the OD600 at the
time at which the sample was taken, mean-centered and Pareto scaled to generate
relative concentration changes. Error bars represent standard error (n = 4).
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Figure S7: Pathway introduced to E. coli W3110hnsΔ93 to enable the production
of 3-hydroxybutyrate (3HB). Intermediates of the glycolysis pathway that would
otherwise have accumulated as described in the main text were instead channelled
towards 3HB production via acetyl-CoA. This was achieved by the introduction, on
the plasmid pTrctesBphaAB, of genes for the (over-) expression of a β-ketothiolase
(PhaA) and an acetoacetyl-Coenzyme A (-CoA) reductase (PhaB) from Cupriavidus
necator, and thioesterase B (TesB) from E. coli. PhaA condenses two molecules of
acetyl-CoA to form acetoacetyl-CoA. This is then reduced by PhaB before the CoA
moiety is removed by TesB to leave 3HB.
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Supplementary table
Table S1. 13C flux analysis in exponentially growing E.coli W3110 wild-type and
hnsΔ93 mutant strains.
Glycolysis (%)
W3110 wt
78.5
W3110hnsΔ93 78.1
PP Pathway (%)
21.0
21.1
ED Pathway (%) Total (%)
0.5
100
0.8
100
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