1. Introduction
Clostridium thermocellum is an anaerobic
thermophilic bacteria that is able to degrade
cellulosic biomass and convert it to biofuels.
This process, however, is not as fast as it
needs to be for industrial use.
The metabolic pathway must be
engineered in order to increase the rate
of conversion by making the change in
free energy (∆G) more negative at key
steps in the pathway.
The key step that this work focuses is the
conversion of phosphoenolpyruvate (PEP)
to pyruvate.
Materials and methods
Three mutant strains of C. thermocellum
were previously engineered, each with
differently altered pathways for the
conversion of PEP to pyruvate.
The three mutant strains (LL1138, LL1163
and LL1251) and the wild type strain
(LL345) of C. thermocellum were cultured
on minimal media at 55˚ C. C-13 labelled
acetate was used to measure the ratio of
forward progression and backward
progression of the reactions (J+/J-).
Escherichia coli grown on C- 13 labelled
glucose was used as an internal standard to
measure relative metabolite concentration
via LC/MS.
From this data, ∆G can be calculated using
the equation: ∆G = ∆G˚’ + RT ln Q = -RT
ln(J+/J-)
Acknowledgments
This research was made possible
by University of Wisconsin –
Madison and the IBS-SRP program,
funded by the NSF. Thanks to Colin
Purrington for the poster template.
Research Goal: Analyze the metabolism of mutant C. thermocellum strains to investigate how the changes affected
the phenotype.
Results
Discussion
Glycolytic Reversibility
Contrary to most organisms, in which
the reactions of upper glycolysis display
essentially 100% forward flux, the
reactions in the EMP pathway of C.
thermocellum display some extent of
reverse flux. This is particularly unique
in the uppermost reactions of
glycolysis[3].
Secret Pathways
In LL1251 the only way for labeled
carbon to make its way back through the
pathway to oxaloacetate is in reverse
from pyruvate to PEP and then forward
to oxaloacetate. Because the enzyme
responsible for PEP-> pyruvate (PYK) in
LL1251 is relatively less reversible than
those used in other strains (PPDK or
PYK and PPDK together), it would be
expected for less label to be found in
oxaloacetate in LL1251. This however is
not the case [4]. This suggests that there
are additional pathways present in this
mutant involving these metabolites that
have yet to be elucidated.
Increase GDP and GTP in LL1163
Compared to the other strains, there is a
considerably higher relative
concentration of GDP and GTP in
LL1163[5]. This is particularly of
interest because GTP production is
linked to conversion of PEP to
oxaloacetate.
Jordan Brown1, Dave Stevenson2, Daniel Amador-Noguez2
1-Department of Botany-Microbiology (Genetics), Ohio Wesleyan University
2-Department of Bacteriology, University of Wisconsin-Madison
References
1) Park, Junyoung O., Sara A. Rubin, Yi-Fan Xu, Daniel
Amador-Noguez, Jing Fan, Tomer Shlomi, and Joshua D.
Rabinowitz. "Metabolite Concentrations, Fluxes and Free
Energies Imply Efficient Enzyme Usage." Nature Chemical
Biology Nat Chem Biol (2016): n. pag. Print.
2) Flamholz, A., E. Noor, A. Bar-Even, W. Liebermeister, and
R. Milo. "Glycolytic Strategy as a Tradeoff between Energy
Yield and Protein Cost." Proceedings of the National
Academy of Sciences 110.24 (2013): 10039-0044. Print.
3) Olsen, Daniel G., Manuel Hörl, Tobias Fuhrer, Jingxuan
Cui, Marybeth I. Mahoney, Daniel Amador-Noguez, Liang
Tian, Uwe Sauer, and Lee R. Lynd. "Conversion of
Phosphorenolpyruvate to Pyruvate in Clostridium
Thermocellum." *Currently under Review and Not Yet
Published (n.d.): n. pag. Print.
4) Chinn, Mari, and Veronica Mbaneme. "Consolidated
Bioprocessing for Biofuel Production: Recent Advances."
EECT Energy and Emission Control Technologies (2015): 23.
Web.
Further Research
C. thermocellum contains the gene for
oxaloacetate decarboxylase (ODC), an
enzyme that catalyzes the conversion of
oxaloacetate to pyruvate. However, there
is no ODC expression in wild type C.
thermocellum. It would be of use to
investigate this, and attempt to express
this gene to create a novel and
potentially more efficient pathway for
the PEP to pyruvate conversion.
Gibbs Energy
Concentration Flux Ratio
Figure 1: The relation between free energy, concentration and flux ratio1
Figure 2: Different PEP to pyruvate pathways in the different strains3
70%
75%
80%
85%
90%
95%
100%
345 1138 1163 1251
Aspartate Reversibility
4
3
2
1
0
84%
86%
88%
90%
92%
94%
96%
98%
100%
345 1138 1163 1251
3PG Reversibility
3
2
1
0
Figure 4: Carbon isotope ratios showing unexpectedly high amounts of 2-labeled
carbon in metabolites in LL1251 (Aspartate used as a substitute for oxaloacetate)
0
0.1
0.2
0.3
0.4
0.5
0.6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Relative GDP Concentration
0
0.1
0.2
0.3
0.4
0.5
0.6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Relative GTP Concentration
Figure 5: Relative concentrations of GDP and GTP across the mutant strains
80%
82%
84%
86%
88%
90%
92%
94%
96%
98%
100%
345 F6P 1138 F6P 1163 F6P 1251 F6P
Fructose-6-Phosphate Reversibility
0-Labeled 1-Labelled 2-Labelled 3-Labelled 4-Labelled 5-Labelled 6-Labelled
80%
82%
84%
86%
88%
90%
92%
94%
96%
98%
100%
345 1138 1163 1251
Glucose-6-Phosphate Reversibility
0-Labeled 1-Labelled 2-Labelled 3-Labelled 4-Labelled 5-Labelled 6-Labelled
Figure 3: Carbon isotope ratios in the uppermost metabolites of glycolysis