Poster

iGEM Bielefeld-CeBiTec 2014

Abstract

Ecological power management is currently facing three challenges: the storage of electrical power, the development of renewable energy resources, which are not timely and locally synced with demand, and the increase of atmospheric carbon dioxide due to the use of fossil fuels. We address these challenges by implementing a proof of concept production of the biofuel isobutanol from carbon dioxide. In our project, a bacterial microcompartment from cyanobacteria, the carboxysome, was deployed in Escherichia. coli for carbon dioxide fixation under aerobic conditions. We further engineered E. coli to derive the energy for this process from electricity by implementing electron-mediator uptake and recycling. This ‘electricity’ pathway comprised proteins such as the furmarate reductase. Feasibility was analyzed in a self-constructed bioreactor. Ultimately, we established an isobutanol production pathway by heterologous expression of five genes from E. coli, Bacillus subtilis and Lactococcus lactis.
Universität Bielefeld
References
Atsumi S et al., Nature 451, pp. 86–89, 2008.
Bonacci et al., Proceedings of the National Academy of Sciences, vol. 2, pp. 478 – 483, 2012.
Parikh et al., Protein Engineering, Design & Selection, vol. 19, pp. 113-119, 2006.
Park et al., Journal of Bacteriology, vol. 181, pp. 2403-2410, 1999.
Stolzenberger et al., Journal of Bacteriology, vol. 195, pp. 5112 - 5122, 2013.
Acknowledgements
Prof. Dr. Jörn Kalinowski
Dr. Christian Rückert
Manuel Wittchen
Nils Lübke
Timo Wolf
Working groups
Fermentation Technology
Microbial Genomics and Biotechnology
Cellular and molecular Biotechnology
RE = redox equivalent Med = mediator
RE
Medox
Medred
a
n
o
d
e
c
a
t
h
o
d
e
CO2
product synthesis
-
-
-
2H2O
O2
4H+
CO2 xation
ATP
respiratory chain
isobutanol
biofuel
biofuel
biofuel
Figure 1: Schematic illustration of our project.
BIELEFELD 2014
GEM
.
Within our project we aim to produce isobutanol by using
electricity for the generation of redox and energy equivalents and
carbon dioxide as a carbon source. In Escherichia coli this task is
seperated into three parts shown below. In addition we
developed an antiobiotic-free selection system shown on the
right.
Abstract
Beside public events we cooperated
with SYNENERGENE.
According to the problem analysis we
developed several application
scenarios and technomoral vignettes
during our project. This enabled us to
have another view on our project and
led to adjustments during the wet lab
work.
Policy and Practices
Fixation of Carbon Dioxide (CO2)
Eight of the eleven enzymes involved in the Calvin cycle already exist in E. coli (Figure
6). For the CO2 xation the phosphoribulokinase (PrkA), the ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO) and the sedoheptulose-1,7-bisphosphatase
(SBPase) need to be expressed heterologous. The activity of the RuBisCO could be
validated in vitro (Figure 7).
rMFC
biofuel
CO2
biofuel
CO2
biofuel
CO2
CO2
CO2
biofuel
biofuel
biofuel
CO2
Figure 2: Application scenario
for a wind power station.
Antibiotic Free Selection
An antibiotic-free selection system was implemented by using
the complementation of the D-alanine auxotrophy in the
E. coli strain DH5α ∆alrdadX. It turned out that the
transformation eciency is about three times higher
compared to the classical selection with chloramphenicol
(Cm). Figure 12: Comparision of the
transformation eciency.
Isobutanol Production
biofuel
The aim was the production of an industrially relevant
product. We decided to implement the isobutanol
production pathway (Figure 10).
The steps in the conversion of pyruvate to
2-ketoisovalerate can be executed by proteins
existing in E. coli (IlvIH, IlvC and IlvD). The native
protein IlvIH is replaced by the AlsS from Bacillus
subtilis to increase the isobutanol production and the
AdhA from Lactococcus lactis is used.
With our
approach we achieved a production of about 56 mg
isobutanol per liter medium.
The carboxysome is
a protein-enveloped
microcompartment
encapsulating the
RuBisCO and the
carbonic anhydrase.
The advantage of the
microcompartment is
the concentration of carbon dioxide in its lumen,
which allows ecient carbon dioxide xation
under aerobic growth conditions (Figure 8).
D-ribulose 1,5-bisphosphate
(RBP)
HCO3
-
RBP
2x
3-phosphoglycerate
HCO3
-
HCO3
-
CO2
RuBisCO
CO2
carbonic
anhydrase (CA)
carboxysome
RBP
Figure 8: Mechanism of CO2 xation
within the carboxysome in E. coli.
The correct carboxysome assembly was veried
using a translational fusion of one shell protein
coding sequence
with gfp (Figure 9).
Figure 6: Reactions of the Calvin cycle.
Figure 9: Fluorescence images taken
through a structured illumination
microscope. Expression of E. coli KRX with
dierent plasmids, encoding the
carboxysome, constitutive gfp expression
and the carboxysome lacking the essential
shell associated protein CsoS2.
reverse Microbial Fuel Cell
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n
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e
-
-
-
Our aim was to generate redox equivalents by supplying electrons from the
cathode through mediators like neutral red or bromphenol blue. To engineer
an electrophilic strain by enabling the cells for electron uptake, various genetic
modications (Figure 3) needed to be implemented.
To carry out our experiments we
designed a reverse microbial fuel cell
(rMFC). Such a system (Figure 4) is
suitable for the investigation of mediator
redox-characteristics and indirect
electron transfer into electrotrophes.
To evaluate the electron
uptake we used a
potentiostat for sensitive
measurements.
Figure 4: Our electrobiochemical reactor system.
Figure 5:
Constructed E. coli strain ΔdcuB::oprF with
expressed fumarate reductase (Frd;
BBa_K1465102) shows a higher electron
uptake by cultivation in the
electrobiochemical reactor compared to the
E. coli wild type.
Figure 10: Isobutanol production pathway.
pyruvate
2-acetolactate
2,3-dihydroxyisovalerate
2-ketoisovalerate
isobutyraldehyde
isobutanol
IlvC - Ketol-acid reductoisomerase
Escherichia coli
IlvD - Dihydroxyacid dehydratase
Escherichia coli
KivD - α-ketoisovalerate decarboxylase
Lactococcus lactis
AdhA - Alcoholdehydrogenase
Lactococcus lactis
AlsS - α-acetolactate synthase
Bacillus subtilis
Figure 11:
Our designed constructs
and the maximum specic
production rate of cultures
carrying them.
Additionally the maximum
amount of produced
isobutanol is visualized.
The dynamic modeling approach containing ordinary
dierential equations indicated possible optimizations.
Stronger expression of kivD and adhA could improve
product synthesis (Figure 12).
Figure 12: Predicted changes in the concentration of the metabolites over time.
0 2 4 6 8 10 12
0
2
4
6
8
10
time [h]
concentration [mM]
pyruvate
2-acetolactate
2,3-dihydroxyisovalerate
2-ketoisovalerate
isobutyraldehyde
isobutanol
IlvC
IlvD
KivD
AdhA
AlsS
Annika Fust1, Birte Hollmann1, Boas Pucker1, David Wollborn1, Janina Tiemann1, Julian Droste1, Sandra Brosda1, Sebastian Blunk1,Simon Riedl1, Tore Bleckwehl1; 1CeBiTec Bielefeld
Figure 7: Decrease of the substrate RBP
(blue) and an increase of the product
3-phosphoglycerate (red) over time course.
biofuel
CO2
c
a
t
h
o
d
e
-
-
-
NRRed
NROx
Sdh
FAD+
FADH2
DcuB
Frd
succinate
fumarate
oprF
dcuB frd
Citric acid cycle
Sdh
Sdh
DcuB
DcuB
Sdh
DcuB
DcuB
Sdh
DcuB
Frd
OprF
outer membrane inner membrane
NR = neutral red OprF = porine Frd= fumarate reductase Sdh = succinate dehydrogenase DcuB = C4-dicarboxylate antiporter
0.0
0.5
1.0
1.5
2.0
specific electron uptake
E. coli wild type electrophilic E. coli strain
[1020 electrons g-1 CDW]
Figure 3: Principle of electron transfer to E. coli. The mediator (neutral red) is reduced at the
cathode and crosses the outer membrane through constitutive expressed outer membrane
porine (OprF). It serves as an electron donor for the fumarate reductase (Frd). Fumarate is reduced
into succinate, which would be reoxidized again by the succinate dehydrogenase (Sdh). In this
step FADH2 is restored. The antiporter dcuB is knocked out to avoid any succinate export.
CO
Decrease of the substrate RBP
(blue) and an increase of the product
3-phosphoglycerate (red) over time course.
RuBisCO
ribulose-5P
ribulose-1,5P2
glycerate-3P
phosphoribulokinase
sedoheptulose-
1,7-bisphosphatase
fructose-6P
glyceraldehyde-3P
pyruvate
CO2
xylose
heterologous
E. coli
+ adhA
- adhA
max. specic production rate
normalized to the OD600 [mg/(l·h)]
0 1 2 3 4
RBS alsS RBS ilvC RBS ilvD RBS kivD
Ptac RBS adhA
Ptac RBS alsS RBS ilvC RBS ilvD RBS kivD
- adhA:
+ adhA:
+ adhA
- adhA
max. amount of
isobutanol [mg/l]
0 20 40 60
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