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Metabolic Engineering 13 (2011) 76–81
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Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben
Manipulating redox and ATP balancing for improved production
of succinate in E. coli
Amarjeet Singh a, Keng Cher Soh b, Vassily Hatzimanikatis b, Ryan T. Gill a,n
a
b
Department of Chemical and Biological Engineering, University of Colorado, UCB 424, Boulder, CO 80309, USA
Laboratory of Computational Systems Biotechnology, Ecole Polytechnique Federale de Lausanne (EPFL), CH 1015 Lausanne, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 October 2010
Accepted 21 October 2010
Available online 30 October 2010
Redox and energy balance plays a key role in determining microbial fitness. Efforts to redirect bacterial
metabolism often involve overexpression and deletion of genes surrounding key central metabolites, such
as pyruvate and acetyl-coA. In the case of metabolic engineering of Escherichia coli for succinate
production, efforts have mainly focused on the manipulation of key pyruvate metabolizing enzymes.
E. coli AFP111 strain lacking ldhA, pflB and ptsG encoded activities accumulates acetate and ethanol as well
as shows poor anaerobic growth on rich and minimal media. To address these issues, we first deleted
genes (adhE, ackA-pta) involved in byproduct formation downstream of acetyl-CoA followed by the
deletion of iclR and pdhR to activate the glyoxylate pathway. Based on data from these studies, we
hypothesized that the succinate productivity was limited by the insufficient ATP generation. Genomescale thermodynamics-based flux balance analysis indicated that overexpression of ATP-forming PEPCK
from Actinobacillus succinogenes in an ldhA, pflB and ptsG triple mutant strain could result in an increase in
biomass and succinate flux. Testing of this prediction confirmed that PEPCK overexpression resulted in a
60% increase in biomass and succinate formation in the ldhA, pflB, ptsG mutant strain.
& 2010 Elsevier Inc. All rights reserved.
Keywords:
Escherichia coli
Redox ratio
NADH/NAD +
Succinate
Fumarate
Metabolic engineering
NZN111
AFP111
1. Introduction
Escherichia coli performs mixed acid fermentation in the
absence of exogenous electron acceptors (Neidhardt and Curtiss,
1996). The primary fermentation products are acetate, lactate and
formate, while ethanol and succinate are formed in minor quantities (Gupta and Clark, 1989; Matjan et al., 1989). Pyruvate serves
as an important branch point for carbon flux distribution (Arita,
2004; Fell and Wagner, 2000). In wild-type strains of E. coli growing
anaerobically, most of the pyruvate flux is handled by two primary
enzymes of fermentative metabolism—lactate dehydrogenase
(ldhA) and pyruvate-formate lyase (pflB); accounting for the
production of acetate, ethanol, formate and lactate. E. coli
NZN111 (DldhA, DpflB) was created to redirect the carbon flux
towards the formation of succinic acid. This mutant, however, is
incapable of growth on glucose in rich or minimal media under
anaerobic conditions (Bunch et al., 1997; Gupta and Clark, 1989;
Stols and Donnelly, 1997) and is known to accumulate high levels of
pyruvate and NADH (Vemuri et al., 2002a). Intracellular redox
ratios (NADH/NAD + ) as high as three times that of the wild-type
E. coli have been observed in NZN111 (Singh et al., 2009). The
inability to synthesize acetyl-coA and/or to regenerate NAD + via
n
Corresponding author: Fax: + 1 303 492 4341.
E-mail address: [email protected] (R.T. Gill).
1096-7176/$ - see front matter & 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymben.2010.10.006
pyruvate reduction are cited as the likely causes of the growth
defect of this strain (Stols and Donnelly, 1997; Stols et al., 1997).
A variety of metabolic engineering strategies including those
focused toward the overexpression of pyruvate metabolizing
enzymes have been pursued for improving succinate production in
E. coli (Chatterjee et al., 2001; Millard et al., 1996; Sanchez et al., 2005;
Stols and Donnelly, 1997; Vemuri et al., 2002a). Other strategies
include providing additional reducing power (Chatterjee et al., 2001;
Hong and Lee, 2002; VanderWerf et al., 1997), as well as creative
combinations of aerobic and anaerobic metabolism (Sanchez et al.,
2006; Vemuri et al., 2002a, b) to attain optimal succinate yields on
glucose by activating the glyoxylate pathway and the reductive TCA
cycle (Vemuri et al., 2002a). Evolutionary engineering strategies have
also been successful. By employing a dual metabolic engineering and
evolutionary approach, Jantama et al. (2008) constructed an E. coli
strain capable of overproducing succinic acid on minimal media in
single batch fermentation. A spontaneous ptsG mutant of NZN111,
with improved fitness and restored fermentation capability, was
reported by Donnelly et al. (1998) as E. coli AFP111. Inactivation of
ptsG is thought to increase the PEP pool, which is then diverted to the
reductive TCA cycle via the action of PEP-carboxylase (PPC)
(Chatterjee et al., 2001; Lin et al., 2005; Vemuri et al., 2002a). The
redistribution of the carbon flux resulting from the ptsG mutation in
AFP111, however, came at the cost of succinate yield. AFP111
produces succinate, ethanol and acetate in a 1:0.5:0.5 ratios, presumably to maintain redox cofactor balances (Chatterjee et al., 2001;
Donnelly et al., 1998). The succinate yield and productivity was
A. Singh et al. / Metabolic Engineering 13 (2011) 76–81
increased by the overexpression of the anaplerotic pyruvate carboxylase (pyc) gene from Rhizobium etli in AFP111, although the formation
of acetate and ethanol could not be completely eliminated (Vemuri
et al., 2002a, b). In the studies reported herein, we observed that
deletion of the acetate forming enzymes acetate kinase and phosphotransacetylase (ackA-pta) completely abolished growth in the initial
microaerobic phase in microaerobic–anaerobic dual-phase fermentation in our AFP111 equivalent strain AD32 (a pflB, ldhA, ptsG triple
mutant). PTA mutants have widely been reported to not grow under
anaerobic conditions and show reduced growth rates in aerobic
conditions (Wolfe, 2005). Formation of acetate also results in ATP
generation, which we suspected could be limiting anaerobic growth in
this strain as the formation of succinate via reductive TCA cycle does
not generate any ATP.
PEP is converted to OAA as the first step in succinate production. In
E. coli, this step is catalyzed by PEP-carboxylase (ppc), while in
Actinobacillus succinogenes, this step is catalyzed by the ATP-generating
PEP-carboxykinase (Kim et al., 2004; Laivenieks et al., 1997;
VanderWerf et al., 1997). Prior studies have shown that PEP-carboxylase overexpression can significantly enhance succinate production in
E. coli (Millard et al., 1996), while PEP-carboxykinase overexpression
only enhances succinate production in an E. coli ppc mutant(Kim et al.,
2004; Millard et al., 1996). These prior studies suggested that this is
explained by the roughly 100-fold lower Km towards bicarbonate of the
PEP-carboxylase relative to PEP-carboxykinase enzyme. These results
also suggested that the PEP to OAA reaction can operate under the
physiological conditions in E. coli, thus promoting this strategy for the
use in succinate production strains. Here, we further assessed the
feasibility of this reaction using Thermodynamics-based flux balance
analysis (TFBA) with the iAF1260 E. coli genome-scale metabolic model
(Feist et al., 2007b). The model confirmed the reaction could proceed in
the ATP-generating direction at elevated extracellular CO2 concentrations. The model also predicted higher biomass and succinate yields as
a result of increased ATP formation. Based on these data, we
investigated the effect of PEPCK overexpression in E. coli strains
engineered for succinic acid production. Overexpression of PEPCK
was indeed observed to improve growth, glucose consumption, and
succinic acid production in an ldhA, pflB and ptsG triple mutant strain.
To further improve succinate yield, we deleted the ethanol forming
alcohol dehydrogenase (adhE) enzyme and observed an increase in
succinate yield. However, byproduct formation could not be completely eliminated as the deletion of ackA-pta completely abolished
microaerobic growth.
77
University, New Haven, CT. Plasmid pAsPCK carrying the PEPCK gene
from A. succinogenes under the control of the inducible lac promoter
was provided by the Dr. C. Vielle’s laboratory at the Michigan State
University (MSU), Lansing, MI. E.coli phosphoenol-carboxylase (ppc)
was PCR amplified and cloned under the control of its natural promoter
into a low copy vector pSmart-LC-Kan from Lucigen Corp. The ASKA
clone plasmid carrying the E.coli PPC under the control of inducible lac
promoter (JW3928) was also utilized for the overexpression studies
(Kitagawa et al., 2005).
2.2. Microaerobic–anaerobic dual-phase fermentation
Single bacterial colonies were grown in a 15 ml plastic tube
containing 5 ml of Luria-Bertani (LB) medium until it reached its
exponential growth at an OD600 nm of 0.4–0.6. The culture was
then diluted to a new OD600 nm of 0.005–0.01 to be used as an
inoculum for the fermentation studies in LB medium supplemented
with 50 mM of sugar source. Glucose, sorbitol and gluconate were
purchased from the Fisher scientific. 10 g/L NaHCO3 was added to
the media for the dual purpose of carbon dioxide source and
buffering agent. The cells were grown in the absence of air
headspace by filling the culturing tubes with the media to the
top to enable rapid anaerobiosis. The cultures were grown at 37 1C
and at an agitation rate of 200 rpm. Separate tubes were run for
each data point to avoid oxygen exposure during sampling.
2.3. Analytical techniques
Cell growth was monitored by measuring the optical density
(OD) at 600 nm (UV–vis spectrophotometer, Shimadzu Corp.).
To analyze the fermentation culture, 1 mL aliquots were centrifuged at 13000 rpm for 2 min and the supernatant was filtered
through a 0.2 mm syringe filter. Samples were analyzed using HPLC
Agilent 1100 series Chemstation equipped with ICSep Coregel 64H
HPLC column (Transgenomics, Omaha, NE). 50 ml of sample was
loaded into the column operated at 50 1C and ran isocratically with
8 mM H2SO4 sat a flow rate of 0.6 ml/min. In the analysis, the
concentrations of organic acids such as succinate, lactate, acetate
and formate were quantified from the signals obtained at 210 nm
by the UV–vis detector (Eiteman and Chastain, 1997). The concentrations of the metabolites were determined using a standard
curve constructed using HPLC grade reagents purchased from
Sigma-Aldrich Co. Glucose concentrations were monitored by YSI
2700 Select Biochemistry Analyzer.
2. Materials and methods
2.1. Strains and plasmids
All mutations were made in E. coli BW25113 (D(araD-araB)567,
DlacZ4787(::rrnB-3), lambda , rph-1, D(rhaDrhaB)568, hsdR514).
Deletion strains were constructed following the method developed
earlier (Datsenko and Wanner, 2000). The kanamycin resistance
cassette was amplified from plasmid pKD13 by PCR using primers
with flanking homologous regions for the target gene. The purified PCR
product was electroporated into host E. coli strain harboring l-Red
recombinase induced off the plasmid pKD46 using 10 mM arabinose.
The resulting kanamycin resistant colonies were screened for the
desired gene knockout by the PCR amplification and subsequent
sequencing. Primers for this confirmation step were designed to bind
300–400 by upstream and downstream respectively of the target gene.
Plasmid pCP20 carrying the FLP-recombinase was subsequently used
to excise the kanamcin selection marker from the knockouts strain. All
plasmids were cured by propagating the strains at 43 1C before
preparing for the deletion of the next target gene. Strains and plasmid
stocks were obtained from the E. coli Genetic Stock Center at Yale
2.4. Thermodynamics-based flux balance analysis using iAF1260
E. coli genome-scale metabolic model
The latest genome-scale E. coli metabolic model, iAF1260 (Feist
et al., 2007a) was used to perform the analysis reported here. This
model was defined as the wild-type (WT), while the AD32 strain
was simulated by constraining the reactions for ldhA, pflB, ptsG to
zero. Thermodynamics-based Flux Balance Analysis (TFBA) generates thermodynamically feasible flux distributions in a given
metabolic network as compared with conventional Flux Balance
Analysis (FBA) which can produce distributions that violate
thermodynamics (Henry et al., 2007). A minimal set of metabolites
were allowed to be exported out of system (in this case: acetate,
formate, malate, hydrogen gas, ethanol, lactate and succinate). The
in-silico media was set to glucose minimal media under anaerobic
conditions. Model assumptions include (i) maintenance energy
remains the same as per aerobic conditions at 8.39 mmol-ATP/
gDW hr, (ii) biomass composition for anaerobic conditions simulated is the same as per aerobic condition, (iii) glucose uptake was
fixed at 10 mmol/gDW hr for ease of comparison of yield and
78
A. Singh et al. / Metabolic Engineering 13 (2011) 76–81
Table 1
Redox balance and the theoretical succinate and ATP yields for the engineered succinic acid production strains of E. coli.
Strains
AD12
AD32
AD32
AD216
AD483
AD346
AD568
AD725
a
b
c
Relevant deletions
ldhA,
ldhA,
ldhA,
ldhA,
ldhA,
ldhA,
ldhA,
ldhA,
pflB
pflB,
pflB,
pflB,
pflB,
pflB,
pflB,
pflB,
ptsG
ptsG, ppc
ptsG, ppc
ptsG, ppc, ackA-pta
ptsG, ppc, adhE
ptsG, ppc, adhE, ackA-pta
ptsG adhE, ack-ptA pdhR, iclR pAsPCK
PEPCK
No
No
No
Yes
Yes
Yes
Yes
Yes
Theoretical yield (mol / mol glucose)
Experimental data (g/L h)
ATP generation
Net NADH
Growth rate
Glucose
consumption
Succinate
generation
0
0.5
1
2
2
2
2
1.4
2a
0b
0
2c
2c
2c
2c
0
0.074
0.091
0.078
0.093
0.012
0.067
0.013
0.107
No growth
0.104
0.008
0.056
0
0.083
0.09
No growth
No growth
0.08
It is assumped that only the succinate pathway is active consistent with the reported data.
1:0.5:05 ratio for succinate:acetate:ethanol is assumed consistent with the reported experimental data.
It is assumed that the presence of PEPCK would drive the carbon flux solely through the succinate branch.
(iv) cytoplasmic pH was fixed at 7.2, with periplasmic and
extracellular pH fixed at 7.
3. Results and discussion
E. coli fitness is closely tied to its ability to balance reducing
equivalents across various pathways while simultaneously generating all required biosynthetic precursors. E. coli strains lacking ldhA
and pflB encoded activities suffer from an inability to synthesize
sufficient acetyl-coA, accumulation of pyruvate, and formation of
undesired by-products acetate and ethanol, all of which lead to
incomplete flux through the desired succinate pathway (Bunch
et al., 1997; Matjan et al., 1989; Stols and Donnelly, 1997; Vemuri
et al., 2002a). An additional mutation in the ptsG restores fermentative growth on glucose in complex media. This strain produces
succinate, acetate and ethanol in a molar ratio of 1:0.5:0.5. It is
thought that pyruvate dehydrogenase complex maintains a low
level of activity under laboratory anaerobic conditions, thus
enabling the conversion of pyruvate to acetyl-CoA. Ethanol is then
produced via adhE with acetate arising from ackA-pta. With a ratio of
1:0.5:0.5, this pathway is NADH balanced and nets 0.5 moles of
ATP/mol of glucose consumed (Table 1). To maximize succinate
yield, it is essential to remove byproduct formation; however, sole
production of succinate through the reductive TCA pathway is
neither NADH balanced nor does it lead to ATP generation (Fig. 1).
Thus, removal of byproduct formation requires a complementary
strategy that yields ATP. Our strategy was to the use the
ATP-generating PEPCK enzyme in combination with deletions in
ethanol and acetate forming pathways to improve succinate
production. Using TFBA, conversion of PEP to OAA and ATP by
PEPCK enzyme was found to thermodynamically feasible at extracellular CO2 concentrations greater than 1.96 mM. Moreover, the
model also predicted a 15–20% increase in succinate productivity
and yield. The fermentation media was thus supplemented with
10 g/L sodium bicarbonate to favor the PEPCK reaction.
3.1. ATP production limits fermentative growth of E. coli AD32 (DldhA,
DpflB, DptsG)
Despite a positive overall ATP balance (Table 1), the deletion of
either adhE or ackA-pta (or both; AD483) was observed to completely abolish fermentative growth of AD32 (BW25113 ldhA ,
pflB , ptsG ) (Fig. 2a). This result suggests that approximately
0.5 mol ATP/mol glucose is required for maintenance under these
conditions and in this genetic background. To test this inference, we
deleted the ppc gene required for anaerobic succinate production.
Fig. 1. Central carbon metabolism of E. coli. Target reactions for the improved
succinic acid production are highlighted in red. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article).
This strain has the ability to be redox balanced as well as ATP
positive, which is not possible for the succinate production strain
alone. As expected, we observed that the growth was maintained in
the Dppc strain albeit at a lower final cell density (Fig. 2a).
A. Singh et al. / Metabolic Engineering 13 (2011) 76–81
Fig. 2. (a) Effect of various gene deletions on growth profile on LB media
supplemented with 50 mM glucose in microaerobic–anaerobic conditions.
(b) Effect of the carbon sources of differing oxidation state on the growth of AD
12 (BW25113 ldhA , pflB ) and AD32 (BW25113 ldhA , pflB , ptsG ) after 24 h on
LB media supplemented with 50 mM glucose.
To further explore the interrelationship between redox and ATP
balancing, we tested the growth of these mutants on sorbitol
(Oxidation state¼ 1), glucose (Ox state¼ 0) and gluconate
(Ox state¼1). Biochemically, sorbitol metabolism results in
3 mol/mol NADH compared with 2 mol/mol for glucose and
1 mol/mol for gluconate, respectively. The substitution of glucose
with sorbitol lead to a 44% decrease in cell growth for AD32
(BW25113 ldhA , pflB , ptsG ) as opposed to AD12 (BW25113
ldhA , pflB ) which grew to over four fold higher cell density
(Fig. 2b). Since succinate appears to be the primary fermentative
pathway in AD12, excess NADH would lead to redox balancing and
higher growth. The cause of lower cell growth of AD32 on sorbitol is
unclear; we speculate that excess NADH would upset the flux
distribution among the three end-products succinate, acetate and
ethanol. Higher cell density of AD12 in sorbitol also suggests that its
growth is not limited by acetyl-coA requirement for biosynthesis.
3.2. Effect of the overexpression of A. succinogenes PEPCK
Our observations suggest that at least 0.5 moles of ATP per mole
of glucose is required for the growth of AD32, which is apparently
accomplished through conversion of 50% of the carbon to acetate
79
Fig. 3. (a) Effect of pAsPCK on growth and fermentation profile in the microaerobic–
anaerobic dual-phase fermentation. (a) Open and closed squares represent anaerobic growth profiles for AD32 (BW25113 ldhA , pflB , ptsG ) and AD216 (AD32
pAsPCK) in LB + 10 g/L glucose, respectively. (b) Glucose consumption (squares),
succinate (triangles) and acetate (circles) production for AD32 (open symbols) and
AD216 (closed symbols).
and ethanol as opposed to succinate. We reasoned that overexpression of an enzyme that could tie ATP production to succinate
formation would increase the fitness of succinate production
strains. PEP-carboxykinase (PEPCK) from A. succinogenes, which
catalyzes the formation of oxaloacetate (OAA) from PEP along with
the generation of an ATP, has been previously cloned into an
expression plasmid (pAsPCK) and shown to complement the an
E.coli K12 PEP-carboxylase (Dppc) mutant (Kim et al., 2004). The
transformation of the pAsPCK in AD12 (BW25113 ldhA , pflB ) and
AD32 (BW25113 ldhA , pflB , ptsG ) did not significantly increase
cell growth (data not shown), presumably due to high activity of
the native phoephoenolpyruvate carboxylase (ppc), as previously
reported (Kim et al., 2004; Millard et al., 1996). It has been shown
that the kinetic properties of the E. coli PPC are superior to those of
PEPCK with respect to bicarbonate. Specifically, E. coli PPC has a Km
of 0.19 and 0.1 towards PEP and bicarbonate anion, respectively,
(Kai et al., 1999). In comparison, the Km towards bicarbonate for
PEPCK is expected to be closer to that of PEP carboxykinases from
E. coli (13 mM) and Anaerobiospirillum succiniciproducens, which
have been measured at about two orders of magnitude greater
(17 mM) than E. coli PPC (Millard et al., 1996).
To address this issue, we deleted the native E. coli ppc and
transformed the pAsPCK plasmid into strain AD32 (designated
AD216). Indeed, we observed that overexpression of A. succinogenes
PEPCK in the absence of the native ppc gene increased growth and
80
A. Singh et al. / Metabolic Engineering 13 (2011) 76–81
Fig. 4. Growth of AD32 (BW25113 ldhA , pflB , ptsG ) derivatives harboring pAsPCK plasmid on LB media supplemented with 50 mM glucose after 24 h in microaerobic–
anaerobic conditions.
succinate production in the microaerobic–anaerobic dual-phase
fermentation (Fig. 3a). AD216 consumed 50% more glucose in
72 h and produced 60% more succinate (5.97 g/L) titers compared
to AD32 (Fig. 3b) although the yield remained roughly the same
at 0.78 (70% of the theoretical maximum, note that this improvement is significant as AD32 has the same disrupted activity in
ldhA, pflB, and ptsG as previously described AFP111). Acetate
constituted 20% of the fermentation product. This observation
suggests that ATP limits cell growth but not the flux distribution in
this strain, as would be expected based on the redox and ATP
balance.
3.3. Efforts to increase theoretical yield of succinic acid production
We expected that diverting the flux towards succinate formation via ATP-forming PEPCK would eliminate the need for the ATP
producing acetate pathway, and thus could be used to further
increase succinate formation. The effect of the presence of PEPCK
was thus studied in strains lacking ethanol and acetate pathways.
As expected, the presence of PEPCK had no effect on the strains
carrying a native PPC, thus the studies were continued in a ppc
deletion background. The strain carrying the adhE deletion, AD346
was able to grow anaerobically upon transformation with pAsPCK
(Fig. 4). The growth of AD346 was similar to AD32, thus channeling
the carbon flux towards succinate formation could eliminate the
need for NADH oxidation via the ethanol pathway. However, the
deletion of acetate forming genes, ackA-pta in AD483 abolished
microaerobic and anaerobic growth even in the presence of pAsPCK
plasmid (Fig. 4). As the derivatives of AD32 (BW25113 ldhA , pflB ,
ptsG ) appear to have pyruvate to acetyl-coA conversion activity,
we suspected that acetate formation would be essential in such
strains in the absence of a functional glyoxylate pathway to avoid
acetyl-coA accumulation.
A 71:29 ratio split in the carbon flux towards reductive TCA
cycle and the glyoxylate pathway respectively would lead to redox
balanced succinate production with a molar yield of 1.71 mol/mol
glucose (Vemuri et al., 2002a). Since the glyoxylate pathway is
typically active during aerobic conditions, optimized dual-phase
aerobic/anaerobic fermentations have been successfully employed
to attain high yields of succinate formation at impressive productivities (Sanchez et al., 2006; Vemuri et al., 2002b). With these
studies in mind, we sought to investigate if the deletions of pdhR
and iclR would increase cellular fitness of our engineered strains.
pdhR is the anaerobic repressor of the pyruvate dehydrogenase
complex while iclR is the repressor of the glyoxylate pathway
genes. The deletions of pdhR and iclR leads to the constitutive
expression of the PDHc (Haydon et al., 1993) and the glyoxylate
pathways genes (Sunnarborg et al., 1990), respectively. However,
these two deletions had no effect on the fitness on the AD32 and its
derivatives, suggesting that neither the conversion of pyruvate to
acetyl-CoA nor the glyoxylate pathway is limiting in our strains.
In summary, cellular fitness of the succinate production strains
appears to be limited by sub-optimal acetyl-coA metabolism.
While the formation of acetyl-coA from pyruvate is essential for
biosynthesis, subsequent formation of acetate decreases the overall yield. The engineered strains were observed to be sensitive to
ackA-pta mutation, underscoring the rigid control of acetyl-coA
metabolism. Future engineering efforts should focus on modulating the pyruvate to acetyl-coA conversion to enable biosynthesis as
well as achieve the delicate split of the carbon flux between the
reductive TCA and glyoxylate pathways.
Acknowledgments
KCS was supported by the Swiss National Science Foundation.
VH was supported by funding from Ecole Polytechnique Fédérale
de Lausanne (EPFL), SystemsX.ch and DuPont.
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