Metabolic Engineering 13 (2011) 328–335
Contents lists available at ScienceDirect
Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben
Succinate production from different carbon sources under anaerobic
conditions by metabolic engineered Escherichia coli strains
Jian Wang a, Jiangfeng Zhu b,c, George N. Bennett d, Ka-Yiu San e,f,n
a
College of Agricultural and Biological Engineering, Jilin University, Changchun, PR China
Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, PR China
c
Key Laboratory of Biofuels, Chinese Academy of Sciences, Qingdao, PR China
d
Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA
e
Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA
f
Department of Bioengineering, Rice University, MS 142 6100, Main Street, Houston, TX 77005, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 September 2010
Received in revised form
26 January 2011
Accepted 18 March 2011
Available online 30 March 2011
Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a
variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future
industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on
sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains
Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for
sucrose uptake and catalyzation. Succinic acid production by this modified strain and the
SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of
glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic
acid efficiently from all combinations of different carbon sources tested with minimal byproduct
formation and with high molar succinate yields close to that of the maximum theoretic values. The
molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the
mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher
productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a
1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose
pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity
as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation
rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic
phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This
study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and
sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of
succinic acid by our metabolic engineered E. coli strain.
& 2011 Elsevier Inc. All rights reserved.
Keywords:
Escherichia coli
Fermentation
Sucrose
Glucose
Fructose
Conjugation
Succinate
1. Introduction
Succinic acid is a member of the C4-dicarboxylic acid family. It
has drawn much interest because it has been used as a precursor
of many industrially important chemicals in the food, chemical,
and pharmaceutical industries (Hong and Lee, 2002; Song and Lee
2006).
Sustainable production of fuels and chemicals is driven by the
depletion of existing oil reserves, increasing oil prices, and the
need to control atmospheric concentrations of greenhouse gases
such as carbon dioxide. In a report from the U.S. Department of
Energy (USDOE), succinic acid was considered as one of the 12 top
n
Corresponding author at: Department of Bioengineering, Rice University, MS
142 6100, Main Street, Houston, TX 77005, USA. Fax: þ 1 713 348 5877.
E-mail address: [email protected] (K.-Y. San).
1096-7176/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymben.2011.03.004
chemical building blocks manufactured from biomass (Werpy and
Petersen, 2004). The prospective economical and environmental
benefits of a bio-based succinate industry have motivated
research and development of succinate-producing organisms.
Many bacteria have been reported to have the ability to produce
succinate as a major fermentation product. The list includes
Actinobacillus succinogenes (Guettler et al., 1996), Anaerobiospirillum succiniciproducens (Samuelov et al., 1991), Bacteroides fragilis
(Isar et al., 2007), Corynebacterium glutamicum (Okino et al.,
2005), Mannheimia succiniciproducens (Lee et al., 2003), Saccharomyces cerevisiae (Raab et al., 2010), and engineered Escherichia
coli (Cox et al.2006; Singh et al., 2011).
However, bio-based succinate is still facing the challenge of
becoming cost competitive against petrochemical-based alternatives (James et al., 2007). In order to develop a bio-based industrial
production of succinic acid, it will be important to grow the cell in
a low cost medium, and the working strain optimally should be
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
able to catalyze a wide range of low-cost sugar feedstock to
produce succinic acid in good yields so that the cheapest available
raw material can be used. It has been reported that good succinate
production candidate bacteria, A. succinogenes (Guettler et al.,
1996; Li et al., 2010), M. succiniciproducens (Lee et al., 2003; Kim
et al., 2004b; Lee et al., 2010), A. succiniciproducens (Lee et al.,
1999, 2008), can produce succinic acid from a broad range of
carbon sources such as glucose, sucrose, maltose, lactose, xylose,
and fructose. However, as one of the most promising bacteria for
industrial succinate production, reports on E. coli almost exclusively use glucose as the carbon source (Chatterjee et al., 2001;
Gokarn et al., 1998, 2000; Hong and Lee 2000, 2002; Kim et al.,
2004a, b; Millard et al., 1996; Sánchez et al., 2005a, b, 2006; Singh
et al., 2011; Stols and Donnelly, 1997; Vemuri et al., 2002),
whereas only a few reports have considered alternative carbon
sources (Donnelly et al. 2004; Lin et al., 2005; Andersson et al.,
2007; Blankschien et al., 2010). Considering the prices and availability of various carbon feedstocks, generally sucrose is lower in
price than glucose. For example, from 1991 to 2005 raw bulk cane
sugar price was around 10 cents per pound (Shapouri and Salassi,
2006) with molasses in the US about $100 per ton in 2008 (USDA:
Sugar and Sweeteners: Recommended Data) and high fructose
corn sirup at 24 cents per pound in 2004–2006 (USDA: Sugar and
Sweeteners: Recommended Data).
Since the use of inexpensive carbohydrates is an effective
method among other possible alternatives to reduce the cost of
fermentation process, we thus constructed E. coli sucrose utilization strains and compared their succinate production capability
on glucose, fructose, sucrose, a mixture of glucose and fructose, a
mixture of glucose, fructose and sucrose and a sucrose hydrolysis
solution which can be potentially obtained from sugar cane,
molasses, or high fructose corn sirup. The strains created showed
great potential for large-scale anaerobic production of succinate
from various carbon sources.
329
converts pyruvate to oxaloacetate (OAA). Plasmid pUR400 containing the scrK, Y, A, B, and R genes from E. coli K12 which
converts sucrose to b-D-fructose and a-D-glucose 6-phosphate
was kindly provided by Dr. Kurt Schmid from the Universität
Osnabrück, Germany.
Luria broth (LB) media supplemented with the appropriate
antibiotics was used in the conjugation experiments. Conjugation
experiments were conducted at 37 1C. E. coli SBS550MG containing the chloramphenicol resistance marker and E. coli K12 containing tetracycline marker were grown in 20 ml LB and
incubated aerobically at 37 1C (250 rpm) to an OD600 of 0.4–0.6.
To ensure that none of the resistance markers had been lost,
100 ml of each strain was plated on LB agar plates containing
the appropriate antibiotic (E. coli K12: 35 mg/ml tetracycline;
SBS550MG: 35 mg/ml chloramphenicol) and incubated overnight
at 37 1C. From the remaining culture, cells were mixed at a ratio of
1 ml K12 (donor strain): 9 ml SBS550MG (recipient strains) and
incubated 3 h at 37 1C. The conjugation mixture (100 ml of each)
was diluted in sterilized water and serial dilutions (10 1, 10 2,
and 10 3) were made, then the dilutions were plated on LB agar
plates containing 35 mg/ml tetracycline and 35 mg/ml chloramphenicol. All plates were incubated at 37 1C. Single colonies were
restreaked on fresh plates to confirm antibiotic resistance. Then
the SBS550MG pUR400 strain was transformed with plasmid
pHL413 carrying the Lactococcus lactis pycA gene. Single colonies
were restreaked on plates containing the mixture of ampicillin
(100 mg/ml) and tetracycline at a reduced concentration (20 mg/ml).
2.2. Fermentation media
2. Materials and methods
All fermentations used a complex medium containing (g/L):
tryptone, 20; yeast extract, 10; K2HPO4 3H2O, 0.90; KH2PO4,
1.14; (NH4)2SO4, 3.0; MgSO4 7H2O, 0.30 and CaCl2 2H2O, 0.25.
The medium was supplemented with 1.0 mg/L biotin and 1.0 mg/L
thiamine. The media also contained 100 mg/L ampicillin. Carbon
sources (fructose, sucrose, a mixture of glucose and fructose, a
mixture of glucose, fructose and sucrose, and sucrose hydrolysis
solution) were added depending on the experiment.
2.1. Bacterial strains and plasmids
2.3. Inoculum preparation
Table 1 listed the strains and plasmids used in this study. The
genes encoding metabolic enzymes such as lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE), acetate kinasephosphotransacetylase (ack-pta) and the IclR transcriptional
repressor (iclR), the repressor of the aceBAK operon, were inactivated as described previously (Sánchez et al., 2005a, b, 2006).
Plasmid pHL413 contains a pyc gene from L. lactis (Lin et al.,
2004a,b), which encodes the enzyme pyruvate carboxylase that
A single colony grown overnight on a LB agar plate was used to
inoculate a 150 ml shake flask caped with a foam stopper
containing 10 ml of LB medium with the appropriate antibiotics
(100 mg/ml ampicillin for SBS550MG pHL413; 100 mg/ml ampicillin and 20mg/ml tetracycline for SBS550MG pHL413 pUR400)
and grown aerobically at 37 1C with shaking at 250 rpm for 12 h
in Innova 43R Incubator Shakers (New Brunswick Scientific,Edison, NJ). Cells were harvested by centrifugation and then washed
twice with LB medium before inoculating at 1.5% v/v into the
bioreactor.
Table 1
List of strains and plasmids used in this study.
Strain
Genotype
Reference or
source
SBS550MG
ldhA, adhE,iclR,
ack-pta::Cm
Sánchez et al.
(2005a, b)
SBS550MG
pHL413
SBS550MG
pUR400
SBS550MG
pHL413 pUR400
Plasmids
pHL413
pUR400
This study
This study
Pyruvate carboxylase from Lactococcus
lactis cloned in pTrc99A, ApR
scrK, Y, A, B, and R genes,Tcr
Lin et al.
(2004a,b)
Schmid et al.
(1988)
2.4. Fed-batch bioreactor experiments
Dual-phase fermentations which comprise an initial aerobic
growth phase followed by an anaerobic production phase were
conducted. The initial media volume was 600 ml in a 1.0 L New
Brunswick Scientific (Edison, NJ) Bioflo 110 fermenter. Once the
initial 2 g/L of sugar was consumed, 2 g/L of sugar was added to
the bioreactor. In the case of using fructose as the carbon source,
fructose was supplemented two times (2 g/L each time) upon
sugar depletion besides the initial 2 g/L of fructose during the
aerobic growth phase. The inlet airflow and the agitation speed
were maintained at 1.0 L/min and 500 rpm during the aerobic
growth phase, respectively, if not mentioned otherwise. After the
additional sugar was depleted and the pH increased to 7.4–7.5,
330
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
oxygen-free CO2 was sparged at 0.2 L/min to maintain anaerobic
conditions, and the agitation speed was reduced to 250 rpm. The
pH was maintained at 7.0 with 2.0 M Na2CO3. A concentrated
sugar solution was added to the bioreactor when the sugar
concentration dropped. The temperature was maintained at
37 1C in both aerobic and anaerobic phases.
A sample of the inoculated media at time zero was removed
from reactor for analysis and samples were withdrawn aseptically
through a sampling port and filtered through a 0.2 mm filter and
immediately injected into the HPLC to determine sugar and
product concentrations.
2.5. Sucrose hydrolysis
Sucrose solution containing 500 g/L sucrose with 0.01 (v/v)
sulfuric acid was treated at temperatures of 100 1C for 30 min.
The treated sucrose solutions were cooled to room temperature
and stored at 4 1C before it was used for the fermentation studies.
2.6. Analytical techniques
The culture samples were diluted with 0.15 MNaCl, and the
optical density was measured at 600 nm with a spectrophotometer (Bausch & Lomb, Rochester, NY, Spectronic 1001).
A correlation of 3 OD to 1 g/l cell dry weight can be used. For
extracellular metabolites analysis, 1 ml of culture sample was
centrifuged, and the supernatant was then filtered through a
0.25 mm pore-size syringe filter for HPLC analysis. The HPLC
system (Shimadzu-10A Systems, Columbia, MD) was equipped
with a cation exchange column (HPX-87H, BioRad, Hercules, CA),
a UV detector (Shimadzu SPD-10A), and a differential refractive
index (RI) detector (Waters 2410, Milford, MA). A 0.5 ml/min
mobile phase using 2.5 mM H2SO4 solution was applied to the
column. The column was operated at 55 1C. Standards were
prepared for succinate, acetate, and pyruvate for both the RI
detector and UV detector, and calibration curves were created.
Succinate and acetate were measured by the RI detector and
pyruvate was measured by the UV detector at 210 nm. Sucrose was
eluted with a 2.5 mM H2SO4 solution at a flow rate of 0.4 ml/min
and at a temperature of 25 1C and was measured by the RI
detector.
3. Results
Fermentations with strain SBS550MG pHL413 and SBS550MG
pHL413 pUR400 using sucrose, glucose, fructose, and mixtures of
glucose/fructose and glucose/fructose /sucrose, and sucrose
hydrolysis solution were performed and analyzed. The metabolic
network of succinate production conducted by SBS550MG
pHL413 and SBS550MG pHL413 pUR400 using glucose, fructose,
and sucrose as carbon sources is shown in Fig. 1. Fed-batch
fermentation of E. coli SBS550MG pHL413 and SBS550MG
pHL413 pUR400 using glucose, fructose, and sucrose as carbon
sources revealed distinct differences between the different substrates as can be seen in Table 2.
3.1. Succinate production of E. coli SBS550MG pHL413 on fructose
Fructose was used as the carbon source for comparison to
glucose (Sánchez et al., 2005a; Martı́nez et al., 2010). The
SBS550MG pHL413 biomass yield was lower using fructose than
using glucose or sucrose during the aerobic phase. Fructose was
added three times because of the low biomass yield of fructose,
but the OD was only 7.71 when 6 g/L fructose was consumed and
the culture was changed to the anaerobic phase.
Fig. 1. Anaerobic glucose, fructose, and sucrose metabolism of SBS550MG pHL413
and SBS550MG pHL413 pUR400 for maximal succinate production.
When fermented on fructose, the strain produced 304.8 mM of
succinate at 63.5 h (Fig. 2). The yield was 1.86 mol/mol fructose
which is significantly higher compared to glucose. The productivity is 0.82 g/L/h. While the biomass of SBS550MG pHL413 was
much lower on fructose than on glucose, the specific rate of
succinate production was higher (0.16 g/L/h/OD).
3.2. Succinate production of E. coli on a mixture of glucose and
fructose (1:1)
A mixture of glucose and fructose was used as the carbon
source during the aerobic phase. SBS550MG pHL413 grew better
in the mixture of glucose and fructose than in fructose alone.
Fig. 3 shows the production of succinate by strain SBS550MG
pHL413 during glucose plus fructose fermentation. From the
graph it is observed that during co-utilization of glucose and
fructose, glucose is consumed preferentially to fructose.
The strain produced 187.3 mM of succinate at 23.5 h. The yield
was 1.74 mol/mol hexose which is higher than on glucose but
lower than on fructose as expected. The highest productivity was
obtained (1.21 g/L/h) and specific rate was slightly lower than in
fructose (0.13 g/L/h/OD), Fig. 3.
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
331
Table 2
Summary of fermentation parameters for SBS550MG pHL413 and SBS550MG pHL413 pUR400.
Strain
Carbon source consumed
(mM)
Agitation speed in
aerobic phase
(rpm)
Yield on
sucrosea
mol/mol
Yield on
glucoseb
mol/mol
Productivity
(g/L/h)
Specific productivity
rate (g/L/h/OD)c
SBS550MG
pHL413
pUR400
SBS550MG
pHL413
pUR400
SBS550MG
pHL413
pUR400
SBS550MG
pHL413
SBS550MG
pHL413
SBS550MG
pHL413 pUR400
SBS550MG
pHL413
pUR400
Sucrose
110.17
400
3.33
–
0.46
0.055
Sucrose
124.35
500
3.15
–
0. 48
0.047
Sucrose
120.18
800
2.05
–
0.60
0.058
Fructose
245.54
glucose/fructose
87.22/51.39
sucrose/glucose/fructose
60.00/52.49/27.19
Sucrose hydrolysis solution
sucrose/glucose/fructose
73.70/27.87/ 1.85
500
–
1.86
0.82
0.160
500
–
1.74
1.21
0.131
500
–
1.67
0.85
0.079
500
–
1.67
0.86
0.082
100
80
60
40
20
0
0
10
20 30 40
time/h
50 60
70
9
8
7
6
5
4
3
2
1
0
concentration/mM
concentration/mM
120
OD600
a
The molar succinate yield was calculated as moles of succinate formed in the anaerobic phase divided by moles of the sucrose utilized (the residual hexose was lower
than 10 mM for the 400 and 500 rpm experiments, and about 30 mM for the 800 rpm experiment).
b
When hexose or sucrose/hexose mixtures were used as the carbon source the molar yield was based on hexose. One mole of sucrose equals two moles hexose.
c
The specific succinate productivity during the anaerobic phase was calculated on the basis of the cell concentration at the moment of taking sample.
350
300
250
200
150
100
50
0
0
10
20
30 40
time/h
50
60
70
50
40
30
20
10
0
0
5 10 15 20 25 30 35 40 45
time/h
14
12
10
8
6
4
2
0
concentration/mM
concentration/mM
60
OD600
Fig. 2. Succinate production on fructose by SBS550MG pHL413. Concentration of fructose and OD (a) and concentration of succinate and essential byproduct (b). Symbols
used: fructose (&), OD (~), succinate (B), pyruvate (þ ), formate (D), and acetate ( ).
250
200
150
100
50
0
0
5 10 15 20 25 30 35 40 45
time/h
Fig. 3. Succinate production on the mixture of glucose and fructose by SBS550MG pHL413. Concentration of glucose and fructose and OD (a) and concentration of
succinate and essential byproduct (b). Symbols used: fructose (&), glucose (m), OD (~), succinate (B), pyruvate (þ ), formate (D), and acetate ( ).
3.3. Succinate production of E. coli on sucrose
From the viewpoint of industrial succinate production it would
be advantageous to grow E. coli on sucrose-based substrates.
However, most E. coli strains are able to consume glucose and
fructose, but not sucrose, as a carbon source for biomass and
succinic acid production due to the lack of an invertase
(Tsunekawa et al., 1992). Schmid et al. (1988) reported an E. coli
which metabolizes sucrose via expression of the 70 kb pUR400
plasmid which encodes the scrK, Y, A, B, and R genes and a
tetracycline resistance cassette. The scrK gene encodes an ATPdependent fructokinase, the scrY gene encodes a sucrose-specific
porin of the outer membrane, the scrA gene codes for enzyme IIscr
of the phosphotransferase system for sucrose uptake, and the scrB
gene encodes an intracellular b-D-fructofuranoside fructohydrolase which cleaves sucrose 6-phosphate into b-D-fructose and
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
a-D-glucose 6-phosphate. The regulon is controlled by the negative regulator ScrR.
Since E. coli SBS550MG cannot use sucrose as a carbon source,
the pUR400 plasmid was conjugated into E. coli strain SBS550MG
and designated SBS550MG pUR400. Then the pHL413 plasmid
was transformed to SBS550MG pUR400 and the strain was
designated SBS550MG pHL413 pUR400.
Sucrose was used as the carbon source during the aerobic
phase at 500 rpm. The strain produced 276.5 mM succinate at
96 h (Fig. 4) with minimal byproduct accumulation. The molar
succinate yield was 3.15 mol/mol sucrose.
It was observed that in sucrose fermentation, free fructose or
glucose in the media did not accumulate during the early period
of the fermentation. This was due to the slow hydrolysis of
sucrose. During the middle period of the fermentation, the
concentrations of glucose and fructose in the medium increased
rapidly as the concentration of sucrose dropped sharply. This was
due to the fact that the hydrolysis of sucrose was faster than the
utilization of glucose 6-phosphate and fructose. When SBS550MG
pHL413 pUR400 was fermented on sucrose, though glucose and
fructose were accumulated at the same time, a similar pattern
emerged in that glucose excreted from the cells was utilized
preferentially to fructose excreted from the cells. The slow
hydrolysis speed during the early period resulted in a succinate
productivity of only 0.48 g/L/h and a specific rate of only
0.047 g/L/h/OD (at 500 rpm stirring) (Fig. 4). In view of these
results, a fermentation using glucose plus fructose and sucrose as
the carbon source was hypothesized to perform better than using
sucrose alone.
3.4. Succinate production of E. coli on a mixture of glucose, fructose,
and sucrose
80
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70 80 90
time/h
16
14
12
10
8
6
4
2
0
OD600
concentration/mM
In order to compare the mixed sugar fermentation with the
sucrose fermentation and to reach a similar cell density, sucrose
was used during the aerobic phase before switching to a mixture
of glucose, fructose, and sucrose during the anaerobic phase.
Fermentation of the strain on glucose, fructose, and sucrose
produced 255.6 mM succinate at 46.8 h. The yield was 1.67 mol/
mol hexose which was lower compared to fructose (1.86 mol/mol
fructose) but higher than that on glucose (Sánchez et al., 2005a).
The co-utilization of glucose with fructose and sucrose was
characterized by an increase in succinate productivity as compared to the culture grow on fructose or sucrose alone. The
productivity was 0.85 g/L/h which was 1.76-fold that of the
corresponding sucrose culture. The specific rate was 0.079 g/L/h/
OD. When SBS550MG pHL413 pUR400 was fermented on the
glucose–fructose–sucrose mixture, glucose was utilized preferentially to sucrose and sucrose was utilized preferentially to
fructose (Fig. 5).
3.5. Succinate production of E. coli on a sucrose hydrolysis solution
Sucrose is a dimer composed of two sugar monomers, b-Dfructose and a-D-glucose. In commercial processes, sucrose is
hydrolyzed to the converted sugars, glucose, and fructose, using
acids, enzymes, or cation resins. Under strong acidic conditions,
sucrose glycosidic bonds hydrolyze rapidly at temperatures
greater than 160 1C (Clarke et al., 1997; L’Homme et al., 2003),
but typical commercial acid convert processes operate below
120 1C to minimize fructose and glucose degradation. Fructose
and glucose are known to degrade at temperatures above 106 1C
and pH below 2.0. These conditions are encountered in dilute acid
pretreatment of lignocellulosic biomass. The primary degradation
pathway is a dehydration of the sugar to 5-hydroxymethylfurfural
(5-HMF), which can hydrolyze and further degrade to levulinic
and formic acid (Clarke et al., 1997; Mosier et al., 2002; Qian et al.,
2005a,b). When a 500 g/L sucrose solution was treated with a
40.5% (v/v) sulfuric acid, 5-HMF was formed (data not shown).
When a 500 g/L sucrose solution with 0.01 (v/v) sulfuric acid was
concentration/mM
332
350
300
250
200
150
100
50
0
0 10 20 30 40 50 60 70 80 90
time/h
40
35
30
25
20
15
10
5
0
0
10
20
30
time/h
40
50
16
14
12
10
8
6
4
2
0
concentration/mM
concentration/mM
Fig. 4. Succinate production on sucrose by SBS550MG pHL413 pUR400. Concentration of sucrose and OD (a) and concentration of succinate and essential byproduct (b).
(500 rpm) Symbols used: fructose (&), glucose (m), sucrose (’), OD (~), succinate (B), pyruvate (þ ), formate (D), and acetate ( ).
300
250
200
150
100
50
0
0
10
20
30
time/h
40
50
Fig. 5. Succinate production on a mixture of glucose, fructose, and sucrose by SBS550MG pHL413 pUR400. Concentration of glucose, fructose and sucrose and OD (a) and
concentration of succinate and essential byproduct (b). Symbols used: fructose (&), glucose (m), sucrose (’), OD (~), succinate (B), pyruvate (þ ), formate (D), and
acetate ( ).
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
3.6. Effect of aeration level during cell growth on succinate
production
0
5 10 15 20 25 30 35 40 45
time/h
18
16
14
12
10
8
6
4
2
0
OD600
concentration/mM
The effect of the aeration level during the aerobic phase on cell
growth and succinic acid production was studied under agitation
rates of 400, 500, and 800 rpm on sucrose. Varying dissolved O2
concentration had an effect on the final biomass. Raising the
dissolved O2 concentration in the media had a positive effect on
cell growth. A maximum OD of 18.16 was obtained under
concentration/mM
800 rpm at 6.4 h when 4 g/L sucrose was consumed. An OD of
14.80 under 500 rpm at 8 h and an OD of 12.87 under 400 rpm at
9.5 h were obtained at the end of aerobic phase. This showed that
reducing the oxygen concentration resulted in a lower growth
rate and in a delay in sugar uptake. However, during the
anaerobic phase, the OD dropped quickly from 18.16 to 6.72 after
41.7 h at 800 rpm agitation which was more rapid than that of
400 rpm (Figs. 7 and 8).
SBS550MG pHL413 pUR400 showed a greater succinate yield
in the anaerobic phase when the preceding aerobic phase
occurred at the oxygen-transfer rate of 400 rpm than when
aerobic growth occurred at the high oxygen-transfer rate
(800 rpm) (Figs. 7, 8 and Table 2). The strain produced
267.8 mM succinate by 94.5 h (Fig. 7), the molar succinic acid
yield was 3.33 mol/mol sucrose under an agitation rate of
400 rpm, produced 178.0 mM of succinate at 49 h (Fig. 8), the
succinic acid yield was 2.05 mol/mol sucrose under an agitation
rate of 800 rpm due to the accumulation of hexoses and other
byproducts. These results suggest that the physiological role of
oxygen is central to establishing succinate productivity during the
anaerobic phase. High oxygen-transfer rates during the entire
growth phase result in a lower sugar uptake rate and the
accumulation of pyruvate (at 25.6 h, 38.2 mM pyruvate was
accumulated, then it was decreased to 4.05 mM), lactate
(7.05 mM), formate (51.2 mM) and acetate (49.0 mM) in the
anaerobic phase. The accumulation of these byproducts was much
larger than that seen at 400 rpm agitation (pyruvate 2.35 mM,
lactate 0 mM, formate 2.59 mM, and acetate 10.8 mM) and at
500 rpm agitation. Overall, low oxygen-transfer rate during aerobic growth is better for succinate production though the productivity was higher under an agitation rate of 800 rpm (0.60 g/L/h)
than that under 400 rpm (0.46 g/L/h) because the initial OD in the
anerobic phase was higher in the 800 rpm experiment. This was
similar to the results reported by Martı́nez and collaborators
(Martı́nez et al., 2010). They showed that the dissolved oxygen
(DO) level decreased considerably faster in the low aeration case,
treated at a temperature of 100 1C for 30 min a ratio of 123.7
sucrose to 35.5 glucose to 27.1 fructose was formed.
Sucrose was used as the carbon source during aerobic phase to
reach a similar OD and to compare the previous sucrose and
mixed carbon (sucrose/glucose/fructose) fermentations. The
strain produced 212.8 mM succinate at 40.2 h (Fig. 6). The
mixture of sucrose, glucose, and fructose resulted in a yield of
1.67 mol/mol hexose which is similar to that on sucrose/ glucose/
fructose mixture as carbon source. An interesting observation was
that the productivity (0.86 g/L/h) of SBS550MG pHL413 pUR400
fermented on sucrose hydrolysis solution was 1.78-fold higher
than on sucrose as the sole carbon source, which was similar to
the fermentation on the mixture of glucose, fructose, and sucrose.
The specific rate was 0.082 g/L/h/OD which was also similar
to the fermentation on the sucrose/glucose/ fructose mixture.
The sulfuric acid pretreatment of sucrose was economically
favorable because of the significant improvement in succinic acid
productivity.
Pyruvate, formate and acetate accumulated as undesirable coproducts during the fermentation process. None of their concentrations was higher than 9 mM which not only increased the yield
but would also decrease the cost of purification. Most of the
pyruvate was excreted during the first 2–4 h of the anaerobic
phase as succinic acid was produced. The pyruvate concentration
decreased in all cases to a final concentration of approximately
0–2.2 mM. Acetate levels increased slowly during all fermentations.
60
50
40
30
20
10
0
333
250
200
150
100
50
0
0
5 10 15 20 25 30 35 40 45
time/h
0
20
40
60
time/h
80
14
12
10
8
6
4
2
0
100
concentration/mM
70
60
50
40
30
20
10
0
OD600
concentration/mM
Fig. 6. Succinate production on sucrose hydrolysis solution by SBS550MG pHL413 pUR400. Concentration of glucose, fructose and sucrose and OD (a) and concentration of
succinate and essential byproduct (b). Symbols used: fructose (&), glucose (m), sucrose (’), OD (~), succinate (B), pyruvate (þ), formate (D), and acetate ( ).
300
250
200
150
100
50
0
0
20
40
60
time/h
80
100
Fig. 7. Succinate production on sucrose by SBS550MG pHL413 pUR400. Concentration of sucrose and OD (a) and concentration of succinate and essential byproduct (b).
(400 rpm) Symbols used: fructose (&),glucose (m), sucrose (’), OD (~), succinate (B), pyruvate (þ ), formate (D), and acetate ( ).
0
10
20
30
time/h
40
50
20
18
16
14
12
10
8
6
4
2
0
concentration/mM
70
60
50
40
30
20
10
0
OD600
J. Wang et al. / Metabolic Engineering 13 (2011) 328–335
concentration/mM
334
200
180
160
140
120
100
80
60
40
20
0
0
10
20
30
time/h
40
50
Fig. 8. Succinate production on sucrose by SBS550MG pHL413 pUR400. Concentration of sucrose and OD (a) and concentration of succinate and essential byproduct (b).
(800 rpm) Symbols used: fructose (&), glucose (m), sucrose (’), OD (~), succinate (B), pyruvate (þ ), formate (D), and acetate ( ).
leading to a microaerobic condition before the system was
switched completely to anaerobic. The high aeration experiment,
on the other hand, never reached a microaerobic condition before
switching to the anaerobic phase. This microaerobic period may
have contributed to differences in cell metabolism, reflected as
differences in metabolic profiles, gene expression profiles and
final product yield and productivity. Martı́nez and collaborators
also found that the level of pyc mRNA in the low aeration case at
the time of transition was 2.2-fold higher than in the high
aeration case (Martı́nez et al., 2010). It was reported that Pyc is
an important factor in succinate yield, likely because Pyc directs
the carboxylation of pyruvate to oxaloacetate decreasing pyruvate
accumulation. (Vemuri et al., 2002; Lin et al.2004a, 2004b;
Gokarn et al., 1998, 2000).
4. Discussions
An examination of Table 2 shows that there was considerable
variation in yields from different substrates. This observation
suggests that the fluxes through different pathways are influenced by the nature of the substrate, including its entry point into
central metabolism, as well as its oxidation state, which would
lead to a different end product patterns. Succinate yields of
defined E. coli strains were affected by the type of PTS-transportable sugar serving as the carbon source. Fructose is transported
into the cell and phosphorylated to fructose-1-phosphate by a
PEP-dependent phosphotransferase system. It then enters glycolysis after phosphorylation to fructose-1, 6-bisphosphate (Kotrba
et al., 2001; Parche et al., 2001). Sucrose-6-phosphate was
identified as the primary product of the phosphoenolpyruvate
(PEP)-dependent sucrose transport (Schmid et al., 1982). Plasmid
pUR400 mediates the ‘‘peripheral’’ metabolism of sucrose in E. coli
K-12 by furnishing the sucrose-specific, membrane-bound
compound EIISCR of the PTS and the hydrolyzing enzyme
sucrose-6-phosphate hydrolase. The host contributes the general
PTS proteins, including EI and HPr, an additional factor, EIIIglu,scr,
which is required for the activity of the sucrose transport system.
The primary intracellular product of sucrose-6-phosphate hydrolysis is fructose which is converted to fructose-6-phosphate by an
ATP-dependent kinase (Ferencd and Koruberg,. 1974). The highest
succinate yields were observed with fructose as a carbon source
as compared to glucose and sucrose for SBS550MG pHL413 and
SBS550MG pHL413 pUR400. The succinate yields were roughly
similar between the glucose/fructose mixture, the glucose/fructose/sucrose mixture, and the sucrose hydrolysis solution.
SBS550MG pHL413 and SBS550MG pHL413 pUR400 showed
large differences with respect to succinate productivity on the
carbon sources glucose, fructose, and sucrose. Succinate productivity was much higher on glucose and fructose mixture as
compared to other carbon sources with sucrose allowing for the
lowest succinate productivity. Furthermore, it should be noted
that the strain SBS550MG pHL413 produced the highest level of
succinate, when grown on fructose.
The carbon source did not show an influence on the formation
pattern of by-products. Pyruvate, formate and acetate were the
major by-products. The concentration of each was low. Elimination of these by-products represents a further opportunity to
increase yield.
Sucrose as a carbon source for E. coli has received little
attention. The high yield and productivity of cells on a sucrose
hydrolysis solution has the potential to be used in industrial
production processes. Furthermore, the observation enhances the
idea that has a wide range of confectionery and other wastes are
accessible as possible feedstocks to produce succinate by E. coli
SBS550MG pHL413 and SBS550MG pHL413 pUR400. Although
this study primarily focused on the conversion of fructose and
sucrose to succinate, it is well known that E. coli has the native
ability to metabolize all hexose and pentose sugars that are
constituents of plant cell walls (Asghari et al., 1996).
Acknowledgments
We wish to thank Dr. K. Schmid (Universität Osnabrück,
Germany) for providing the E. coli K-12 strain containing the
pUR400 plasmid. The work was supported by the Jilin Province
Science and the Technology Development Foundation of China
under Grant no. 20090161, Fundamental Research Funds for the
Jilin Universitiy.
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