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Biochemical Engineering Journal 40 (2008) 312–320

Metabolic engineering of Escherichia coli for


the production of malic acid
Soo Yun Moon a , Soon Ho Hong c , Tae Yong Kim a,b , Sang Yup Lee a,b,∗
a Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program),
BioProcess Engineering Research Center, Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST),
335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea
b Center for Systems and Synthetic Biotechnology, Institute for the BioCentury,

KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea


c School of Chemical Engineering & Bioengineering, University of Ulsan,

29 Mugeo-dong, Nam-gu, Ulsan 680-749, Republic of Korea


Received 7 October 2007; received in revised form 23 December 2007; accepted 7 January 2008

Abstract
Malic acid is a C4 -dicarboxylic acid and an intermediate of tricarboxylic acid (TCA) cycle. It has been widely used in the polymer, food and
pharmaceutical industries. Metabolic flux analysis was performed to find a strategy for enhanced malic acid production in Escherichia coli. The
simulation results suggested that the amplification of phosphoenolpyruvate (PEP) carboxylation flux allowed increased malic acid production.
Since the PEP carboxylase of E. coli converts PEP to oxaloacetate without generating ATP, thus losing the high-energy phosphate bond of PEP, the
PEP carboxykinase, which generates ATP during this conversion, was chosen. However, the E. coli PEP carboxykinase catalyzes the reaction that
converts oxaloacetate to PEP rather than the desirable opposite reaction. Thus, we cloned the PEP carboxykinase (enconded by the pckA gene) of
Mannheimia succiniciproducens, which converts PEP to oxaloacetate as a favorable reaction. The pta mutant E. coli strain WGS-10 harboring the
plasmid p104ManPck containing the M. succiniciproducens pckA gene was constructed and cultured at 37 ◦ C. The final malic acid concentration
of 9.25 g/L could be obtained after 12 h of aerobic cultivation.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Malic acid; Mannheimia succiniciproducens; Recombinant DNA; Fermentation; Glucose; Protein

1. Introduction or fumaric acid. Enzymatic conversion of fumaric acid using


fumarase also allows malic acid production [1–3]. However,
Malic acid is a member of C4 -dicarboxylic acid family, and these two processes require rather expensive chiral resolu-
an intermediate of the tricarboxylic acid (TCA) cycle (Fig. 1). tion agent, complex reaction processes, or expensive enzyme,
Industrially, malic acid has been employed for the prepara- which make them difficult to be used practically [1]. Fer-
tion of food additives and synthesis of various fine chemicals mentation of Aspergillus species and Schizophyllum commune
[1–3]. Malic acid can be produced by various methods such also allows production of malic acid from renewable feed-
as extraction from plants, enzymatic conversion and chem- stocks [3,4], but the productivity of malic acid is rather low
ical synthesis. Malic acid extraction from fruit has been a at 0.1–0.6 g/L/h [1,4]. Thus, the development of more effi-
traditional method of its production, but this method has no prac- cient malic acid production system is required to make the
tical use due to the small production capacity [3]. Malic acid biological malic acid production process economically feasi-
can be chemically synthesized by hydration of either maleic ble.
Metabolic engineering [5,6] can be employed to re-design
the metabolic network for the enhanced production of desirable
∗ Corresponding author at: Center for Systems and Synthetic Biotechnology, metabolites. Recently, various computational tools developed
Institute for the BioCentury, KAIST, 373-1 Guseong-dong, Yuseong-gu, Dae-
for metabolic flux analysis (MFA) are allowing estimation
jeon 305-701, Republic of Korea. Tel.: +82 42 869 3930;
fax: +82 42 869 8800. of intracellular fluxes, which can be used to decipher the
E-mail address: leesy@kaist.ac.kr (S.Y. Lee). metabolic characteristics under given genetic and environmen-

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.01.001
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S.Y. Moon et al. / Biochemical Engineering Journal 40 (2008) 312–320 313

Fig. 1. Central metabolic pathways in E. coli. Enzymes encoded by the genes shown are: aceE/F, pyruvate dehydrogenase multienzyme complex; acnA/B, aconi-
tase; adhE/C, alcohol dehydrogenase; fumA/B/C, fumarase; gltA, citrate synthase; icdA, isocitrate dehydrogenase; ldhA, d-lactate dehydrogenase; mdh, malate
dehydrogenase; pckA, phosphoenolpyruvate carboxykinase; ppc, phosphoenolpyruvate carboxylase; pta/ackA, phosphate acetyltransferase/acetate kinase; ptsG, an
phosphotransferase system enzyme; pykA, pyruvate kinase; sfcA, malic enzyme; sdhA/B/C/D, succinate dehydrogenase; sucA/B-lpdA, ␣-ketoglutarate dehydrogenase
complex; sucC/D, succinyl-CoA synthetase.
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314 S.Y. Moon et al. / Biochemical Engineering Journal 40 (2008) 312–320

tal conditions and consequently to suggest the target genes 2.2. Plasmids
to be manipulated for the enhanced production of metabolites
[7–9]. The plasmids used in this study are also listed in Table 1.
In this study, we report a new strategy for the production of To integrate the Cmr , Knr and Tcr genes into the chromo-
malic acid in Escherichia coli. The phosphoenolpyruvate (PEP) some of wild type E. coli W3110, the gene replacement vector
carboxykinase of Mannheimia succiniciproducens MBEL55E, pKO3 [11] was used. The plasmid pKO3 contains a temperature-
which was isolated from bovine rumen, was overexpressed for sensitive origin of replication and Cmr marker for positive
the enhanced malic acid production in recombinant E. coli selection of chromosomal integration. First, the ldhA, pta and
[10]. The effects of introducing the M. succiniciproducens adhE genes were cloned by PCR from E. coli XL1-Blue (Strata-
PEP carboxykinase gene into E. coli was also evaluated by gene Cloning Systems, La Jolla, CA). The PCR primers (Table 2)
MFA. were designed based on the reported E. coli genome sequence
[12]. PCR was performed using a PCR Thermal Cycler MP
2. Materials and methods TP3000 (Takara Shuzo Co., Shiga, Japan) and the High Fidelity
PCR System (Boeringer Mannheim, Mannheim, Germany). The
2.1. Bacterial strains amplified ldhA, pta and adhE genes were inserted into the
BamHI, SmaI and BamHI sites of pUC19 to construct pUCldhA,
The strains used in this study are listed in Table 1. Cells were pUCpta and pUCadhE, respectively [13]. The Cmr and Tcr gene
routinely grown in Luria Bertani (LB) medium containing 10 g/L were also amplified by PCR from pACYC184, and the Knr gene
of tryptone, 5 g/L of yeast extract and 10 g/L of NaCl. E. coli from pACYC177. The amplified Cmr gene was cloned into the
strain W3110, WGS-3 (W3110 ldhA::Cm pta::Kn adhE::Tc) KpnI site of pUCldhA, which is present in the middle of the ldhA
and WGS-10 (W3110 pta::Kn) were used for malic acid pro- gene, to make pUCldhACm. The amplified Knr gene was cloned
duction. The mutant strain WGS-3 lost the activity of lactate into the BamHI site of the pta gene in pUCpta to make pUCp-
dehydrogenase, phosphotransacetylase, and alcohol dehydro- taKn. The amplified Tcr gene was cloned into the NcoI site of
genase by the insertion of chloramphenicol resistance (Cmr ), the adhE gene in pUCadhE to construct pUCadhETc. The Cmr
kanamycin resistance (Knr ) and tetracyclin resistance (Tcr ) gene inserted ldhA gene, Knr inserted pta gene and Tcr inserted adhE
in the middle of the ldhA, pta and adhE genes, respectively. The gene were cloned into the BamHI, SmaI, and BamHI sites of the
mutant strain WGS-10 lost the activity of phosphotransacety- replacement vector pKO3 to make pKOldhACm, pKOptaKn and
lase by the insertion of the Knr gene in the middle of the pta pKOadhETc, respectively.
gene. The procedure for disruption of these genes is described The pckA gene, which encodes PEP carboxykinase was
below. cloned by PCR from both E. coli XL1-Blue and M. succinicipro-

Table 1
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristics Reference of source

Strains
E. coli
XL1-Blue supE44 hsdR17 recA1 endA1 gyrA96 thi relA1 lac F [proAB+ lacIq lacZΔM15 Tn10(tetr )] Stratagenea
W3110 F− mcrA mcrB IN(rrnD− rrnE) 1λ− Lab stock
WGS-3 W3110 (ldhA::Cm pta::Kn adhE::Tc) This study
WGS-10 W3110 (pta::Kn) This study
M. succiniciproducens MBEL55E [10]
Plasmids
pUC19 APr ; cloning vehicle New England Biolabs
pUCldhA pUC19 derivative; E. coli ldhA gene This study
pUCpta pUC19 derivative; E. coli pta gene This study
pUCadhE pUC19 derivative; E. coli adhE gene This study
pUCldhACm pUC19 derivative; Cmr ; E. coli ldhA gene This study
pUCptaKn pUC19 derivative; Knr ; E. coli pta gene This study
pUCadhETc pUC19 derivative; Tcr ; E. coli adhE gene This study
pKO3 Cmr ; replacement vector; temperature-sensitive origin of replication [11]
pKOldhACm pKO3 derivative; Cmr ; E. coli ldhA gene This study
pKOptaKn pKO3 derivative; Knr ; E. coli pta gene This study
pKOadhETc pKO3 derivative; Tcr ; E. coli adhE gene This study
p10499A APr ; pTrc99A derivative; gntT104 promoter [14,15]
p104ColiPck p10499A derivative; E. coli pckA gene This study
p104ManPck p10499A derivative; M. succiniciproducens MBEL55E pckA gene This study
a Stratagene Cloning System, La Jolla, CA, USA.
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S.Y. Moon et al. / Biochemical Engineering Journal 40 (2008) 312–320 315

Table 2
The primers used for PCR
Primer Primer sequence Target gene Template used

Primer 1 5 -CGGGATCCATGAAACTCGCCGTTTATAGC-3 ldhA E. coli XL1-Blue chromosome


Primer 2 5 -CGGGATCCTTAAACCAGTTCGTTCGGGC-3
Primer 3 5 -TCCCCCGGGGACGCGCGCATTTCTAAACT-3 pta E. coli XL1-Blue chromosome
Primer 4 5 - TCCCCCGGGGCGCAGTTAAGCAAGATAATC-3
Primer 5 5 -CGGGATCCTCTCAGGGTGGTATCGGTG-3 adhE E. coli XL1-Blue chromosome
Primer 6 5 -CGGGATCCTTAAGCGGATTTTTTCGCTTTTT-3
Primer 7 5 -CGGGATCCGGTACCAGCACTTCACTGACACCCTC-3 Cmr pACYC184
Primer 8 5 -CGGGATCCGGTACCACTTATTCAGGCGTAGCACC-3
Primer 9 5 -CGGGATCCAAAGCCACGTTGTGTCTCAAA-3 Knr pACYC177
Primer 10 5 -CGGGATCCTTAGAAAAACTCATCGAGCATC-3
Primer 11 5 -CATGCCATGGATTCTCATGTTTGACAGCTTATC-3 Tcr pACYC184
Primer 12 5 -CATGCCATGGTCCGTTAGCGAGGTGCCG-3
Primer 13 5 -CGGAATTCATGCGCGTTAACAATGGTTTG-3 pckA E. coli XL1-Blue chromosome
Primer 14 5 -GCTCTAGATTACAGTTTCGGACCAGCCG-3
Primer 15 5 -CGGAATTCATGACAGATCTTAATCAATTAAC-3 pckA M. succiniciproducens MBEL55E chromosome
Primer 16 5 -TGCTCTAGATTATGCTTTAGGACCGGCAG-3
a Restriction enzyme sites are underlined.

ducens [10]. The amplified E. coli and M. succiniciproducens 2.3. Gene replacement
pckA genes were inserted into the EcoRI and XbaI sites of
p10499A to construct p104ColiPck and p104ManPck, respec- First, E. coli strain W3110 was transformed with pKOptaKn
tively (Fig. 2). Plasmid p10499A contains a constitutive gntT104 by electroporation to construct WGS-10 (W3110 pta::Kn). The
promoter [14,15]. cells were plated on prewarmed LB plates containing 30 ␮g/mL

Fig. 2. Construction of plasmids p104ColiPck and p104ManPck.


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316 S.Y. Moon et al. / Biochemical Engineering Journal 40 (2008) 312–320

Cm, and incubated at 30 ◦ C. From the plate, one to five colonies WGS-10, WGS-10 (p104ColiPck) and WGS-10 (p104ManPck)
were picked and transferred into 10 mL of LB broth, and sub- strains. For the flux analysis of WGS-10 (p104ManPck), PEP
cultured four to five times at 42 ◦ C to allow recombination to carboxylase reaction was replaced with reversible PEP car-
occur. The diluted culture broth was plated on LB plate contain- boxykinase. The glucose uptake rate was normalized to be
ing 30 ␮g/mL Km at 30 ◦ C. The colonies obtained on LB-Km 10 mmol/gDCW/h. MFA was carried out using the program
plate were replica plated onto the LB-Cm plate to find those package MetaFluxNet (http://mbel.kaist.ac.kr/mfn/index.html;
colonies that lost the plasmid pKOptaKn. The gene replace- [18]).
ment was confirmed by PCR using the primers flanking the
targeted gene. Similarly, WGS-10 was transformed with pKOad- 3. Results and discussion
hETc and pKOldhACm by electroporation for the construction
of mutant strain WGS-3 (W3110 ldhA::Cm pta::Kn adhE::Tc). 3.1. Prediction of optimal malic acid production pathway
The correct gene replacement was also confirmed by PCR.
MFA was carried out to predict the optimal malic acid
2.4. Fermentation production pathway. Even though malic acid is a metabolite,
favorably produced under anaerobic condition, just like suc-
Fermentations were carried out at 37 ◦ C in a 5 L biore- cinic acid, simulation condition was not restricted to anaerobic
actor (BioFlo 3000, New Brunswick Scientific, Edison, NJ) condition considering higher cellular growth rate under aero-
containing 3 L of R/2 medium supplemented with 20 g/L glu- bic condition (Table 3). The oxygen uptake rate was limited
cose. The R/2 medium contains per liter: KH2 PO4 , 6.75 g/L; to be below an average oxygen uptake rate of E. coli, which is
(NH4 )2 HPO4 , 2 g/L; citric acid, 0.85 g/L; MgSO4 ·7H2 O, 20 mmol/gDCW/h [19]. To obtain feasible solutions, constraints
0.7 g/L; trace metal solution (5 M HCl; FeSO4 ·7H2 O, on the rates of metabolites uptake and excretion were applied
10 g/L; CaCl2 , 2 g/L; ZnSO4 ·7H2 O, 2.2 g/L; MnSO4 ·5H2 O, (Table 3).
0.54 g/L; CuSO4 ·5H2 O, 1 g/L; (NH4 )Mo7 O24 ·4H2 O, 0.1 g/L; To predict optimum intracellular metabolic flux distribution
Na2 B4 O7 ·10H2 O, 0.02 g/L), 5 mL/L. The pH was controlled allowing high malic acid production and good cell growth, the
at 6.7 with 5 M NaOH. Dissolved oxygen (DO) level was malic acid production rate was altered from the minimum to the
maintained over 40% of oxygen saturation during cultivation. maximum value during the linear programming-based optimiza-
Ampicillin (Ap), Km and Cm were added at concentrations of tion using the maximizing the growth as an objective function.
100, 30 and/or 30 ␮g/mL, respectively, depending on the plas- MFA results showed that 19 mmol/gDCW/h of malic acid flux
mid and strains employed. can be achieved with no cellular growth, and it linearly decreased
as biomass flux increased (Fig. 3). The reaction catalyzed by the
2.5. Analytical procedure reversible PEP carboxykinase was introduced into the E. coli
Cell growth was monitored by measuring the optical den-
sity at 600 nm (OD600 ). Fermentation products in the medium Table 3
Constraints on the specific rates of metabolite uptake and excretion
were analyzed by high-performance liquid chromatography
(Hitachi chromatography system, Tokyo, Japan) equipped with Basic constraints
an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad Glucose uptake <10a
Oxygen uptake b <200
Laboratories, Herculules, CA) and a refractive index detector (L-
NH3 uptake >0
3300, Hitachi chromatography system). The column was eluted Phosphate uptake >0
isocratically with 5 mM H2 SO4 . H2 SO4 uptake >0
CO2 uptake >0
2.6. Metabolic flux analysis CO2 excretion >0
ATP requirement for maintenance c >76

The in silico flux response analyses were performed using the Additionalconstraints
genome-scale metabolic model E. coli MBEL979 consisting of WGS-10 (p10499A)
Acetic acid excretion 0.9
979 metabolic reactions and 814 metabolites (144 extracellular Lactic acid excretion 0.2
metabolites and 670 intermediates), which is a slightly modified Malic acid excretion 0
network of iJR904 reported by Palsson and coworkers [16,17]. In WGS-10 (p104ColiPck)
order to examine the correlation between the malic acid produc- Acetic acid excretion 0
tion rate and the cell growth rate, the malic acid production rate Lactic acid excretion 0
Malic acid excretion 1.5
was perturbed from its minimum value to the maximum value WGS-10 (p104ManPck)
using an objective function of the maximization of the growth Acetic acid excretion 0
rate. The flux profile graph is generated by comparing the malic Lactic acid excretion 0
acid production rate on Y-axis and the growth rate on X-axis. In Malic acid excretion 7.1
order to investigate the flux distribution of the engineered strains a Specific rate (mmol/gDCW/h).
on malic acid production, constraints-based flux analysis was b [19].
c [18].
performed using exponential growth phase fermentation data of
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Fig. 4. Time profiles of the culture OD600 () and concentrations of glucose
(g/L, 䊉), acetic acid (g/L, ), and malic acid (g/L, ) during the batch fermen-
tation of WGS-10 harboring p104ManPck from 20 g/L of glucose.
Fig. 3. Phenotypic phase plane for biomass formation rate and malic acid pro-
duction rate. Dotted and solid lines represent the solution spaces without and
obic fumarase and fumarate reductase are not expressed. Under
with the introduction of the pckA gene. Symbols represent real points of WGS10
(p10449A) (), WGS10 (p104ColiPck) () and WGS-10 (p104ManPck) (). anaerobic condition, malic acid is converted to succinic acid by
anaerobic fumarase and fumarate reductase, and succinic acid
is produced as a final product. To realize and verify the pro-
metabolic network because the original metabolic network con- posed malic acid pathway, the M. succiniciproducens pckA gene,
tains only the irreversible PEP carboxykinase reaction, which encoding PEP carboxykinase, was overexpressed in recombi-
converts oxaloacetate to PEP. It has generally been suggested nant E. coli. Also, as a comparison, the E. coli pckA gene was
that the PEP carboxykinase is more appropriate than PEP car- also overexpressed in E. coli.
boxylase since PEP carboxykinase produces ATP during PEP
conversion to oxaloacetate. When PEP carboxykinase working 3.2. Fermentation
towards carboxylation reaction was introduced, the maximum
cell growth flux and malic acid flux increased by 1.4 and Fermentations of E. coli W3110, WGS-10 harboring
5%, respectively. The solution space was expanded, suggesting p104ColiPck or p104ManPck were carried out, and the results
enhanced cell growth and malic acid production by the introduc- of which are summarized in Table 4. No malic acid was pro-
tion of the reversible PEP carboxykinase into E. coli (Fig. 3). duced in E. coli W3110 regardless of the plasmid introduced.
Based on these MFA results, aerobic production of malic To direct more carbon flux to malic acid pathway and to pre-
acid using the strains with amplified PEP carboxykinase activ- vent the production of acetic acid, the pta gene mutant strain E.
ity was proposed as the most efficient malic acid pathway. By coli WGS-10 was constructed. When the E. coli pckA gene was
overexpression of the PEP carboxykinase, PEP will be con- overexpressed in WGS-10, 1.42 g/L of malic acid was produced.
verted to oxaloacetate without losing high energy phosphate On the other hand, the final malic acid concentration reached up
bond, and will be further converted to malic acid by reversible to 9.25 g/L by the overexpression of the M. succiniciproducens
malate dehydrogenase and TCA cycle. Under aerobic condition, pckA gene. These results suggest that M. succiniciproducens
fumarase and succinate dehydrogenase are expressed, and TCA PEP carboxykinase is more suitable for malic acid production
cycle functions to convert oxaloacetate to malic acid via succinic by efficiently converting PEP to oxaloacetate. This result is con-
acid. Produced malic acid is not converted further to succinic sistent with our previous report that M. succiniciproducens is an
acid but is accumulated under aerobic condition because anaer- efficient succinic acid producer using PEP carboxykinase as a

Table 4
Results of fermentation
Strain Plasmids Cell concentration (OD600 ) Malic acid concentration (g/L) Malic acid yield Malic acid productivity (g/L/h)

W3110 p10499A 6.80 ± 0.3 0 0 (0) 0


W3110 p104ColiPck 6.79 ± 0.3 0 0 (0) 0
W3110 p104ManPck 5.88 ± 0.3 0 0a (0 b ) 0
WGS-3 p10499A 7.37 ± 0.3 0 0 (0) 0
WGS-3 p104ColiPck 7.44 ± 0.3 0 0 (0) 0
WGS-3 p104ManPck 5.58 ± 0.3 9.62 ± 0.9 0.61 ± 0.03 (0.82 ± 0.04) 0.69 ± 0.03
WGS-10 p10499A 7.74 ± 0.3 0 0 (0) 0
WGS-10 p104ColiPck 7.85 ± 0.3 1.42 ± 0.1 0.11 ± 0.01 (0.15 ± 0.01) 0.09 ± 0.002
WGS-10 p104ManPck 6.30 ± 0.3 9.25 ± 0.9 0.56 ± 0.03 (0.75 ± 0.04) 0.74 ± 0.03
a Mass yield (g malic acid/g glucose).
b Molar yield (mole malic acid/mole glucose).
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key enzyme providing high flux towards the reductive TCA cycle was 0.75 mol/mol glucose, and no by-product, such as acetic
[20]. The final cell concentration did not show much difference acid, was produced.
in spite of the large increase of final malic acid concentration, Even though, no significant by-products were detected, we
suggesting that the growth of the recombinant E. coli was not decided to further inactivate the ldhA and adhE genes, in addition
negatively affected by the amplification of the PEP carboxylation to the pta gene already knocked-out, for its use in fed-batch cul-
flux. tures where cells can encounter microaerobic condition. When
The time profiles of cell growth and concentrations of E. coli and M. succiniciproducens PEP carboxykinase were
glucose, malic acid and acetic acid obtained with WGS-10 overexpressed in this pta, ldhA and adhE mutant strain WGS-
(p104ManPck) are presented in Fig. 4. Glucose was completely 3, further enhancement of malic acid productivity and yield
consumed in 12 h of cultivation. At the end of fermentation of were not observed, even though lactic acid and ethanol were
WGS-10 (p104ManPck), 9.25 g/L of malic acid was produced not produced (Table 4). At the end of fermentation of WGS-
with the productivity of 0.74 g/L/h (Table 4). Malic acid yield 3 (p104ManPck), the concentrations of malic and acetic acids

Fig. 5. The calculated values of the intracellular fluxes (mmol/gDCW/h) in recombinant E. coli WGS-10 (p10499A)/WGS-10 (p104ColiPck)/WGS-10 (p104ManPck).
Abbreviations are: G6P, glucose-6-phosphate; D6PGL, gluconolactone-6-phosphate; D6PGC, 6-phospho gluconate; RL5P, ribulose-5-phosphate; X5P, xylurose-5-
phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP,
glyceraldehyde-3-phosphate; DHAP, dihydroxyacetonephosphate; GBP, 1,3-bisphosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; ACA, acetyl-CoA;
CIT, citrate; ICIT, isocitrate; AKG, ␣-ketoglutarate; SUCOA, succinyl-CoA; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate.
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were 9.62 and 0.86 g/L, respectively. The malic acid productivity WGS-10 (p104ManPck) was altered to favor malic acid for-
obtained with WGS-3 (p104ManPck) was 0.69 g/L/h, which is mation by the introduction of M. succiniciproducens pckA
slightly lower than that obtained with WGS-10 (p104ManPck). gene.
We believe that these two recombinant strains can be employed Altered metabolism of WGS-10 (p104ManPck) is also pre-
for enhanced malic acid production by fed-batch culture, which sented in Fig. 3, which shows the MFA results obtained
will be our future study. with WGS10 (p10449A), WGS10 (p104ColiPck) and WGS-
10 (p104ManPck). WGS10 (p10449A) is located on the X-axis
3.3. Metabolic flux analysis because no malic acid was produced. WGS10 (p104ColiPck)
is located on the dashed line suggesting that WGS10
MFA was carried out based on the fermentation data of (p104ColiPck) produced as much malic acid as it can since
WGS-10 (p10499A), WGS-10 (p104ColiPck) and WGS-10 the dashed line represents the metabolic feasible space of E.
(p104ManPck) to see if intracellular metabolic fluxes are reori- coli system. In case of WGS-10 (p104ManPck), it is located
ented as intended. The typical E. coli metabolic network, which in the expanded solution space (dark grey area) indicating
has irreversible PEP carboxykinase reaction, was employed that the metabolism of WGS-10 (p104ManPck) was altered
for the analysis of metabolic fluxes in WGS-10 (p10499A) beyond the original metabolic limit of WGS10 (p104ColiPck)
and WGS-10 (p104ColiPck) strains. For the analysis of the and produced more malic acid. The recombinant strain WGS-10
metabolic fluxes in WGS-10 (p104ManPck), the reversible PEP (p104ManPck) and its further derivative WGS-3 (p104ManPck)
carboxykinase reaction was used instead of the original irre- should be useful for the production of malic acid by fermentation
versible PEP carboxykinase reaction. The results of MFA for from renewable biomass.
WGS-10 (p10499A), WGS-10 (p104ColiPck) and WGS-10
(p104ManPck) are shown in Fig. 5.
4. Conclusion
MFA results showed that malic acid flux and PEP carboxy-
lation flux slightly increased by the overexpression of the E.
For the enhanced production of C4 -component such as malic
coli pckA gene. Only a small amount of malic acid was pro-
acid and succinic acid, reorientation of carbon flux from the
duced, which was not inconsistent with the previous report that
C2 - and/or C3 -compounds to C4 -compounds is required [23,24].
the overexpression of the E. coli pckA gene did not lead to the
In this study, MFA was used to predict the optimal malic acid
enhanced production of succinic acid [21]. However, it has also
production pathway and consequently a rational metabolic engi-
been reported that the activities of PEP carboxylase and PEP car-
neering strategy. More reliable in silico flux distribution could
boxykinase are correlated with each other; the activity of PEP
be determined by considering both malic acid production rate
carboxylase is proportional to the activity of PEP carboxyki-
and growth rate during the simulation.
nase while that of glyoxylate shunt is reverse proportional to
Even though the calculated metabolic flux values do not
the activity of PEP carboxykinase [22]. Therefore, it might be
represent the actual flux values, they are useful for under-
possible that the overexpression of E. coli pckA gene elevated
standing the whole metabolic characteristics and for developing
the expression level or activity of PEP carboxylase. Then, the
metabolic engineering strategies. According to the MFA result,
PEP carboxylation flux was increased by the action of enhanced
PEP carboxykinase genes of M. succiniciproducens and E.
PEP carboxylase activity. Nonetheless, the extent of increase
coli were overexpressed for the possible increase of the flux
was rather small, and the overall metabolic flux distribution of
from the C3 -component PEP to the C4 -component oxaloacetate
WGS-10 (p104ColiPck) was not much different from that of
[10,25,26]. Recombinant E. coli strains WGS-10 and WGS-3
WGS-10 (p10499A). These results suggest that the introduction
harboring p104ManPck, thus equipped with the M. succinicipro-
of the E. coli pckA gene did not allow flux redistribution towards
ducens pckA gene, were able to produce malic acid efficiently
enhanced malic acid formation.
under aerobic condition. Considering higher cell growth rate
On the other hand, WGS-10 (p104ManPck) showed differ-
under aerobic condition, these strains may be applied for the
ent flux distribution from those of WGS-10 (p10499A) and
production of malic acid to a high concentration with high
WGS-10 (p104ColiPck). One of the most notable changes
productivity by fed-batch culture. Furthermore, the strategy of
was the activation of PEP carboxykinase flux. In WGS-10
metabolic engineering reported here may be useful for devel-
(p10499A) and WGS-10 (p104ColiPck), which have a typical
oping strains for the production of other metabolites from
E. coli metabolic network, the flux from PEP to oxaloacetate
renewable resources.
was mediated only by PEP carboxylase. MFA result proposed
that PEP carboxylation was catalyzed by the introduced M. suc-
ciniciproducens PEP carboxykinase in WGS-10 (p104ManPck). Acknowledgements
Oxaloacetate thus produced was further converted to malic acid
in WGS-10 (p104ManPck). On the other hand, malic acid was This work was supported by the Genome-Based Integrated
converted to oxaloacetate in WGS-10 (p10499A) and WGS- Bioprocess Development Project of the Ministry of Science
10 (p104ColiPck) strains. MFA results proposed that pentose and Technology through the Korea Science and Engineering
phosphate (PP) pathway and amino acid biosynthesis fluxes Foundation (KOSEF). Further supports by the LG Chem Chair
were decreased while glycolytic flux increased in WGS-10 Professorship and Center for Ultramicrochemical Process Sys-
(p104ManPck). These results suggest that the metabolism of tem (KOSEF) are appreciated.
Author's personal copy

320 S.Y. Moon et al. / Biochemical Engineering Journal 40 (2008) 312–320

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