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Enzyme and Microbial Technology 45 (2009) 491–497

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

mprovement of succinate production by overexpression of a cyanobacterialarbonic anhydrase in Escherichia coli

an Wanga,b, Qiang Lia,b, Wangliang Lia, Jianmin Xinga,∗, Zhiguo Sua

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, ChinaGraduate University of Chinese Academy of Sciences, Beijing 100049, China

r t i c l e i n f o

rticle history:eceived 1 May 2009eceived in revised form 27 July 2009ccepted 6 August 2009

a b s t r a c t

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacteriumAnabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, theactivity of CA was 21.8 U mg−1 protein, whereas non-detectable CA activity was observed in the controlstrain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg−1

−1 −1

eywords:uccinatescherichia coliarbonic anhydrase (CA)icarbonate

protein to 1.13 U mg protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol molglucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just0.19 mol mol−1 glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucosephosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be furtherincreased to 1.26 mol mol−1 glucose. It could be concluded that the enhancement of the supply of HCO3

−

ssion

arbon dioxide in vivo by ecaA overexprecoli.

. Introduction

Succinate and its derivatives are valuable chemicals exten-ively used in food, agricultural and pharmaceutical industries1]. Traditionally, succinate is produced via petroleum-basedhemical processes. Nowadays, green technology is becoming ariving force in the chemical industry. Renewable feedstocks haveeen used to produce succinate through the fermentative way2].

Escherichia coli, a facultative anaerobe, primarily ferments glu-ose to ethanol, formate, acetate and lactate with only detectablemounts of succinate under anaerobic condition [3]. Nevertheless,etabolic flux analysis revealed that the maximum achievable suc-

inate molar yield in E. coli is 1.647 [4]. Furthermore, E. coli isenetically engineered with relative ease, and has the advantagesf fast growth and simple requirements for nutrients. So it has beentudied as one of the most promising strains for succinate pro-

uction through metabolic engineering these years. The succinateormation pathway of E. coli is shown in Fig. 1. The carboxyla-ion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA) catalyzedy PEP carboxylase (PEPC) is considered as the most important

∗ Corresponding author at: Institute of Process Engineering, Chinese Academyf Sciences, P.O. Box 353#, Zhongguancun Bei-er-tiao 1, Haidian District, Beijing00190, China. Tel.: +86 10 62550913; fax: +86 10 62550913.

E-mail addresses: danwang088@gmail.com (D. Wang),mxing@home.ipe.ac.cn (J. Xing).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.08.003

is an effective strategy for the improvement of succinate production in E.

© 2009 Elsevier Inc. All rights reserved.

reaction. 1 mol CO2 is assimilated in this step to form the first C4metabolites OAA. This step was improved for better utilization ofthe PEP pool. One strategy is to overexpress native PEP carboxy-lase (ppc) [5], heterologous ppc [6] and PEP carboxykinase (pepck)[7–8] to enhance the carboxylation of PEP. Another is to block thecompetition pathways of the PEP carboxylation, such as to inacti-vate pyruvate:formate lyase (pfl) and lactate dehydrogenase (ldh)[9–10], glucose phosphotransferase (ptsG) [11], alcohol dehydro-genase (adh) [12], and phosphate acetyltransferase–acetate kinase(pta–ack) [13].

However, few literatures reported the better utilization of CO2for succinate synthesis in E. coli. Although MgCO3 or NaHCO3 wereadded to the culture medium in some works [9,11–13] and pureCO2 was also used [11–13], the real form of initial CO2 fixationin E. coli was seldom considered. In fact, the active substrate forPEPC is not CO2, but the chemically less reactive bicarbonate anionHCO3

− [14]. Compared with CO2, a non-polar micromolecular,HCO3

− is hard to permeate the cell membrane. Furthermore, thehydration/dehydration reaction speed between CO2 and HCO3

− isrelatively slow [15–17]. So there might not be sufficient HCO3

−

spontaneously made in vivo to meet the biosynthetic needs. Theenzyme carbonic anhydrase (CA, EC.4.2.1.1) encoded by cynT orcan in E. coli catalyze the interconversion of CO2 and HCO3

− as:

H+ + HCO3

− ↔ CO2 + H2O [15]. However, cynT is part of the cynoperon, which can be expressed only when induced by cyanate[18]. Can expression is largely influenced by the growth rate and thegrowth cycle [19]. It is difficult to sustain the increasing demand ofHCO3

− in the succinate fermentation. Thus, special strategy should

492 D. Wang et al. / Enzyme and Microbial Technology 45 (2009) 491–497

F al succE sferasp pyc, pa

bb

sbcico

dgpt(apvHk

2

2

tht[S[tp

ig. 1. Mixed-acid anaerobic metabolism of E. coli. Thick arrows represent the centr. coli. ppc, PEP carboxylase; ldh, lactate dehydrogenase; ptsG, glucose phosphotranta, phosphate acetyltransferase; ack, acetate kinase; adh, alcohol dehydrogenase;nhydrase.

e adopted to enhance the supply of HCO3− in vivo. Then CO2 can

e better utilized.In this paper, the supply of HCO3

− was enhanced in vivo foruccinate synthesis by overexpressing a heterogeneous CA encodedy cyanobacterium Anabaena sp. 7120 ecaA gene. The ecaA gene washosen for its more highly evolution adaptively for CO2 assimilationn photosynthetic organism, compared to can in heterotrophic E.oli. It was also chosen for the relative ease of genetic manipulationf prokaryotic organism, compared to higher plants.

The expression plasmid pET-ecaA was constructed and intro-uced into E. coli BL21 (DE3). The effects of CA on the cell growth,lucose consumption kinetics and the distribution of fermentationroducts were explored. Furthermore, the effect of the concentra-ion of magnesium carbonate on succinate production in E. coli BL21DE3) bearing ecaA was examined. Overexpression of ecaA was alsopplied to an E. coli K-12 mutant strain DC1515 deficient in glucosehosphotransferase (ptsG), lactate dehydrogenase (ldh) and pyru-ate:formate lyase (pfl). The strategy of enhancing the supply ofCO3

− in vivo for succinate production in E. coli, to the best of ournowledge, has not been reported.

. Materials and methods

.1. Strains and plasmids

The strains, plasmids and oligonucleotides used for polymerase chain reac-ion (PCR) amplification were shown in Table 1. E. coli strain BL21 (DE3) [F− ompTsdSB (rB

− mB−) gal dcm (DE3)] was purchased from Novagen, Merck. Cyanobac-

erium Anabaena sp. 7120 came from the Pasteur Institute in France. DC1515pflA::Cam ldhA::Tn10 ptsG400::Kan in W1485] was kindly donated by Prof. Clark,outhern Illinois University. Manipulation of DNA was done as described before20]. The ecaA gene was amplified with primers ca1 and ca2 using cyanobac-erium Anabaena sp. 7120 genomic DNA as the template. The sequences of theserimers were as follows—ca1:5′-AACCGCGGATCCATGAGTAGTAC-3′ (BamHI restric-

inate forming pathway. Dot arrows represent the heterogenous gene introduced toe; pfl, pyruvate:formate lyase; frd, fumarate reductase; fhl, formate hydrogenlyase;yruvate carboxylase; mae, malic enzyme; pepck, PEP carboxykinase; ecaA, carbonic

tion site underlined); ca2: 5′-GGACAGAGTCGACTTAAATGGCTTC-3′ (SalI restrictionsite underlined). The PCR was performed for 30 cycles of denaturation for 30 s at94 ◦C, annealing for 40 s at 48 ◦C, and extending for 50 s at 72 ◦C. The single prod-uct was isolated, purified and ligated into the expression plasmid pET-21a(+) withBamHI and SalI excision to give plasmid pET-ecaA. Then the plasmid was introducedinto a common E. coli BL21 (DE3) to form a recombinant designated as SD131. Plas-mid pTrc-ecaA, which can be expressed in an E. coli K-12 mutant strain DC1515, wassimilarly constructed. The ecaA gene was amplified with primers ca3 and ca4 usingthe same DNA template and ligated into the expression plasmid pTrchisB with EcoRIand HindIII excision to give plasmid pTrc-ecaA. The sequences of primers used wereas follows—ca3: 5′-AGGGAATTCAGGGAAATGAGTAGTAC-3′ (EcoRI restriction siteunderlined); ca4: 5′-AGGCAAGCTTTATGACATAGCAACAC-3′ (HindIII restriction siteunderlined). The PCR was performed using the same conditions as before. The strainDC1515 harboring plasmid pTrc-ecaA was designated as SD133. Parental strainsBL21 (DE3) carrying the corresponding backbone plasmid pET-21a(+) and DC1515carrying the plasmid pTrchisB were constructed as the control strains SD130 andSD132. Each PCR fragment was verified by DNA sequencing at the Shanghai SangonBiological Engineering Technology & Service Co., Ltd. E. coli Top 10 was used forpropagation and amplification of plasmids used in this work.

2.2. Fermentation mediums and growth conditions

All E. coli strains were cultured in Luria-Bertani (LB) medium at 37 ◦C. Thetransformed colonies were selected from LB plates containing the appropriateantibiotic(s). Antibiotics were included as necessary at the following concentra-tions: 50 �g ml−1 ampicillin, 50 �g ml−1 kanamycin, 10 �g ml−1 tetracycline and30 �g ml−1 chloramphenicol. A seed inoculum of 200 �l from an overnight 5-mlculture was added to a 100-ml flask containing 20 ml of LB medium with the appro-priate antibiotic(s) to facilitate aerobic growth. When OD600 reached 1.0, 1 ml of thisculture was inoculated to a 50-ml sealed serum tube. The tube contained 10 ml of LBmedium supplemented with 0.2 g MgCO3 (added to the tube before autoclaving tomaintain the pH and supply CO2), the appropriate antibiotic(s), 0.1 mM isopropyl-�-

d-thiogalactopyranoside (IPTG), and approximately 15 g glucose per liter. The initialpH of the medium was set to be pH 7.0. The tubes were sealed airtight, allowing noexchange of gas with the outside atmosphere. The headspace was not replaced withCO2. This setup which allowed the cells to grow faster in the presence of oxygenwas preferred in the fermentation experiments. It helps generating biomass beforeanaerobic fermentation takes place. The cultures were grown in a rotary shaker at

D. Wang et al. / Enzyme and Microbial Technology 45 (2009) 491–497 493

Table 1Strains, plasmids and oligonucleotides used in this study.

Strains Description Source/restriction site

BL21(DE3) F− ompT hsdSB (rB− mB

−) gal dcm (DE3) Novagen, MerckDC1515 pflA::Cam ldhA::Tn10 ptsG400::Kan in W1485 Clark, unpublishedAnabaena sp.7120 Providing ecaA gene Pasteur Institute in FranceSD130 BL21 (DE3) harboring pET-21a(+), SD131 control strain This studySD131 BL21 (DE3) harboring pET-ecaA, overexpressing CA This studySD132 DC1515 harboring pTrchisB, SD133 control strain This studySD133 DC1515 harboring pTrc-ecaA, overexpressing CA This study

PlasmidspET-21a(+) Expressing vectors with T7 promoter Novagen, MerckpTrchisB Expressing vectors with Trc promoter InvitrogenpET-ecaA ecaA gene cloned from Anabaena sp. 7120 under the T7 promoter of pET-21a(+) This studypTrc-ecaA ecaA gene cloned from Anabaena sp. 7120 under the Trc promoter of pTrchisB This study

Oligonucleotides′ ′

′′

3svMaa

2

bEcbtfustaaimpcotodNipotetat8pS

2

f(fHaai1c5a

ca1 5 -AACCGCGGATCCATGAGTAGTAC-3ca2 5′-GGACAGAGTCGACTTAAATGGCTTC-3′

ca3 5′-AGGGAATTCAGGGAAATGAGTAGTAC-3ca4 5′-AGGCAAGCTTTATGACATAGCAACAC-3

7 ◦C and 150 rpm for 38 h. For anaerobic growth studies, cultures were grown inerum tubes lacking MgCO3 in a 1:1 mixture of LB medium and M9 medium (to pro-ide buffering) supplemented with glucose and the appropriate antibiotic(s). ThegCO3 concentration was varied from 0 to 50 g l−1 for the investigation of carbon-

te effects; otherwise it was 20 g l−1. Samples for analysis were removed at intervalsnoxically with a syringe.

.3. Enzyme activity assay

To analyze enzyme activities, cell extracts of the E. coli strains were preparedy washing the cell pellets with appropriate buffer (50 mM Tris–HCl (pH 8.0), 1 mMDTA, 1 mM EGTA, 0.05% (v/v) NP40 and 0.1 mM DTT) and disrupting the suspendedells by sonicating at 100 W for 10 min at 1-s interval on ice. Cell debris was removedy centrifugation at 10,000 × g for 10 min at 4 ◦C. The supernatant was further cen-rifuged at 10,000 × g at 4 ◦C for 20 min and the resulting supernatant was usedor the assay of enzyme activity. The CA activity was measured electrometricallysing a veronal buffer according to the method of Wilbur [21]. Briefly, 500 �l ofupernatant was added to 5 ml of buffer in a water-jacketed reaction vessel cooledo 2 ◦C. The reaction was started by the addition of 4.5 ml of CO2-saturated water,nd the catalyzed hydration reaction was measured by following the drop in pH ofpproximately 1 unit as a result of the production of H+. CA activity was expressedn Wilbur–Anderson (WA) units per mg of protein and was calculated using the for-

ula [(t0/t − 1) × 10]/mg protein, where t0 and t represent the time required for theH to change from 8.0 to 7.0 in a buffer control and cell extracts, respectively. The PEParboxylase (PEPC) activity was assayed by monitoring the decrease in absorbancef NADH of 340 nm using malate dehydrogenase as a coupling enzyme, modifica-ion of Kodaki’s method [22]. The 1-ml reaction mixture for PEPC analysis consistedf 50 mM HEPES (pH 7.3), 5 mM PEP, 10 mM MgCl2, 5 mM NaHCO3, 4 U of malateehydrogenase, 0.2 mM NADH and 25 �l of cell extract. The extinction coefficient forADH at 340 nm was 6.22 cm−1 mM−1. 1 U of PEPC activity was defined as the activ-

ty of oxidizing 1 �M NADH per min at 30 ◦C. Moreover, the cell extract was partiallyurified to separate the PEPC from the CA by ion-exchange chromatography for rig-rous assessment of each. The pH of the cell extract was adjusted to 6.5, and thenhe cell extract was applied to a CM Sepharose FF column (1.0 cm × 4.0 cm, GE, USA)quilibrated with Buffer A (50 mM sodium phosphate, pH 6.5). It was eluted withhe same buffer until the fraction P0 containing PEPC had been completely elutednd pooled. Elution was continued with the same buffer containing 1 M NaCl andhe fraction P1 containing CA was pooled. The pH of the fractions was adjusted to.0. Electrophoresis and enzyme activity assay of the two fractions were done. Therotein concentration was measured by Lowry’s method (modified Lowry reagent,igma), using bovine serum albumin as standard.

.4. Analytical methods

Cell growth was monitored by the optical density at 600 nm and was trans-ormed into dry cell weight (DCW) using the coefficient as: dry cell massg l−1) = 0.48 × OD600 [23]. Glucose consumption and products (succinate, lactate,ormate, acetate and ethanol) formation during fermentation were analyzed byPLC using a Bio-Rad Aminex HPX-87H ion-exchange column (7.8 mm × 300 mm)nd HP1200 chromatography working station system equipped with UV absorbance

nd refractive index detectors. Samples of anaerobic culture were removed anox-cally and centrifuged at 10,000 × g for 10 min. Each supernatant was diluted with0 volumes of 5 mM H2SO4, and 20 �l of the diluted sample was injected. Theolumn was eluted isocratically at a rate of 0.6 ml min−1 with 5 mM H2SO4 under5 ◦C. The approximate retention time were as follows: glucose, 8.9 min; succiniccid, 11.5 min; lactic acid, 12.5 min; formic acid, 13.65 min; acetic acid, 14.86 min;

BamHISalIEcoRIHindIII

ethanol, 21.4 min. Glucose concentrations were also monitored by using a SBA sen-sor machine (Institute of Microbiology, Sandong, China). The expression of ecaAwas visualized on a 12% sodium dodecyl sulfate polycrylamide gel with CoomassieBrilliant Blue staining.

3. Results and discussion

3.1. Construction and characterization of the plasmids

Plasmid pET-ecaA, containing the ecaA gene from Anabaena sp.7120 under the control of the T7 promoter, was constructed. Gelelectrophoresis of the BamHI and SalI digesting samples was doneto identify the positive clone. The plasmid pTrc-ecaA with ecaAgene under the control of the Trc promoter was also character-ized by EcoRI and HindIII excision. The approximately 800-bp smallsegment was the same length of ecaA (refer to NCBI sequenceBA000019) as expected, which showed that the PCR product wasindeed ligated to the target plasmid. The integrity of the cloned ecaAand open reading frame was further checked by sequencing. TheSDS-PAGE was done of SD131 and SD133 induced by 0.1 mM IPTG.Soluble crude extracts were prepared from fermentative mediumwithout MgCO3 under anaerobic conditions every 2 h. A band ofincreasing intensity around the 30 kDa size range was observed onthe gel upon Coomassie Brilliant Blue staining, which was consis-tent with the molecular weight of CA predicted from its nucleotidesequence by software Dnaman (Version 5.2.2, Lynnon BioSoft) (datanot shown). So the heterogenous ecaA gene can be expressed in E.coli successfully.

Plasmids pET-ecaA and pTrc-ecaA were further characterizedby determining the activities of CA under anaerobic conditions.The PEPC activities of the recombinant strains were also analyzed.From Table 2, it can be seen that the CA activity of strain SD131was 21.8 U mg−1 protein. It was 5.7-fold higher than the activity ofSD133, which was 3.84 U mg−1 protein. Control strains BL21 (DE3)harboring the plasmid pET-21a(+) and DC1515 harboring the plas-mid pTrchisB showed non-detectable CA activities as expected.Overexpression of ecaA not only increased the activity of CA, butalso increased the activity of PEPC. The PEPC activities of strainSD131 and SD133 were 1.13 and 0.86 U mg−1 protein, respectively,whereas their activities were approximately 0.2 U mg−1 protein inthe control strains. Increased CA activity can serve to enhance the

supply of HCO3

− and increased PEPC activity showed an increasedcarboxylation capacity.

The activity of PEPC in the recombinant E. coli strains increaseswith the ecaA overexpression. But these were measured in cellextracts having overexpressed carbonic anhydrase, which may

494 D. Wang et al. / Enzyme and Microbial

Table 2CA and PEPC activities in U mg−1 of total protein from anaerobic cultures of strains6 h after induced by 0.1 mM IPTG.

Strain Activity (U mg−1)

CAa PEPCb

SD130 ND 0.20 ± 0.02SD131 21.8 ± 0.09 1.13 ± 0.15SD132 ND 0.21 ± 0.02SD133 3.84 ± 0.06 0.86 ± 0.11

CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase.Medium contained glucose approximately 13.6 g l−1 in LB medium.All experiments were performed a minimum of three independent sets, and the datarepresent mean ± SD.ND, not detected (less than 0.01 U mg−1).

a Calculated as Wilbur–Anderson (WA) units per milligrams of protein, using theformula [(t /t − 1) × 10]/mg protein, where t and t represent the time required fort

c

hrfoihw(bEeffifretucasEimaPcgpi

TC

CAN

0 0

he pH to change from 8.0 to 7.0 in a buffer control and cell extracts, respectively.b Calculated as �mol NADH used per milligrams of protein per minute when

oupled with malate dehydrogense.

ave an effect on PEPC enzyme assay. So further study was car-ied out to partially purify the cell extract to separate the PEPCrom the CA by ion-exchange chromatography, and the assessmentf each fraction was done to determine whether the differences due to the presence of CA or the in vivo conditions generatedigher expression of the PEPC. The ion-exchange chromatographyas done based on the difference of isoelectric points for PEPC

5.56) and CA (7.15), predicted from their nucleotide sequencesy ProtParam tool (http://www.expasy.org/tools/protparam.html).lectrophoresis of the individual fractions derived from ion-xchange chromatography showed that nearly 90% CA was purifiedrom the cell extract and concentrated in the P1 fraction (elutionraction) (data not shown). Enzymatic activities analyses are shownn Table 3. Although the PEPC had a higher specific activity in P0raction (breakthrough fraction) due to the partial removal of impu-ities, the total activity was nearly the same as in the crude cellxtract. So it was not owing to the presence of CA in the extracthat PEPC activity in vitro increased. The possible reason may bepper regulation of DNA transcription or mRNA translation, whichhanged with the organism demand and indicated a metabolicdaptation to the genetic disturbance [24]. Millard et al. found thatuccinate production can be enhanced by overexpression of ppc in. coli [5]. In this study, the increased availability of HCO3

− and thencreased PEPC activity which are caused by ecaA overexpression

ay together contribute to the improvement of the succinate yieldt the end of the anaerobic fermentation. Whether the increased

EPC activity caused by ecaA overexpression is sufficient for suc-inate production or should PEPC activity be further enhanced byene overexpression and then co-express ppc with ecaA for bettererformance? This question is what we are currently investigat-

ng. Other anaplerotic enzymes such as malic enzyme (ME) and

able 3A and PEPC activities in individual fraction of cell extracts derived from ion-exchange ch

Fraction Total protein (mg) Specific activity (U mg−1)

CAa PEPCb

SD131 total cell extract 10.29 ± 0.66 21.8 ± 0.09 1.13 ± 0.15SD131 P0 5.74 ± 0.29 3.67 ± 0.11 1.89 ± 0.22SD131 P1 3.85 ± 0.09 51.06 ± 0.12 NDSD133 total cell extract 12.62 ± 0.82 3.84 ± 0.06 0.86 ± 0.11SD133 P0 8.18 ± 0.55 0.53 ± 0.09 1.25 ± 0.12SD133 P1 2.84 ± 0.23 15.02 ± 0.45 ND

A, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase.ll experiments were performed a minimum of three independent sets, and the data repD, not detected (less than 0.01 U mg−1).a Calculated the same as Table 2.b Calculated the same as Table 2.

Technology 45 (2009) 491–497

pyruvate carboxylase (PYC) are all HCO3− required, and research

work has shown that they can enhance C4 metabolism while beingoverexpressed in E. coli [6,10,12]. If ecaA can co-express with them,adequate HCO3

− can be supplied to these enzymes and the velocityof CO2 fixation may be accelerated. However, it is not easy work toco-express genes since they must be expressed at approximatelybalanced levels to avoid the accumulation of toxic intermediatesor bottlenecks that result in growth inhibition or suboptimal yields[25].

3.2. Cell growth and glucose consumption of SD131

To further characterize the effect of ecaA overexpression, thecell growth and glucose consumption of strain SD130 and SD131were examined without MgCO3. From Fig. 2a, it can be seen thatecaA overexpression lengthened the duration of the lag phase forthe recombinant strain. However, by the end of the fermentation,the cell density of SD131 reached 2.0 and the dry cell weight calcu-lated was 0.96 g l−1. The dry cell weight was 117% higher than thatof control strain which was 0.82 g l−1 calculated from the cell den-sity. Considering the increased HCO3

− supplement and increasedPEPC activity caused by ecaA overexpression all led to more activecell metabolism: producing abundant anaplerotic chemicals (suchas OAA and succinate et al.) and key biosynthetic blocks (such asarginine, nucleotide base and fatty acid) [19], the lag phase shouldbe shortened. But beyond the expectation, the overexpression ofecaA caused the growth retardation. This was not due to the forma-tion of inclusion bodies, since a moderate IPTG concentration wasused in this study to avoid the formation of inclusion bodies in thecells. Possibly it was solely due to ribosome load caused by proteinoverexpression [26]. From Fig. 2b, it can be seen that the glucoseconsumption rate was not obviously affected by overexpression ofecaA. The recombinant strain SD131 can consume 13.6 g glucose in16 h the same as the control. From Fig. 2c, it can be seen that the pHvalue of the fermentation broth decreased through the anaerobicculture, but could still remain above 5.8 at the end as a result of pHbuffering.

3.3. Batch fermentation performance of SD131

Batch fermentations were performed with strain SD131 toexplore the effect of ecaA overexpression on the metabolites distri-butions. Due to the buffering of MgCO3, the pH values at the end ofthe fermentations were maintained about 6.0. The deduced molaryields of different metabolites based on the consumed glucose and

the calculated C4 metabolism ratio, O/R ratio and the succinateproductivity are shown in Table 4. The succinate yield of SD131reached 0.391 mol mol−1 glucose. It was 2.1-fold higher than thatof the control strain. The C4 metabolism ratio (C4 product per sumof C2 + C3 + C4) of SD131 was 20.8%, which was 10.1% for SD130.

romatography.

Total activity (U) Purification fold Recovery (%)

CA PEPC CA PEPC CA PEPC

224.32 ± 0.93 11.63 ± 1.54 1 1 100 10021.06 ± 0.62 10.82 ± 1.26 – 1.67 9.39 93.04

196.58 ± 0.46 ND 2.34 – 87.63 –48.46 ± 0.76 10.85 ± 1.39 1 1 100 100

4.32 ± 0.71 10.22 ± 0.95 – 1.45 8.91 94.1942.66 ± 1.29 ND 3.91 – 88.03 –

resent mean ± SD.

D. Wang et al. / Enzyme and Microbial

Fig. 2. The cell growth and glucose consumption of the strains SD130 and SD131.(a) Growth kinetics curve; (b) Glucose consumption curve; (c) The pH values of themedium. The fermentative culture is 1:1 LB and M9 supplemented with 13.6 g l−1

glucose. Filled square, SD130; filled circle, SD131.

Table 4Effect of ecaA overexpression on the production of fermentation products.

Straina Yield (mol of product per mol of glucose)

Succinate Lactate Formate Acetate Etha

SD130 0.186 ± 0.021 0.455 ± 0.012 0.768 ± 0.019 0.720 ± 0.024 0.48SD131 0.391 ± 0.029 0.717 ± 0.109 0.445 ± 0.009 0.356 ± 0.013 0.41SD132 0.935 ± 0.027 0.036 ± 0.008 0.004 ± 0.001 0.522 ± 0.011 0.48SD133 1.262 ± 0.043 0.035 ± 0.008 0.002 ± 0.001 0.403 ± 0.013 0.38

All experiments were performed a minimum of three independent sets, and the data repra Medium contained glucose approximately 13.6 g l−1 and magnesium carbonate 20 g l−b C4 metabolism ratio represents the C4 molar yield per sum of C4 + C3 + C2 molar yieldc O/R ratio is the ratio of oxidation products per reduction product, which is calculated

carbohydrate (CH2O)x and expressing each 2H in excess as −1 value and each 2H shortagis −2.

d Succinate productivity is calculated as the maximum concentration of succinate in th

Technology 45 (2009) 491–497 495

The succinate productivity calculated based on the maximum suc-cinate concentration in the medium of SD131 was 0.116 g l−1 h−1,while it was 0.058 g l−1 h−1 for the control strain SD130. Theseresults indicated that the recombinant strain overexpressing ecaAcan significantly shift the metabolic fluxes to succinate production,showing potential ability for industrial use. Furthermore, as a directsubstrate of PEPC, HCO3

− promoted the velocity of the carboxyla-tion of PEP.

From Table 4, it can be seen that the yield of acetate, formateand ethanol decreased. In E. coli, the total quantity of formate pro-duced should be the same as the sum of ethanol and acetate underanaerobic fermentations [27]. The table actually just shows theextracellular formate that has not been processed to CO2 and H2by formate hydrogen lyase. But it could be calculated that the ecaAoverexpression decreased the total formate yield. The hydrolysisof formate to CO2 seems to be increased in SD131, too. The higheryield of lactate in SD131 compared to SD130 was unexpected. Per-haps part of the reduction of the flux through pyruvate–formatelyase flows to the pathway of lactate. It indicated that the intro-duction of ecaA to E. coli cannot reduce all the by-products level,some other metabolic improvement is needed, such as introduc-ing the pyruvate carboxylase (pyc) or knocking out the ldh gene toreduce the lactate production.

There is less need to worry about the CO2 fixation in naturalsuccinate producing bacteria Anaerobiospirillum succiniciproducens[28], Actinobacillus succinogenes [29] and Mannheimia succinicipro-ducens [30], because in these strains, the key enzyme for PEPcarboxylation is PEP carboxykinase, which utilizes CO2 as the co-substrate [31,32]. But in E. coli, PEP accepts CO2 in the form ofHCO3

−, which should be improved in vivo due to causes explainedin the introduction. It is well known that the level of CO2 in themedium exerts a significant effect on the succinate production ofthe natural succinate producing bacteria, e.g., A. succinogenes’ succi-nate yields increased with increasing CO2 [33]. Thus, the enhancedsuccinate production under improved HCO3

− supply through ecaAovexpression in E. coli was understandable.

3.4. Effect of magnesium bicarbonate concentration on succinateproduction of SD131

The aim of overexpression of ecaA is to strengthen the supplyof HCO3

− in the cytoplasm, so the effect of the concentration ofmagnesium carbonate as CO2 source was explored. The magnesium

carbonate of the medium varied from 0 to 50 g l−1 to investigate theeffects on the engineered strain SD131 and the control strain BL21(DE3) harboring pET-21a(+). From Fig. 3, it can be seen that in gen-eral, both strains showed greater succinate production as carbonateavailability increased, although the dependency on the carbonate

pH C4 metabolismratio(%)b

O/R balancec Succinateproductivity(g l−1 h−1)d

nol

8 ± 0.0016 6.15 ± 0.03 10.1 1.023 0.058 ± 0.0047 ± 0.016 6.08 ± 0.06 20.8 1.002 0.116 ± 0.0096 ± 0.021 6.06 ± 0.05 47.2 0.966 0.228 ± 0.0063 ± 0.016 6.02 ± 0.02 60.6 1.676 0.313 ± 0.011

esent mean ± SD.1 in LB medium..by assigning an arbitrary O/R value of 0 to compound with empirical formula of a

e as +1 value; i.e., formate is +1, acetate is 0, lactate is 0, succinate is +1 and ethanol

e medium divided by the time reached the peak value.

496 D. Wang et al. / Enzyme and Microbial

FpsB

casswamdp2cmd1SMTC

productivity of 1.3 g l−1 h−1 in a 2.5-l reactor while harboring pyc

ig. 3. Effect of ecaA overexpression with MgCO3 supplementation on succinateroduction in E. coli strains. (a) Succinate yield; (b) Succinate concentration. Filledquare, SD130; filled circle, SD131. (c) The pH values at the end of the fermentation.lack, SD130; light gray, SD131.

oncentration was variable. But when the magnesium carbon-te concentration was >20 g l−1, succinate production of the twotrains decreased. As the carbonate concentration was increased,uccinate production increased more in the control strain SD130,ith 0.823 mmol l−1 succinate with no addition of carbonate

nd 9.46 mmol l−1 with 20 g l−1 MgCO3, the yield of which wasore than 10 times increased. The strain SD131 showed less

ependent on MgCO3 supplementation for succinate production,roducing 5.66 mmol l−1 succinate with no carbonate added and9.70 mmol l−1 with 20 g l−1 MgCO3, just 5-fold increased. Espe-ially under the condition of lower concentration of MgCO3 in theedium, SD131 showed greater succinate producing ability, pro-

ucing 11.963 mmol l−1 (0.203 mol mol−1) with 5 g l−1 MgCO3 and6.873 mmol l−1 (0.223 mol mol−1) with 10 g l−1 MgCO . The strain

3D130 just produced 1.613 mmol l−1 (0.024 mol mol−1) with 5 g l−1

gCO3 and 2.690 mmol l−1 (0.036 mol mol−1) with 10 g l−1 MgCO3.hese results indicated that ecaA overexpression can enhance theO2 fixation under conditions of both lower and higher concen-

Technology 45 (2009) 491–497

trations of CO2, providing the recombinant strain SD131 metabolicflexibility on the reliance of CO2. More specifically, ecaA overex-pression increased HCO3

− supply when a certain amount of CO2was provided, which enabled anaplerotic reaction head to succinateproduction.

From Fig. 3c, it can be seen that at the end of the fermentation,the pH values can remain above 5.8 with MgCO3 addition, whileit dropped to around 4.6 in the medium without MgCO3. Exces-sive MgCO3 was not utilized at the end of the batch tests whenthe MgCO3 concentration was >10 g l−1. There were only nuance inpH values between SD130 and SD131 when a certain quantity ofMgCO3 added. So the effect of pH can be neglected when comparingthe effect of MgCO3 concentration on the two strains.

We used MgCO3 here, while Kwon et al. [34] used NaHCO3 intheir study, who reported that bicarbonate concentration in themedium was proportional to the C4 metabolism enhancement in E.coli overexpressing NADP-dependent malic enzyme (MaeB). SinceHCO3

− is hard to permeate the membrane, the NaHCO3 used intheir study could not directly supply HCO3

− for MaeB, which isHCO3

− required. The NaHCO3 may release CO2 the same as MgCO3does in our study due to acid formation in the fermentation. ThenCO2 permeates the membrane and eventually transforms to HCO3

−

in the cytoplasm where PEPC and MaeB located. Increased bicar-bonate or magnesium carbonate concentration indicated increasedCO2 source, therefore the C4 metabolism enhanced with increasedbicarbonate or carbonate level in both studies. Interestingly, glu-cose consumption and succinate production were suppressed tosome extent in the medium with more than 20 g l−1 MgCO3 in thisstudy. Kwon et al. also found the upper limit for bicarbonate addi-tion was 10 g l−1 [34]. Another related previous report was aboutMannheimia succiniciproducens [35], which also indicated that sur-feit carbonate may harm the metabolism of the organism. So it wasimportant to optimize the carbonate concentration in the fermen-tation broth.

3.5. Batch fermentation performance of SD133

Overexpression of ecaA has been shown to be effective inenhancing succinate production in E. coli BL21 (DE3), but thelactate production was still high in SD131. So we constructed pTrc-ecaA which can be expressed in a mutant strain DC1515. Batchfermentations were performed with strain SD133 to investigatethe metabolites distribution affected by ecaA overexpression. Thededuced molar yields of different metabolites, the calculated C4metabolism ratio, O/R ratio and the succinate productivity areshown in Table 4. The succinate yield of SD133 was 1.262 mol mol−1

glucose at the end of the fermentation. It was 1.35-fold higher thanthe yield of the control strain which was just 0.935 mol mol−1 glu-cose. This indicated that overexpression of ecaA also functionedeffectively in strain DC1515 to increase succinate production. TheC4 metabolism ratio of SD133 was 60.6%, which was 47.2% forSD132. The O/R of recombinant strain SD133 was 1.676, which indi-cated that by overexpressing the ecaA gene, the increased HCO3

−

concentration affected the redox state of the cell as well as theflux distribution. The cell preferably utilized the succinate pathwayfor NADH recycling more than the ethanol pathway. So part of theincreased succinate production came at the cost of the decrease inethanol production. The succinate productivity calculated based onthe maximum succinate concentration in the medium of SD133 was0.313 g l−1 h−1, while it was 0.228 g l−1 h−1 for the control strainSD132. E. coli AFP111, which is similar to DC1515, can reach a

gene [36]. However, in this study, the fermentation was executed inserum tubes and the final cell concentration just reached 0.66 g l−1,while it was 12.95 g l−1 in Vemuri et al.’s study. The specific pro-ductivity of succinate for strain SD133 achieved 474 mg g−1 dry cell

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D. Wang et al. / Enzyme and Mic

eight h−1, much higher than that of recombinant AFP111 (about35 mg g−1 dry cell weight h−1).

The succinate yield of SD133 just increased by 35%, while theuccinate yield of SD131 increased by 110%. The values in Table 3bout yields of metabolites showed that pTrc-ecaA affected DC1515n a less obvious way from that pET-ecaA affected BL21 (DE3), whichndicated the plasmid–host interactions were clearly dependent onhe host cell genetic background and the plasmid itself, not justhe gene carrying on the plasmid [37]. Furthermore, DC1515 has

heavier metabolic burden causing by gene inactivation in thearbon metabolic pathway, so it shows less sensitive to geneticanipulation than BL21 (DE3). But as a promising strain for suc-

inate production, SD133 deserves our effort to further increase itsuccinate yield, the next step may be inactivating the acetate andthanol pathway.

. Conclusions

This study demonstrates that it is possible to increase the avail-bility of intracellular HCO3

− through genetic engineering on theetabolic patterns of E. coli and therefore enhance CO2 utilization

n C4 metabolism under anaerobic conditions. We propose thatore rapid equilibration between HCO3

− and CO2 caused by ecaAverexpression enhance the supply of HCO3

− for PEPC enzyme.herefore, the PEP carboxylation can be easily processed and it isikely that the flux from PEP to OAA increases, which eventuallyesults in higher succinate production. Utilization of HCO3

− ratherhan CO2 should be paid more attention to further explore the suc-inate production ability of E. coli. Our results will have a positivempact on using E. coli as an industrial strain to produce succinate.

cknowledgements

This research was supported in part by grants from the Knowl-dge Innovation Program of Chinese Academy of Sciences (Granto. KSCX2-YW-G-021). The authors thank Prof. Clark (Southern

llinois University) for strain DC1515 and kindly guidance.

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