- Open Access
Engineering an electroactive Escherichia coli for the microbial electrosynthesis of succinate from glucose and CO2
© The Author(s) 2019
- Received: 23 May 2018
- Accepted: 20 January 2019
- Published: 28 January 2019
Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO2. Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products.
In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO3+, which produced a succinate yield of 1.10 mol/mol glucose—a 1.6-fold improvement over the parent strain T110.
The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.
- Microbial electrosynthesis
- Bioelectrochemical systems
- CO2 fixation
Energy is a sine qua non of all biosynthetic processes, and is supplied in the form of chemical energy from the metabolism of substrates or from light through photosynthesis. Moreover, certain microbes were recently found to be able to utilize electric energy for the synthesis of chemical compounds [1–3]. The process in which microorganisms utilize electrons from a cathode to reduce carbon dioxide, glucose or other substrates is termed microbial electrosynthesis (MES) . Due to the necessity for the reduction of CO2 emissions and storage of various forms of renewable electricity, research on microbial electrosynthesis techniques is becoming increasingly popular [1, 5–7].
Microbial electrosynthetic processes are performed in a bioelectrochemical system (BES) for biological reductive reactions, which comprises an anode, a cathode and a reference electrode [8, 9]. Electrons derived from the cathode are transformed into reducing equivalents to power biological processes such as fumarate reduction or CO2 fixation, while an oxidation process occurs at the anode at the same time [10–12]. When the electrons derived from the cathode are used by the fermentative microbial catalysts, the fermentation balance shifts toward the production of more reduced metabolic products [4, 10, 11, 13]. More importantly, it may be possible to reduce CO2 and fix it into organic compounds using the reduction power stemming directly from electric energy.
Current MES experiments are mostly based on naturally electroactive microbes, such as S. oneidensis MR-1 [14–16]. While these microbes have high electric activity, the palette of products that can be synthesized is limited to simple compounds such as acetate and formate [10, 17]. Furthermore, there are no mature synthetic biology tools available for these microbes, precluding their engineered into applicable cell factories. Thus, more versatile MES platforms based on model bacteria are needed to expand the capacities of the MES technique.
In this study, the electrically-inactive model microbe Escherichia coli was used to study the key factors involved in microbial electric activity, and was engineered into an electroactive bacterium to perform MES. The electron transfer pathway of S. oneidensis MR-1, which consists of a c-type outer membrane cytochrome (MtrC), a periplasmic c-type cytochrome (MtrA), a non-heme outer membrane β-barrel protein (MtrB), and an inner-membrane associated quinol oxidase (CymA) [14, 18], has been established as a model for biological electron transfer . Therefore, we employed the electron transfer pathway from S. oneidensis in E. coli to make it electrically active and able to utilize electric energy in a biosynthetic pathway for CO2 fixation and production of reductive fermentation products.
Strains, media, and growth conditions
E. coli strains and plasmids used in this research
Strains and plasmids
ATCC8739, a∆ldhA, ∆pflB, ∆ptsI, Ppck*-galP, Ppck*-pck
Tan et al. 
Cm, p15A origin of replication, lacI
Apr, pMB1 origin of replication, lacI
Str, pMB1 origin of replication
Amp, pMB1 origin of replication, lacI
carbonic anhydride (CA) encoding gene (ccaA) and bicarbonate transporter (BT) encoding gene (bicA) in pTrac99A-M
Kan, pBBR1 origin of replication, BBa J23100
pACYC184 derived plasmid expressing the outer membrane protein complex encoding genes mtrA-mtrB-mtrC from S. oneidensis
pTrac99A-apr derived plasmid expressing cymA and fccA from S. oneidensis
pTrac99A-spe and pTrac99A-M derived plasmid expressing fccA from S. oneidensis
pACYC184 derived plasmid expressing the fusion protein of MtrA-RFP
pTac99A-apr derived plasmid expressing fusion protein of CymA-RFP
pTrac99A-apr derived plasmid expressing fusion protein of FccA-RFP
Construction of plasmids and strains
The mtrA, mtrB, and mtrC genes were amplified from S. oneidensis MR-1. The primers pACYC184-K-F and pACYC184-K-R were used for amplifying the plasmid backbone of pMtrABC, including the lacI gene and the trc promoter region. Artificial regulatory part RBS1 was embedded within the primers MtrB-F and MtrC1-F, respectively. The plasmid backbone and DNA fragments were ligated using Golden Gate DNA assembly  to obtain the plasmid pMtrABC. Similarly, the fccA and cymA genes were cloned from S. oneidensis MR-1 and ligated with a pTrac99A-apr backbone using the primers pTrac99A-K-F and pTrac99A-K-R to obtain pFccA-CymA, artificial regulatory part RBS2 was embedded within the primer CymA-F. To construct pFccA, the backbone of the plasmid pFccA was amplified using the primer pair pTrac99A-spe-F and pTrac99A-spe-R from pTrac99A-spe, the lacI gene was obtained from plasmid pTrac99A-M using primers pTrac99A-M-F and pTrac99A-M-R, the FccA gene was amplified from the genome of S. oneidensis MR-1, these three fragments were ligated using Golden Gate assembly. E. coli DH5α (CWBIO, China) was used for cloning and plasmid propagation, while T110 and 8739 served as the chassis strains. pMtrABC and pFccA-CymA have different resistance markers and origin of replication, and are compatible. Primers used in this study are listed in Additional file 1: Table S1.
Microbial electrosynthesis reaction setup
A single colony of the relevant strain was used to inoculate 3 mL of fresh LB medium and cultured overnight at 37 °C under constant orbital shaking at 200 rpm. The resulting seed culture was used to inoculate fermentation medium at 2% (v/v), and anaerobically incubated at 30 °C and 200 rpm in 500-mL (183 mm × 108 mm × 36 mm) anaerobic flasks (ShuNiu, China). After 3 h of cultivation, the culture was induced with 0.1% (v/v) of a 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) solution, sealed, and incubated for another 16 h under the same conditions. After measuring the OD600, the cells were harvested by centrifugation at 3000×g and 15 °C for 5 min. The obtained cell pellet was washed twice with NBS medium (New Brunswick Scientific, USA) and transferred into 99.99% pure platinum electrode clip of three-electrode bioelectrochemical reactors (Wenoote, China) for the microbial electrosynthesis reaction.
Control, measurement and analysis of the electric parameters were conducted using a CHI1030C eight-channel potentiostat (Chenhua Instruments Co., Ltd., Shanghai, China). A CHI115 platinum wire electrode and a CHI111 Ag/AgCl electrode (Chenhua Instruments Co., Ltd., Shanghai, China) were used as the counter electrode and reference electrode, respectively. A 2 × 2 cm carbon cloth pretreated with an 8% H2SO4 solution was nipped using the platinum electrode holders, and was used as the working electrode. The BES was operated in batch mode at room temperature (25 °C), in a 100-mL blue cap bottle (99 mm × 57 mm × 30 mm × 40 mm, ShuNiu, China). The three electrodes were inserted into the bottle via the cap. Pure nitrogen (N2) gas was sparged continuously into the reaction medium in the BES to maintain anaerobic conditions, while the BES chamber was stirred using a magnetic stirrer at 200–250 rpm.
Membrane proteins extraction and samples preparation
To collect membrane-associated MtrA, MtrB, MtrC, CymA and FccA, cell membrane was extracted with the protocol: (1) Prepare the 500 mL-fermentation medium of the strains E. coli T110 and T110(pMtrABC, pFccA-CymA), then the cells are harvested by centrifugation at 3000×g for 10 min, (2) the cell pellet is redissolved using 15 mL PBS buffer (pH 7.0), followed by cell crushing for three times using the high-pressure homogenizer (JNBIO-3000 PLUS, China), (3) the cell suspension is centrifuged at 8000×g for about 60 min, (4) discard the pellet and collect the supernatant for the next step, (5) the membrane proteins are collected by centrifugation at 200,000×g using the ultracentrifuge (Optima L-100XP, Beckman, USA). The preparation of protein sample was as follows: (1) the collected membrane protein is dissolved using the 2.5 mL protein lysate (8 M urea, 1% DTT) and mixed well, (2) the suspension is centrifuged at 10,000×g for 15 min, 18 °C, (3) collect the supernatant into the 2 mL centrifugal tube and repeat this step once, and the samples are stored at − 80 °C for analysis or mass spectrometry.
Analytical techniques and chemical analysis
Samples comprising 1 mL of the reaction mixture were obtained, and the supernatants were used for the analysis of the relevant metabolites including organic acids and residual glucose, using an Agilent 1260 series high performance liquid chromatography (HPLC) system (Agilent, USA), equipped with a refractive index detector and a Bio-Rad Aminex HPX-87H ion exclusion column (300 mm × 7.8 mm, Aminex, USA), which was kept at 35 °C and eluted with 5 mM H2SO4 at a flow rate of 0.5 mL/min; the injection volume was 20 μL . The cathode was poised at − 650 mV in all the experiments. The curve of current versus time (i–t curve) was used to detect current changes during the entire process of BES reactions. The SDS-PAGE was run using the commercially purchased SurePage™ Gels (GenScript, Nanjing). The protein mass spectrometry was performed using the OrbiTrap Fusion LUMOS Tribrid Mass Spectrometer (LC–MS) (Thermo Fisher, USA) and the methods could be referred to references [21, 22].
The significance of differences between mean values of control and test samples was compared by Student’s t-test, using the open-source software “R” (http://cran.r-project.org/). Differences with p < 0.05 were regarded as obvious, p < 0.01 as significant, and p < 0.001 as very significant.
Design and setup of a neutral-red-mediated MEC system for E. coli
Due to the reported toxicity of neutral red to living cells , the optimal concentration was determined to perform MES for E. coli T110. With the OD600 nm value as the evaluation index of living cell number, various concentrations of neutral red (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 mM) were incubated with E. coli T110 with an initial OD of 0.5 for 12 h. Based on the results (Fig. 2b), when the neutral red concentration was in the range of 0.05, 0.1 and 0.5 mM, the cell growth was affected corresponding to the concentrations. In the range of 1 and 5 mM, the cell growth was significantly affected. And in the range of 0, 0.001 and 0.01 mM, the cells could keep up the growth. We have also done the same experiment with E. coli 8739, and the experimental result was as shown in Additional file 1: Fig. S3. E. coli 8739 exhibited a very similar pattern as the E. coli T110 with different concentrations of neutral red, except the final OD600 values were generally higher than that of E. coli T110 in the range of 0, 0.001 and 0.01 mM. Therefore, the optimal neutral red concentration for both strains was 0.01 mM.
Based on the previous experimental results of optimization of neutral red concentrations, the neutral red with a concentration higher than 0.01 mM was toxic to E. coli cells. We selected the neutral red concentrations below 0.01 mM including 0.005 and 0.01 mM, but also above 0.01 mM including 0.02, 0.03 and 0.04 mM, to investigate the effect of different neutral red concentrations on the current with strain E. coli T110 (pMtrABC, pFccA-CymA). The experimental results were shown as in Additional file 1: Fig. S4, that the current increased significantly when the neutral red concentration was raised from 0.005 to 0.01 mM. But in the range from 0.01 to 0.04 mM, the increasing extent was small. Considering both cell growth status and system current level, 0.01 mM neutral red was selected to perform the MES experiments in this work.
Engineering of an electrosynthetically active E. coli with the key elements from S. oneidensis
To determine if the heterologous proteins MtrA, MtrB, MtrC, CymA, and FccA were successfully expressed in E. coli cells and presented in the membrane compartment, three experiments were performed including fluorescent tag location tracing, SDS-PAGE of extracted membrane proteins, and protein mass spectrometry of membrane proteins. To analyze cellular localization of the expressed membrane proteins, RFP protein was used as a reporter protein. The genes of membrane proteins were fused with the rfp gene respectively for making reporter fusions. The strain E. coli T110 without expressing the membrane protein from S. oneidensis was used as the control, which only expressed the RFP reporter protein. And the strain E. coli T110(pMtrA-RFP), T110(pCymA-RFP) and T110(pFccA-RFP), which had MtrA, CymA and FccA fused with RFP respectively were the experimental group, and the constructed plasmid maps were as shown in Additional file 1: Fig. S5. The fluorescence microscope Leica™ DM5000B was used for measuring RFP fluorescence at respective excitation and emission wavelengths of 557 and 590 nm, using a 100× oil-immersion objective. However, the quality of the fluorescence microscopy images is not ideal. We have repeated the practice for a few times, but the best fluorescent images we could obtain was still somewhat vague. Thus, we did not put these images as formal manuscript figures but left them in supplemental files as shown in Additional file 1: Fig. S6. In Additional file 1: Fig. S6a, the control without membrane protein showed RFP filled with the whole cell, indicating that there is no membrane localization occurred. However, in Additional file 1: Fig. S6b–d, we could see that RFP fluorescence was more distributed towards the edge of cells. The fluorescent microscopy results indicated these membrane proteins was possibly located with the membrane compartment. Since the image quality is low, we had to perform the following experiments to illustrate their location status.
SDS-PAGE of proteins associated with membranes were performed for determining the expression of MtrA, MtrB, MtrC, CymA and FccA, and the result was as illustrated in Additional file 1: Fig. S7. We could see that the control strain E. coli T110 almost had no expression of these proteins, whereas, the strain E. coli T110(pMtrABC, pFccA-CymA) had relatively clear bands matched the sizes of MtrA, MtrB, MtrC, CymA and FccA.
The protein mass spectrometry was conducted for further determine the expression of the heterologous membrane proteins derived from S. oneidensis. The result of membrane protein mass spectrometry was as illustrated in Additional file 1: Fig. S8, the detailed identification results of the MtrA, MtrB, MtrC, CymA and FccA proteins were marked in red in Additional file 1: Fig. S8. It was illustrated that while no MtrA, MtrB, MtrC, CymA and FccA were found in the control samples of E. coli T110, all these heterologous proteins scored a “High” in the “Found in Sample” section by the strain E. coli T110(pMtrABC, pFccA-CymA), which indicated the presence of these proteins. With the above three experiments, heterologous proteins MtrA, MtrB, MtrC, CymA and FccA were proved to be successfully expressed and presented in the membrane compartment.
To further improve the engineering strategy and increase the electric activity of other E. coli strains, the representative wild-type strains E. coli ATCC8739 and MG1655 were modified in the same way as T110 (pMtrABC, pFccA-CymA), and MES reactions were performed using the resulting engineered strains under the same conditions. As shown in Fig. 4c and d, the engineered strains E. coli 8739 (pFccA), 8739 (pFccA-CymA), 8739 (pMtrABC), and 8739 (pMtrABC, pFccA-CymA) showed almost the same performance as their counterparts based on the T110 chassis. The succinate production of ATCC8739 (pMtrABC, pFccA-CymA) increased by nearly 88%, and the difference was very significant (p < 0.001). The same was observed for the strains derived from E. coli MG1655 (Fig. 4e, f), whereby the succinate production of MG1655(pMtrABC, pFccA-CymA) was increased by 80% compared with the parent strain, and the increase was highly significant (p < 0.001). A similar trend of increased succinate production and system-wide current of the three groups of strains indicated that this metabolic engineering strategy is probably universally applicable to E. coli.
By calculating the electron balance, as known as the coulombic efficiency, in the MES system (Additional file 1: Table S2), the values of the coulombic efficiency of these processes were at the range of 100–300%, which indicated that the mole value of electrons delivered by the current to the reduced fermentation products did not stoichiometrically account for the increased succinate. In fact, the increased succinate mole value was larger than electrons delivered to the system, similar to the data presented by some other reports [4, 34, 35]. A possible hypothesis was that only a portion of succinate was directly reduced by the accepted electrons, while the other portion of succinate production was due to the cell response to the low redox-potential environment induced by the delivering of the electrons, which led to the increased succinate production from fumarate compared with the condition without electricity in the MES system [4, 34, 35].
E. coli 8739(pMtrABC, pFccA-CymA) was able to perform MES with glucose as the carbon source
Bioelectrochemical techniques offer a promising way to supply reducing power to intracellular electron carriers, such as NADH, by providing electrons through a cathode, enabling the biotransformation and biosynthesis of a number of reduced products [36, 37].
MES fermentation profile of E. coli ATCC8739(pMtrABC, pFccA-CymA)
Concentration (mM) of the following fermentation products
8739 (pMtrABC, pFccA-CymA) without electricity
1.31 ± 0.02a
5.43 ± 0.03
1.06 ± 0.02
32.21 ± 0.50
34.68 ± 0.70
Yield (mol/mol Glucose)
0.02 ± 0.00
0.10 ± 0.00
0.02 ± 0.00
0.58 ± 0.04
0.63 ± 0.05
8739 (pMtrABC, pFccA-CymA) with electricity
2.17 ± 0.01
11.96 ± 0.20
1.60 ± 0.04
24.53 ± 1.00
53.31 ± 1.60
Yield (mol/mol Glucose)
0.04 ± 0.01
0.22 ± 0.01
0.03 ± 0.00
0.44 ± 0.08
0.96 ± 0.08
A successful MES reaction was thus performed using the electroactive E. coli 8739(pMtrABC, pFccA-CymA), and electricity was utilized in the MES system to produce more reduced fermentation products from glucose. The results suggested that the cells in the experimental group supplemented with electricity obtained excess reducing power that was not available to the control group. Therefore, more reduced metabolites were produced to consume the excess reducing power and balance the intracellular redox state [38, 39]. The intracellular reducing power is stored in the form of reduced cofactors such as NADH, FAD and CoQ, indicating that some of the cofactors had accepted electrons and were reduced with supplementation of electricity in the MES system [40–43].
During this electrosynthesis fermentation process, a charge of 69.6 ± 5.5 °C was transferred to the anode, equal to 0.72 ± 0.06 mM of electrons, and the increased reduced products had a total mole amount of 4.55 mM (Table 2). Thus, the coulombic efficiency MES reaction of E. coli 8739(pMtrABC, pFccA-CymA) was calculated to be 632%. Similar to the previous fumarate reduction MES reactions, the coulombic efficiency was larger than 100% again. We hypothesized that the electricity-driven reducing power might affect the intracellular redox potential and hence metabolite profiles, and facilitated a higher production of reduced metabolites products, such as succinate, lactate, ethanol [4, 35, 44, 45].
The electroactive E. coli T110 produced succinate at high yield with CO2 supplementation in the MES system
MES fermentation profile of E. coli T110, T110 (pMtrABC, pFccA-CymA) and T110 (pMtrABC, pFccA-CymA, pBTCA)
Consumed Glucose (mM)
Product concentration (mM) and yields without electricity
Product concentration (mM) and yields with MES
27.28 ± 0.52
16.64 ± 0.36a
13.47 ± 0.12
18.55 ± 0.38
13.91 ± 0.07
Yield (mol/mol glucose)
0.61 ± 0.05
0.48 ± 0.03
0.68 ± 0.05
0.51 ± 0.03
T110 (pMtrABC, pFccA-CymA)
27.78 ± 0.52
15.56 ± 0.40
14.68 ± 0.32
26.39 ± 1.20
11.11 ± 0.75
Yield (mol/mol glucose)
0.56 ± 0.03
0.53 ± 0.02
0.95 ± 0.04
0.40 ± 0.02
T110 (pMtrABC, pFccA-CymA, pBTCA)
27.78 ± 0.52
18.06 ± 0.56
14.10 ± 0.50
30.56 ± 1.26
9.16 ± 0.20
Yield (mol/mol glucose)
0.65 ± 0.08
0.51 ± 0.04
1.10 ± 0.03
0.33 ± 0.01
In this research, an electroactive succinate-producing cell factory, E. coli T110(pMtrABC, pFccA-CymA, pBTCA), was obtained by both internal metabolic-network engineering and the introduction of electroactivity, mainly in the periplasm compartment (Figs. 3 and 5). To achieve the maximal succinate yield from glucose, excess reducing power was needed in the succinate fermentation. Thus, we designed an electrosynthetically active E. coli cell as illustrated in Fig. 3, in which the heterologously expressed MtrABC complex was assembled, which allowed the input of electrons transferred from electron carriers. Afterwards, the electrons were delivered to menaquinone or other biological carriers inside the cell, and transformed into usable reductive power for biosynthesis reactions.
The best strain T110(pMtrABC, pFccA-CymA, pBTCA) was fermented in an MES system supplemented with HCO3+, with neutral red as the electron carrier, and achieved a succinate yield of 1.10 mol/mol glucose—a 1.6-fold improvement over the parent strain T110. This strain is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this research can most certainly be exploited for the engineering of various other cell factories that utilize electricity for bioproduction. Since CO2 fixation is a biosynthesis reaction that requires vast energy inputs, the application of our MES system may provide a solution for the conversion of CO2 into various bio-based chemicals and fuels. A further implementation of this research might enable the commercialization of MES technologies [9, 51].
WZQ and BC planned and performed the experiments, analyzed and interpreted the data. BC, ZX, LJ and WY supervised the study, designed the experiments and analyzed and interpreted the results. WZQ wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
We provide all the necessary data for the publication of the article. All supporting data is present in the article and the supplemental material documents. Specifically, the MES system map and DNA sequence data can be found in Additional file 1.
Consent for publication
I hereby give the Journal of Microbial Cell Factories the right and permission to publish this article.
Ethical approval and consent to participate
This research was financially supported by the Key Deployment Project of the Chinese Academy of Sciences (ZDRW-ZS-2016-3), Grants from the National Natural Science Foundation of China (31522002), Natural Science Foundation of Tianjin (15JCYBJC49400), and the Tianjin Key Technology R&D program of Tianjin Municipal Science and Technology Commission (11ZCZDSY08600).
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