Electrode-assisted acetoin production in a metabolically engineered Escherichia coli strain
© The Author(s) 2017
Received: 24 December 2016
Accepted: 28 February 2017
Published: 14 March 2017
This paper describes the metabolic engineering of Escherichia coli for the anaerobic fermentation of glucose to acetoin. Acetoin has well-established applications in industrial food production and was suggested to be a platform chemical for a bio-based economy. However, the biotechnological production is often hampered by the simultaneous formation of several end products in the absence of an electron acceptor. Moreover, typical production strains are often potentially pathogenic. The goal of this study was to overcome these limitations by establishing an electrode-assisted fermentation process in E. coli. Here, the surplus of electrons released in the production process is transferred to an electrode as anoxic and non-depletable electron acceptor.
In a first step, the central metabolism was steered towards the production of pyruvate from glucose by deletion of genes encoding for enzymes of central reactions of the anaerobic carbon metabolism (ΔfrdA-D ΔadhE ΔldhA Δpta–ack). Thereafter, the genes for the acetolactate synthase (alsS) and the acetolactate decarboxylase (alsD) were expressed in this strain from a plasmid. Addition of nitrate as electron acceptor led to an anaerobic acetoin production with a yield of up to 0.9 mol acetoin per mol of glucose consumed (90% of the theoretical maximum). In a second step, the electron acceptor nitrate was replaced by a carbon electrode. This interaction necessitated the further expression of c-type cytochromes from Shewanella oneidensis and the addition of the soluble redox shuttle methylene blue. The interaction with the non-depletable electron acceptor led to an acetoin formation with a yield of 79% of the theoretical maximum (0.79 mol acetoin per mol glucose).
Electrode-assisted fermentations are a new strategy to produce substances of biotechnological value that are more oxidized than the substrates. Here, we show for the first time a process in which the commonly used chassis strain E. coli was tailored for an electrode-assisted fermentation approach branching off from the central metabolite pyruvate. At this early stage, we see promising results regarding carbon and electron recovery and will use further strain development to increase the anaerobic metabolic turnover rate.
Acetoin production in several approaches
Anoxic, NO3 −
Recently, Bursac et al. studied the electrode-assisted fermentation based production of acetoin by using S. oneidensis as biocatalyst . The authors used lactate as carbon and electron source and achieved a production of 78% of the theoretical production maximum. Nevertheless, the spectrum of usable carbon sources is rather narrow for S. oneidensis. Sufficient growth under anoxic conditions is only sustained with lactate, while typical cheap substrates as for instance glucose or glycerol cannot be anaerobically metabolized.
In this study, an E. coli strain was developed as biocatalyst for an electrode-assisted acetoin production. Hence, the impact of the deletion of four genes encoding enzymes of the mixed acid fermentation pathways was investigated. A high efficient chassis strain from the characterization routine was developed further to establish acetoin production in an electrode-assisted fermentation.
Plasmid and strain construction
Plasmids were constructed using standard methods . Primers and plasmids used in this study are listed in Additional file 1: Table S1 and Additional file 2: Table S2. Strains applied in this study are found in Additional file 3: Table S3. All deletions were constructed in strain JG146 which contains a synthetic operon encoding for cymA and mtrA in the genome. Integration of the cymA–mtrA fragment caused a deletion of the E. coli frd gene locus . The deletion of adhE was performed as described before . The deletion fragment was obtained with primers 1 and 2 using pSG76–CSH as a template. The amplificate was transformed into E. coli, carrying the plasmid pKD46 and integrated in the genome as described in . Cells were spread on LB-agar plates containing chloramphenicol (6–10 µg/ml). Clones were tested by PCR with primers 3 and 4. The chloramphenicol resistance cassette was deleted as described by . For the construction of the ldhA-deletion plasmid, genes were amplified by PCR and cloned in the host vector in four steps. The first step was the ligation of cscRAKB_LguI in the linearized (SmaI) vector pASK43+ leading to plasmid pASK43+ _csc_LguI. The cscRAKB_LguI fragment was obtained from a PCR using primers 5 and 6 and plasmid pKJL124 as template . Secondly, plasmid pASK43+ _csc_LguI was linearized (LguI) and ligated with the PCR product of primers 7 and 8 which comprises the upstream homologous region to ldhA (pASK43+ _csc_ldh-up). Thirdly, the resulting plasmid was used as the template for the reaction with primers 9 and 10. The PCR product was then ligated in the SmaI-digested pASK43+ vector (pASK43+ LguI_csc_ldh-up). In the last construction step, the plasmid was linearized using the LguI sites added with primer 9 and ligated with the PCR product of primer 11 and 12 comprising the downstream homologous region. The plasmid was digested with XbaI resulting in the final deletion fragment containing homologous regions to the up- and downstream areas of ldhA flanking the csc-genes for the sucrose utilization- based selection (pASK43+ _ldh-down_csc_ldh-up). It was used to delete ldhA in the Δfrd ΔadhE mutant via homologous recombination mediated by pKD46. Deletion of pta–ack was performed as described by . First, galK was amplified using the primers 13 and 14 (template JG287) and the first homologous region was amplified via PCR with primer 15 and 16 using genomic DNA of E. coli as template. Primer 13 and 16 were phosphorylated. The fragment containing galK was digested with HindIII and the fragment including the homologous upstream region was cut with NheI. After the ligation into the HindIII and NheI-digested pASK43+ vector, the product was linearized with HindIII and dephosphorylated (alkaline phosphatase, Thermo Fisher Scientific). The second fragment, necessary for homologous recombination, was amplified with the primers 17 and 18. It was integrated in the vector using isothermal in vitro ligation as described by Gibson et al. . The constructed plasmid and the helper plasmid pACBSR  were used for the transformation in E. coli. After induction of pACBSR with arabinose (final concentration 0.2%), cells were plated on M63-agar with galactose as sole carbon source and tested via colony-PCR (Primer 19 and 20).
The production of acetoin requires the activity of acetolactate synthase (alsS) and acetolactate decarboxylase (alsD). Therefore, the plasmid pMAL_alsSD was designed for acetoin production. The insert was obtained using Bacillus subtilis PY79 as template and the primers 21 and 22 for amplification. The amplificate was then ligated into a pMAL vector.
Stc was integrated into the genome using the CRIM-System . The plasmid was constructed and integrated at the phage attachment site P21 as described by . The pEC86 plasmid containing the ccm genes of E. coli was transformed into the production strain, to improve the expression of c-type-cytochromes .
Growth conditions and media
Escherichia coli was routinely grown in LB medium [1% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.5% (w/v) peptone]. Media were supplemented with antibiotics, if necessary. For the selection of the galK insertion, E. coli was grown on M63 agar plates [25 mM (NH4)2SO4, 100 mM KH2PO4, 9 µM FeSO4, 0.1 mM CaCl2, 1 mM MgSO4, thiamine [0.05‰ (w/v)], and agar [2% (w/v)], pH 7] with galactose (0.2%) as sole carbon source. All growth experiments were conducted in independent triplicates. Oxic growth experiments were performed in flasks containing M9 medium (47.6 mM Na2HPO4, 22 mM KH2PO4, 18.6 mM NH4Cl, 8.6 mM NaCl2, 10 mM HEPES) complemented with trace elements (100×: 0.27 mM CoCl2, 0.02 mM CuSO4, 5.66 mM H3BO3, 0.24 mM Fe(II)Cl2, 6.72 mM Na2-EDTA, 0.13 mM MnSO4, 0.22 mM Na2MoO4, 0.13 mM Na2SeO4, 1 mM NaCl, 0.5 mM NiCl, 0.16 ZnSO4), CaCl2 (final concentration: 0.1 mM), MgSO4 (1 mM), casamino acids [0.15% (w/v)], thiamine [0.05‰ (w/v)], and glucose (10 mM). Anoxic growth experiments were conducted in N2-flushed culture flasks. Under anoxic respiratory conditions, DMSO (final concentration: 50 mM) or nitrate (final concentration: 12.5 mM) was added to the medium. Culture flasks were inoculated to an initial optical density (600 nm) of 0.05. Cultures were incubated at 30 °C in a rotary shaker (125 rpm) for 24–96 h. Samples were taken at the beginning and the end of the experiment for end product analysis.
Acetoin production experiments were conducted anoxically in sealed flasks containing M9–medium. Expression of the als-genes was induced with 50 µM IPTG.
For the bioelectrochemical approach, cells were cultured in 20 ml aerobic LB medium supplemented with antibiotics (10 µg/l kanamycin sulfate, 100 µg/l ampicillin, 50 µg/l chloramphenicol) overnight at 37 °C. Afterwards, the cells were transferred to 500 ml sterile LB medium with identical supplementation and again incubated over night at 37 °C. The gained cell mass was harvested by centrifugation at 6000×g for 8 min and resuspended in an equal volume of fresh LB medium. In addition, this culture was supplemented with 14.8 µM anhydrotetracycline for the induction of the cymA–mtrA locus and 1 mM arabinose for the induction of stc. The induction period took at least 4 h. Thereafter, the cells were washed twice and then resuspended in washing buffer . The cells were then transferred to 200 ml of an anoxic phosphate-buffered minimal medium supplemented with 50 µM methylene blue and the necessary inducers and antibiotics . The carbon source was 20 mM glucose. The initial OD600 was adjusted to 6.
Electrode-assisted fermentation experiments
Glucose and formic acid were determined enzymatically. Glucose was quantified using hexokinase and glucose-6-phosphate dehydrogenase. A sample volume of 20 µl was mixed with 180 µl of the reaction buffer (0.75 M triethanolamine, 10 mM Mg2+, 1.1 mM NADP+, 8 mM ATP) containing the enzymes hexokinase (2.1 U/ml) and glucose-6-phosphate dehydrogenase (2.1 U/ml). The absorbance at 340 nm was determined after incubation at 37 °C for 20 min, and the glucose concentration was calculated based on a calibration curve. Formic acid determination was conducted in a 96-well assay with standard concentrations as a reference. Each well was filled with 20 µl sample and 190 µl reaction buffer (final concentration in the assay: 50 mM potassium phosphate buffer pH 7.5, 1 mM pyrazole, 6.3 mM NAD+, and 3.6 U/ml formate-dehydrogenase). Ethanol was quantified via gas chromatography (Clarus 480, Perkin Elmer). The measurement was performed using a ZB-WAXplus column (Phenomenex) and nitrogen as the carrier gas (2.3 ml/min flow at the detector). Gas chromatography parameters (synthetic air flow: 5.5 bar, oven temperatures: 40 °C for 5 min, 5 °C/min up to 150 °C, hold 150 °C for 5 min, 20 °C/min up to 220 °C, and hold for 5 min) were adjusted, and 0.5 µl of the sample was injected (split 1:20). Pyruvate, acetic acid, and succinic acid were determined via HPLC (Elite La Chrome L-2130, L-2200, L-2300, L-2455, Hitachi) according to Sturm-Richter et al. . Acetoin was measured photometrically at 490 nm using the Voges–Proskauer reaction in a 96-well assay (Sigma-Aldrich, Darmstadt, Germany). Standards were used for a calibration curve. A sample volume of 20 µl was used and mixed with the reaction buffer (100 µl H2O, 14 µl 10 mg/ml l-arginine, 12 µl Barritt's reagent A). The Voges–Proskauer reaction was initiated with the addition of 6 µl Barritt's reagent B and the reaction was incubated for 30 min at 37 °C.
Characterization of E. coli mutants under anoxic conditions
Accumulation of acetic acid and pyruvate during anaerobic respiration
The production of mixed acid fermentation end products was of interest since later experiments aimed at the production of acetoin from pyruvate. Hence, the focus of this section will be on the accumulation of pyruvate and acetate in the supernatant growth medium of the individual strains. The concentrations of these two compounds and of the other typical end products of mixed acid fermentation can be found in the supplementary section (Additional file 5: Table S5).
The Δfrd ΔadhE ΔldhA triple mutant accumulated high amounts of acetic acid in the medium during anaerobic respiration with DMSO, while the acetate concentration was very similar to the initial strain under nitrate-reducing conditions. Pyruvate was detected only in small amounts during anaerobic respiration (DMSO: 0.037 ± 0.025 mol pyruvate/mol glucose consumed, nitrate: 0.018 ± 0.008 mol pyruvate/mol glucose consumed). The quadruple mutant (Δfrd ΔadhE ΔldhA Δpta–ack) showed minimal production of acetic acid, while pyruvate was secreted to the medium in high concentrations. During anaerobic growth with DMSO as terminal electron acceptor, 65% of the consumed glucose was converted to pyruvate (1.3 ± 0.12 mol pyruvate/mol glucose consumed). With nitrate as electron acceptor an insignificantly higher yield of 70% (1.4 ± 0.16 mol pyruvate/mol glucose consumed) was achieved. Under oxic conditions pyruvate was secreted to the medium as well, but was consumed after the depletion of glucose (data not shown).
Acetoin production in E. coli JG806 pMAL alsSD
Several studies analyzed the effects of single mutations in genes encoding for key enzymes of the anoxic fermentative metabolism in E. coli [42–44]. Nevertheless, a thorough analysis of the growth capabilities of these mutants under respiratory and fermentative conditions and a subsequent analysis of metabolic end products have so far not been conducted. Therefore, we combined this analysis with our aim to construct a strain that would accumulate pyruvate under anoxic respiratory conditions.
It was surprising to observe that deletion of the fumarate reductase and alcohol dehydrogenase encoding genes already led to a strain that was almost not capable of fermentative growth anymore, although lactate production alone would be suitable to recycle the cofactor NAD+. Still, even the initial strain used in this study produced lactate only in minor quantities under fermentative conditions and the regulatory routines of E. coli apparently do not seem to sustain a flexible production of end products steered by the available possibilities to balance the cellular redox state.
Interestingly, the deletion of frdA-D (fumarate reductase), adhE (alcohol dehydrogenase), and ldhA (lactate dehydrogenase) led to a 1.25-fold increased growth rate under aerobic and decreased growth rates under anaerobic respiratory conditions. Aerobic respiration is accompanied by the almost full oxidation of glucose to carbon dioxide (Additional file 5: Table S5). Side pathways from the canonical routine of glycolysis and citric acid cycle will therefore not lead to faster growth. Nevertheless, conditions in a shaker flask will not provide every individual cell with equivalent shares of oxygen and it seems reasonable to assume that oxygen depletion might lead in some cells to a partial switch to anoxic growth [45, 46]. This would be accompanied with the production of mixed acid fermentation end products and lower ATP yields . Streamlining of the metabolism by deletion of fermentation pathways ensures that all cells have to metabolize glucose directly to carbon dioxide even if oxygen might be the limiting factor. As a consequence the cells will grow faster.
The situation is different under anoxic respiratory conditions. The absence of oxygen triggers a downregulation of the citric acid cycle, which leads to an incomplete oxidation of glucose via fermentative pathways . Therefore, blockage of these pathways reduces the substrate conversion rates and as a consequence also the growth rate. Still at least under nitrate-reducing conditions, evidence was provided that some part of the growth substrate is completely oxidized to carbon dioxide via the citric acid cycle . This is not possible with DMSO instead of nitrate as electron acceptor. DMSO reduction is menaquinol (E°′ = −74 mV) dependent. The low midpoint redox potential of the menaquinone/menaquinol pair hampers respiratory glucose consumption coupled to DMSO reduction, because E. coli cannot use a reverse electron transfer-coupled succinate oxidation within the citric acid cycle. The utilization of nitrate as an electron acceptor sustains succinate oxidation within the citric acid cycle because nitrate reduction is functional with ubiquinone (E°′ = +113 mV) as electron shuttle within the membrane [49, 50]. It might be this reaction within the citric acid cycle that leads to the observed differences in the phenotypes of the mutants compared to the initial strain under respiratory growth with nitrate or DMSO, respectively. Acetate is the main end product under anoxic conditions. Its production is accompanied by the formation of 1 mol ATP per mol acetate. Hence, it is not surprising that a deletion of the ack/pta genes negatively influenced growth of the E. coli strains under anoxic conditions.
The quadruple mutant Δfrd ΔadhE ΔldhA Δpta–ack produced nearly no acetic acid, but accumulated pyruvate instead with a yield of 1.4 mol pyruvate per mol of glucose, which is close the value of 1.6 mol/mol achieved by Causey and colleagues using a strain deficient of the pyruvate oxidase and the ability to gain energy by oxidative phosphorylation . Therefore, this strain was selected for further development towards acetoin production. In this study, acetoin was produced with a yield of 90% with nitrate as the terminal electron acceptor. This represents the highest yield reported for anaerobic acetoin production in E. coli , indicating the efficiency of the engineered metabolism. The studies listed in Table 1 report acetoin production in several approaches. Although it is common to investigate acetoin producing strains in complex media containing significant amounts of yeast extract or other fermentable compounds, the acetoin yields are calculated in consideration of the added glucose only. Nevertheless, the literature is full of examples in which productivities over 100% are reached due to the catabolic consumption of complex media constituents . It is not unlikely to assume that the achievable rates will increase if higher amounts of complex media constituents are added. To our best knowledge, the overall highest efficiency for the fermentation of acetoin was reported by Ui et al. with a value of 96% in an engineered E. coli strain . However, the experiments have been conducted in oxic LB medium, containing 15 g/l of complex organic compounds. In this study, only 1 g/l of casamino acids was supplemented to the medium to allow for the analysis of the coulombic efficiency in the subsequent bioelectrochemical process. Works of Chen et al. and Zhang et al. conducted the fermentation process in a comparable minimal medium under oxic to microaerobic conditions with engineered strains of B. subtilis as the catalyst organism [22, 52]. The achieved yields reach values between 60.5 and 75%. Hence, the yields in this study range well within notable benchmarks.
Recently, Bursac et al. could show acetoin production in an analogous setup. The authors used S. oneidensis as biocatalyst. This organism can conduct an extracellular electron transfer to an electrode surface. This is certainly an advantage regarding the development of electrode-assisted fermentation processes. Nevertheless, the E. coli strain used in this study is able to metabolize a larger spectrum of carbon sources including glucose, sucrose, and glycerol. Moreover, the strain produces acetoin with a similar yield but is still able to connect this metabolism to (slow) growth. Hence, the developed E. coli strain can be applied in a continuous culture, while the S. oneidensis biocatalyst will either have to be replaced or the growth conditions would have to be changed to oxic conditions. In conclusion, both strategies for enabling electrode-assisted fermentations have their advantages, and while the E. coli strain must be developed further for higher electron transfer rates, S. oneidensis would have to be developed in the direction of a broader spectrum of useable growth substrates.
In this study, a potential platform organism for pyruvate-dependent production processes was developed. It accumulates high amounts of pyruvate during anaerobic respiration to the medium broth. The presented process for acetoin production is so far unique and offers the potential for a number of newly efficient biotechnological production routines. Of note, all electrode-assisted fermentations are accompanied with the production of an electrical current as side product. This current can be used to sustain the process or to produce hydrogen on the cathode site via a microbial electrolysis cell approach. We do not want to conceal that we can achieve far high yields but that the production rates have to be improved. Further experiments will have to tackle this limitation, for instance, via random mutagenesis and selection for higher substrate conversion rates.
AF conducted the strain development and the batch experiments. FG was involved in reactor development. SB developed the bioelectrochemical reactor applied in this study and conducted the electrode-assisted fermentation experiments. JG supervised the work in this study and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
We thank Prof. Dr. György Posfai for sharing the plasmids pKD46, pSTKST, and pSG76–CSH for the deletion of genes in E. coli.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated during the current study are available on request.
We are grateful for the financial support from the German Ministry of Education and Research (BMBF) under the Program 03SF0496B.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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