Enzyme-Assisted Microbial Electrosynthesis of Poly(3-hydroxybutyrate

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Enzyme-Assisted Microbial Electrosynthesis of Poly(3hydroxybutyrate) via CO Bioreduction by Engineered Ralstonia eutropha 2

xiaoli chen, Yingxiu Cao, Feng Li, Yao Tian, and Hao Song ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00226 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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ACS Catalysis

EnzymeEnzyme-Assisted Microbial Electrosynthesis of Poly(3Poly(3-hydroxybutyrate) via CO2 Bioreduction by Engineered Ralstonia eutropha Xiaoli Chen‡, Yingxiu Cao‡, Feng Li‡, Yao Tian, Hao Song* Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, SynBio Research Platform, Collaborative Innovation Centre of Chemical Science and Engineering, Tianjin University, Tianjin, 300072, China. ‡

Equal contribution

ABSTRACT: Microbial electrosynthesis (MES) is a promising technology to reduce carbon dioxide using inward electron transfer mechanisms to synthesize value-added chemicals with microorganisms as electro-catalysts and electrons from cathodes as reducing equivalents. To enhance CO2 assimilation in Ralstonia eutropha, a formate dehydrogenase (FDH)-assisted MES system was constructed, in which FDH catalyzed the reduction of CO2 to formate in the cathodic chamber. Formate served as the electron carrier to transfer electrons derived from cathodes into R. eutropha. To enable efficient formation of formate from CO2, neutral red (NR) was used to facilitate the extracellular regeneration of NADH, the cofactor of FDH. Meanwhile, NR also played an essential role of electron shuttle to directly deliver electrons from cathodes into R. eutropha to increase the level of intracellular reducing equivalents, thus facilitating the efficiency of MES. On the other hand, the Calvin–Benson–Bassham (CBB) cycle was further engineered by the heterologous expression of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in R. eutropha, which dramatically strengthened the CBB pathway for CO2 fixation. Upon applying the cathode potential at -0.6 V (vs. Ag/AgCl) in the MES system with the genetically engineered R. eutropha, 485 ± 13 mg/L poly(3-hydroxybutyrate) (PHB) was obtained, which was ~3 times of that synthesized by the control (165 ± 8 mg/L), i.e., the wild-type R. eutropha in the absence of FDH and NR

KEYWORDS: Microbial electrosynthesis, Ralstonia eutropha, formate dehydrogenase, Calvin–Benson–Bassham

cycle, poly(3-hydroxybutyrate)

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such as O2- and NO generated by the inorganic electrocatalytic system were toxic to R. eutropha, which may inhibit the growth of R. eutropha and reduce the yield of the products 29.

Introduction Excessive carbon dioxide emission would bring about climate changes and catastrophes, which motivated the development of many chemical and biological technologies to reduce CO2 emission, including carbon capture and storage 1-2, and CO2 reduction for synthesis of value-added chemicals via electrocatalysis and photocatalysis processes, etc. 3-4. Microbial electrosynthesis (MES) is a promising and sustainable technology to reduce CO2 by feeding electricity to microorganisms to form intracellular reduing equivalents 5-9, thus synthesizing fuels and chemicals including hydrogen 10-12, alcohols 4, 13-14 and hydrocarbons 15-17, etc.

To avoid the potential of ROS toxicity to cell growth, we here adopted a two-chamber bioelectrochemical reactor, in which R. eutropha in the cathodic chamber was separated from the anodic chamber by a proton exchange membrane (PEM) (left panel, Figure 1). Thus, ROS produced in the anodic chamber could not penetrate PEM, avoiding its entry into the cathodic chamber to poison cells. To facilitate the efficiency of CO2 reduction to formate, we constructed an enzyme-assisted NR-mediated MES system by incorporating FDH, which had two advantages over the indium foil cathode for formate formation 29. On the one hand, the cathode potential was poised only at -0.6 V (vs. Ag/AgCl), which was much higher than that poised at -1.6 V (vs. Ag/AgCl) in the system of Liao and co-workers 29, thus electricity power consumption could be tremendously reduced by such low overpentential. On the other hand, the elecron transport from the cathode into R. eutropha was increased since NR played the role of electron shuttle in addition to formate as an electron carrier (right panel, Figure 1).

A number of acetogens including Sporomusa spp. and Moorella thermoacetica could take up electrons directly from electrodes via direct contact-based extracellular electron transfer (EET) mechanims to form intracellular reducing equivalents, which reduced CO2 to synthesize acetate 8, 18. However, genome editing methods for these acetogens were yet established, which significantly hindered the spectrum of available products 19. To overcome this shortcoming, non-electroactive bacteria such as Ralstonia eutropha and Escherichia coli were adopted to indirectly uptake electrons via electron shuttles 7, 20. The electron shuttles such as riboflavin 21-22, methyl viologen 23, quinone analogues 24, and neutral red (NR) 25-26, were extensively adopted to accept electrons from cathodes and deliver into microorganisms to increase the level of intracellular reducing equivalents (such as NADH), thus facilitating the efficiency of MES. Among these employed electron shuttles, NR was commonly used because it had a low standard reduction potential (-525 mV vs. Ag/AgCl) similar to that of NADH/NAD+ (-520 mV vs. Ag/AgCl) 27. Thus, NR facilitated the reduction of CO2 to form formate catalyzed by the NADH-dependent formate dehydrogenase (FDH) using electrons from the cathode with much low overpotential than inorganic electrocatalysts 28-29, thus saving much energy. In addition, NR had high solubility and stability with minor cellular toxicity at low concentrations 30.

Lastly, R. eutropha H16 assimilates CO2 through the Calvin– Benson–Bassham (CBB) cycle, which is a primary carbon fixation pathway in the biosphere 36. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a pivotal role in the CBB cycle. However, RubisCO was an inefficient catalyst that showed a low turnover rate and a strong oxygenation side reaction 37. The kinetic properties of Rubisco from diverse sources vary distinctly 38. Harnessing these variations had the potential to enhance the CO2 assimilation efficiency in the CBB cycle. To raise the carbon fixation rate in the CBB cycle, a number of strategies in regulating and modifying Rubisco were used to improve its catalytic efficiency 39-42, such as improving carboxylation efficiency of RubisCO by protein engineering 43, introducing CO2 concentrating mechanisms 44, and heterologously expressing cyanobacterial RuBisCO along with a carboxysomal protein in tobacco 45, etc. We herein exploited the capacity of CBB cycle by heterologous expression of the key enzyme Rubisco from Synechococcus elongatus PCC7942 in R. eutropha. Thus, the CO2 fixation rate was promoted by engineering the metabolic pathways of R. eutropha. In all, 485 ± 13 mg/L PHB was synthesized in 120 hours in the FDH-assisted NR-mediated MES system with the genetically engineered R. eutropha.

In addition to electron shuttles, hydrogen and fomate generated by inorganic catalysts (such as indium, and cobalt-phosphorus, etc.) could play the role of electron carriers, which drove CO2 fixation for the production of biofuels such as butanol and isobutanol, etc. in R. eutropha 28-29. Silver and co-workers 28, 31 developed an efficient water splitting-biosynthesis system, in which a cobalt phosphate catalyst was used as the anode to drive the oxygen reduction, and a cobalt-phosphorus alloy was used as the cathode to catalyze the hydrogen evolution reaction. Since H2 was the electron carrier, R. eutropha in the cathodic chamber could then oxidize H2 to generate reducing equivalents (e.g., NADPH) and ATP, which were subsequently used to convert CO2 to synthesize poly(3-hydroxybutyrate) (PHB). Nonetheless, H2 as the electron carrier was limited by its lower mass transfer and electron delivery rates, which were caused by the much lower solubility of H2 (0.00016 g/100 g H2O for H2) than CO2 (0.169 g CO2/100 g H2O at room temperature and pressure) in water 32-33. To overcome the low solubility of H2, Liao and co-workers adopted formate as the electron carrier, in which an indium foil cathode was used to catalyze the electrocatalytic reduction of CO2 to formate. Formate was consequently used by the genetically engineered R. eutropha as a substrate to synthesize isobutanol and 3-methyl-1-butanol 29 34-35 . Unfortunately, a number of reactive oxygen species (ROS)

Results and discussion Design and construction of the FDH-assisted MES system The low efficiency of EET between cathodes and microbial cells was the crucial bottleneck in MES process 46. Meanwhile, the low efficiency of the CBB cycle for CO2 fixation in R. eutropha also greatly limited the performance of MES 47. Aiming to promote the EET efficiency and CO2 fixation rate, we designed an enzyme-assisted MES system (Figure 1). A H-shaped dual-chamber bioelectrochemical reactor was used and separated by the PEM. The protons generated from H2O splitting by platinum anode were able to pass through the PEM into the cathodic chamber, while the reactive oxygen species (ROS) (Figure S1) such as O2- and NO generated in the anode reactions was incapable of. Thus, the toxicity of ROS to bacteria 2


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in the cathodic chamber was avoided. The electrons from the carbon cloth cathode were used to reduce the electron shuttle NR, which then delivered into R. eutropha for intracellular NADH regeneration to provide intracellular reducing power for CO2 fixation (Pathway I, Figure 1). Meanwhile, formate was produced from CO2 catalyzed by the NADH-dependent FDH. Subsequently, R. eutropha assimilated formate to produce PHB (Pathway II, Figure 1). Therefore, both formate and NR could serve as the electron carriers, which could significantly facilitate the electron delivery from cathode into R. eutropha. On the other hand, we rationally engineered the CBB cycle by incorporating efficient Rubisco from Synechococcus elongatus PCC7942 into R. eutropha H16 to increase CO2 conversion for the first time.

4 g/L Cs-FDH2 was added. The cathode of the MES was poised at -0.8 V vs. Ag/AgCl, and 0.4 mM NADH was added (Figure S4A). Formate was detected by HPLC under three different NR concentrations (i.e., 0.05 mM, 0.1 mM, and 0.2 mM). The concentration of formate at 6 h was measured to be 0.56 ± 0.03, 0.51 ± 0.03, and 0.53 ± 0.03 mM with 0.05 mM, 0.1 mM, and 0.2 mM NR, respectively. Then, the formate synthesis at three different NADH concentrations (i.e., 0.2 mM, 0.4 mM, and 0.6 mM) with 0.05 mM NR was examined (Figure S4B). The concentration of formate at 6 h was 0.26 ± 0.02, 0.56 ± 0.03, and 0.37 ± 0.02 mM with 0.05 mM, 0.1 mM, and 0.2 mM NR, respectively. Thus, the optimal concentration of NR was identified to be 0.05 mM and the optimal concentration of NADH was 0.4 mM for the reduction of CO2 to formate by Cs-FDH2. The Faradaic efficiency of the reduction of CO2 to formate was calculated to be 33.5 ± 1.5% under such optimal condition.

Selection of purified FDHs and electron shuttles, and optimization of FDH-driven CO2 reduction

Furthermore, we found that the MES system could function only when NR was added in the cathodic chamber. As shown in Figure 2B, upon the addition of 0.05 mM NR in MES, the reduction of NR was visibly observed by the color change in the cathodic cultures from red (the oxidized form of NR, NRox) to yellow (the reduced form of NR, NRred). After centrifuging the cultures, red biomass and yellow supernatant were observed, which indicated electrically reduced NR entered cells and was re-oxidized into oxidized NR via NAD+ reduction within the R. eutropha cells 52.

We screened four NADH-dependent FDHs from three bacteria, Ceriporiopsis subvermispora, Ogataea parapolymorpha and Candida methylica, respectively. These FDHs were suitable for practical applications because they were O2-tolerant and more stable than the NADH-independent FDHs 48. The recombinant four FDHs were expressed in Escherichia coli BL21(DE3) and purified (Figure S2), which were then used to identify their enzyme activities (Table 1). Cs-FDH2 had the highest enzyme activity (0.408 ± 0.016 U/mg), followed by Cs-FDH1 (0.330 ± 0.016 U/mg), while Op-FDH (0.184 ± 0.009 U/mg) and Cm-FDH (0.160 ± 0.008 U/mg) had relatively low enzymatic activities. Thus, the most active Cs-FDH2 was chosen in our MES system.

Subsequently, to evaluate the performance of our MES system, the cell growth and the PHB production were analyzed. The cell growth profiles of R. eutropha showed that the cells remained at a certain cell density (OD600) during the MES operations (Figure S5 and Figure S6A). The PHB production in the presence of NR was significantly enhanced from that in the absence of NR (Figure S6B). Thus, electrons derived from the cathode were evidenced to enter into R. eutropha cells via NR for NADH regeneration, which facilitated the PHB biosynthesis. Meanwhile, addition of the electron shuttles rhodium complex and riboflavin could not lead to the increase in PHB synthesis in the MES process (Figure S6B), which demonstrated neither rhodium complex nor riboflavin could deliver cathode-derived electrons into R. eutropha. Based on the above results, NR functioned as an efficient electron shuttle for intracellular NADH regeneration.

Electron shuttles were required to recycle extracellular NAD+ to NADH by electrons derived from the cathode 49. Such NADH regeneration was to reduce the amount of NADH added in the cathodic chamber. We thus examined three kinds of electron shuttles (i.e., NR, rhodium complex 50, and riboflavin 21), and estimated their capacity for NADH regeneration in the MES system. 0.5 mM NR, 0.5 mM rhodium complex (Rh), and 0.25 mM riboflavin (basically saturated) were used at the potential of -0.8 V vs. Ag/AgCl to reduce NAD+ (The initial concentration of NAD+ was 4 mM) (Figure 2A). As a result, 0.78 ± 0.03 mM NAD+ was reduced within 8 h in the presence of NR, which proved that NR was an efficient electron mediator for NADH regeneration, while Rh achieved 0.59 ± 0.01 mM NADH regeneration at 8 h, and riboflavin was unable to regenerate NADH due to its redox potential (-0.4 V vs. Ag/AgCl) higher than that of the NAD+/NADH (-0.52 V vs. Ag/AgCl). The Faradaic efficiency of the NADH formation could be calculated by the Equation 151: η=

nzF ∫ ୧ୢ୲

Optimization of the FDH-assisted MES system To poise an optimal potential of MES for saving electricity energy, the electrochemical redox reaction of NR was examined by the cyclic voltammetry (CV). There was no redox peak in the cathodic culture of MES in the absence of NR, while a significantly redox peak was observed at ~-0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR (Figure 3A). To reduce NR, the applied cathode potential should be poised in the range of -0.6 to -1.0 V vs. Ag/AgCl in MES. A potential of -0.4 V vs. Ag/AgCl was firstly applied to testify whether it was feasible when the applied potential was higher than that of the NR, which unsurprisingly could not lead to an increase in the PHB biosynthesis in comparison to the control (Figure 3B). Next, the PHB biosynthesis profiles in MES at three different poised potentials (i.e., -0.6 V, -0.8 V and -1.0 V vs. Ag/AgCl) were examined. R. eutropha was initially cultivated in a rich broth for

(Equation 1)

where n was the amount of product (mol); z, the number of transferred electrons (z = 2 for NADH formation from NAD+); F, the Faraday constant (96,485 C/mol); i, the current (A); and t, the time (s). Thus, according to Figure S3 and Equation 1, the Faradaic efficiency of NADH regeneration mediated by the shuttle NR and Rh complex was 45.0 ± 1.2% and 34.8 ± 0.4%, respectively. We then optimized the initial concentrations of NR and NADH for CO2 reduction to formate catalyzed by Cs-FDH2. The working volume of the cathodic chamber was 50 mL, in which 3


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initial growth, in which certain amount of PHB was accumulated in the cell. The cell pellet was then transformed to the minimal medium to examine the PHB biosynthesis from CO2 in MES, where CO2 is the sole carbon source. 0.05 mM NR was replenished in cathodic chamber every 24 h upon exhaustion of active NR. We observed that the amount of PHB was reduced to 156 ± 8 mg/L at open circuit of MES, because PHB was a storage substance in R. eutropha and degraded to maintain the survival of R. eutropha in the course of the MES operation, in which CO2 was the sole carbon source. However, the yield of PHB within 120 h was 241 ± 5, 242 ± 6, and 243 ± 6 mg/L at the poised potential of -0.6 V, -0.8 V and -1.0 V, respectively (Figure 3B). Since similar amounts of PHB were produced at these three potentials, -0.6 V (vs. Ag/AgCl) cathode potential was chosen as the optimal operational potential of MES to reduce the electricity consumption. Notably, formate was synthesized from CO2 reduction by NR-assisted FDH catalysis in our MES system, which showed extremely small overpotential for formate electrocatalysis (only -0.6 V vs. Ag/AgCl poised cathode potential), and saved much electric energy. While formate electrocatlysis for CO2 reduction by precious metals required much higher overpotential, i.e., -1.6 V vs. Ag/AgCl for In foil cathode 29. In addition, the potentiostat setting of -0.6 V (vs. Ag/AgCl) was lower than H2 evolution potential, which circumvented the generation of H2 in the system 33.

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The formate could provide both carbon source and reducing equivalent for R. eutropha. It entered the cells of R. eutropha and was oxidized to form intracellular CO2 and NADH by intracellular formate dehydrogenase (FDH). Then, intracellular CO2 and NADH participated in the CBB cycle (Figure 1). In the operation of the FDH-assisted MES system with the addition of 4 g/L Cs-FDH2 in the cathodic chamber, the PHB yields from CO2 by the Rubisco-overexpressed and the WT R. eutropha were examined. As shown in Figure 5A, 485 ± 13 mg/L PHB was synthesized by the Rubisco-overexpressed R. eutropha, while only 383 ± 8 mg/L PHB was synthesized by the WT R.eutropha. To prove the occurrence of bioelectrocatalysis in the FDH-assisted MES system, cylic voltammetry (CV) was performed in the presence (i.e., turnover) and absence (i.e., non-turnover) of CO2 (the carbon source). Representative CVs for both conditions were shown in Figure S8. The CV current under the turnover condition was much higher than that under the non-turnover condition, which evidenced the occurrence of bioelectrocatalysis.

Conclusions This work developed a novel enzyme-assisted MES system, enabling an efficient bioreduction of CO2 for PHB biosynthesis. On the one hand, we introduced an efficient formate dehydrogenase (Cs-FDH2) selected from four NADH-dependent FDHs from different microbial genura in the MES system to reduce CO2 to form formate, in which NR was incorporated to in vitro regenerate NADH as a cofactor for Cs-FDH2. Formate as the electron carrier could be transferred into R.eutropha for the PHB biosynthesis. Thus, the electrons from cathode could be efficiently transferred to Cs-FDH2 for CO2 bioreduction. Meanwhile, NR could simultanously carry electrons derived from the cathode to deliver into R.eutropha to provide intracellular reducing equivalents (i.e., NADH).

The NR level was further optimized to improve the efficacy of MES. It was observed that 226 ± 6, 201 ± 6, and 191 ± 4 mg/L PHB was accumulated in R. eutropha with 0.05, 0.1, and 0.2 mM NR, respectively. Our experimental data indicated that NR in high concentration would be toxic to R. eutropha. To examine this hypothesis, we monitored the cell growth under different levels of NR (Figure 3D), which showed that 0.1 mM and 0.2 mM NR had a certain inhibition to the cell growth compared with 0.05 mM NR. Enhanced CO2 fixation by engineering CBB cycle In the autotrophic growth mode, R. eutropha fixed CO2 via CBB cycle, the rate of which was mainly limited by the key enzyme Rubisco 53. To further enhance the PHB biosynthesis in the FDH-assisted MES, we incorporated the Rubisco from Synechococcus elongatus PCC7942 into R.eutropha H16 to improve the efficiency of CO2 fixation in the CBB cycle in R. eutropha (Figure 4A).

As a result, 226 ± 6 mg/L PHB was produced with the addition of 0.05 mM NR and the cathode potential was poised at -0.6 V vs. Ag/AgCl in MES, while only 156 ± 8 mg/L PHB was accumulated by the WT R.eutropha at open circuit (Figure 5B). Introducing the key enzyme Rubisco from Synechococcus elongatus PCC7942 (Se7942) into R. eutropha for enhanced CBB cycle led to a PHB yield of 264 ± 12 mg/L. With the incorporation of FDH, 485 ± 13 mg/L PHB could be synthesized in the FDH-assisted MES system. This rationally designed MES system could also be further extended to other microorganisms for CO2 bioredcution and the produciton of many other value-added chemicals.

We firstly constructed a Biobrick compatible plasmid pHG12B, containing the pBAD promoter inducible by L-arabinose (Figure S7A). Then, the plasmid pHG12B-Rubisco (Figure S7B) was constructed for the expression of Rubisco in R. eutropha. As shown in Figure 4B, the heterologous overexpression of Rubisco could accelerate the PHB production with the induction of 0.25% L-arabinose, enabling a yield of PHB of 264 ± 12 mg/L, a further increase from that of the wild-type (WT) R. eutropha strain in the MES (226 ± 6 mg/L). To the best of our knowledge, it was the first time to engineer the CBB cycle in R.eutropha to increase the CO2 fixation.

Materials and methods Genes, Strains, and Growth Conditions. Four different FDH genes were extracted from the NCBI database and optimized for expression in Escherichia coli BL21(DE3) by a Java codon adaption tool (JCAT). The restriction enzyme sites of EcoRI and HindIII were avoided in the codon-optimized sequences. The genes were synthesized by Genewiz (Suzhou, China) (Table S1). The Rubisco gene (including small subunit and large subunit) from Synechococcus elongatus PCC7942 was extracted from the NCBI database and adapted for optimal expression in R.

Improvement of PHB production by the Rubisco-overexpressed R. eutropha in the FDH-assisted MES system 4


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eutropha H16 (ATCC 17699) (Table S1) by JCAT. The restriction enzyme sites of EcoRI, XbaI, SpeI, PstI, AvrII, NdeI, NheI, and XhoI were avoided in the codon-optimized sequences.

by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 5% stacking gel and a 15% separating gel as described by Laemmli 58. Gels were stained with Coomassie Brilliant Blue R-250 and destained with an aqueous mixture of 20% (v/v) ethanol and 10 % (v/v) acetic acid.

All bacterial strains and plasmids used in this study were listed in Table S1. For the genetic manipulations, E. coli Trans1-T1, BL21, and S17-1 were cultured in Luria Bertani (LB) medium at 37oC, and the R. eutropha strains were cultured in rich broth (10 g/L tryptone, 10 g/L yeast extract, 6 g/L beef extract, 5 g/L (NH4)2SO4) at 30oC. For all R. eutropha cultures, 10 µg/mL gentamicin was added in the rich broth. For recombinant strains to maintain plasmids, kanamycin at 50 µg/mL for E. coli and 200 µg/mL for R. eutropha were supplemented in the culture medium, respectively. The FDH genes in all E. coli BL21 strains were induced by 0.5 mM IPTG. The gene expression in R. eutropha was induced by 0.25% L-Arabinose.

Protein concentration was routinely determined by the method of Bradford 59 with bovine serum albumin as the standard. The activity of FDH was measured by using the spectrophotometric method to monitor the rate of NADH generation at 340 nm in the condition of 30°C. The reaction mixture of 1 mL contained 100 mM phosphate buffer (pH 7.0), 0.36 mM NADH, 50 mM NaHCO3, and 3 g/L FDH. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the conversion of 1 µmol of NADH into NAD+ per minute.

Regeneration and Quantification of NADH. To generate NADH, dual-chamber bioreactors (130 mL for each chamber) were separated by the PEM (Nafion-117, from DuPont Inc., USA), which enabled proton transfer. A carbon cloth (CeTech WOS1002, from Rocktek, China) cathode (2.5 cm × 3.0 cm) and a platinum anode (1.0 cm × 4.0 cm) were used. The Ag/AgCl reference electrode (0.2046 V vs. normal hydrogen electrode) was installed in the cathode chamber. The anolyte consisted of 50 mM K2HPO4 and 50 mM KH2PO4. 0.5 mM NR, 0.5 mM Rh, and 0.25 mM riboflavin (basically saturated) were added to the minimal medium (1.5 g/L KH2PO4, 6.74 g/L Na2HPO4•7H2O, 1.0 g/L (NH4)2SO4, 1 mg/L CaSO4•2H2O, 80 mg/L MgSO4•7H2O, 0.56 mg/L NiSO4•7H2O, 0.4 mg/L ferric citrate, 200 mg/L NaHCO3, and pH 7.0) in the cathode chamber, respectively. Rhodium complex was synthesized according to the method of Schmid 60. The potential of -0.8 V vs. Ag/AgCl was applied on the CHI1000C multi-channel electrochemical workstation (CH Instrument, Shanghai, China). The concentration of NADH was quantified by the absorbance at 340 nm with a spectrophotometer (PERSEE, TU-1810).

Plasmid Construction and Transformation. All plasmid constructions were performed in E. coli Trans1-T1. The plasmid to be transformed into R. eutropha was firstly transformed into the plasmid donor strain E. coli S17-1, and then transferred into R. eutropha by conjugation 54. Synthesized FDH genes (Table S3) and plasmid pET-28a-(+) were digested with the restriction enzymes EcoRI and HindIII, respectively. The digested DNA fragments and plasmid were then ligated together and formed the expression plasmids (Table S1). A 200 bp oligonucleotide was synthesized, containing a RBS site, multicloning sites (including EcoRI, XbaI, SpeI, PstI, AvrII, NdeI, NheI, and XhoI), and a T1 terminator. This fragment flanked by an upstream restriction site EcoRI and a downstream suffix PstI was inserted into the plasmid pHG11 that previously constructed by our laboratory 55 and formed the plasmid pHG12. Then the vector pHG12B was constructed by inserting the pBAD promoter 56 (flanked by two BsaI restriction sites at each end) into pHG12B using one-step Golden Gate assembly (see Table S2 for all primer sequences) 57. Finally, the synthesized Rubisco gene (Table S3) was incorporated into pHG12B, forming the resulting expression plasmid.

Setup of MES. R. eutropha strains were firstly activated 24 h in rich broth. Then, 2 mL bacterial cultures were inoculated into 100 mL fresh broth. After shaking at 30°C for 12 h at 200 rpm, the cells were harvested by centrifugation at 6000 rpm for 5 min and washed three times with the minimal medium to remove organic substrates. The cell pellets were then resuspended and the culture concentrations were adjusted to equal levels (OD600 = 1.5) with minimal medium. A carbon cloth cathode (working, 2.5 cm × 3.0 cm), a platinum anode (counter,1.0 cm × 4.0 cm), and an Ag/AgCl reference electrode were used in dual-chamber MESs. 50 mM K2HPO4 and 50 mM KH2PO4 were added to the anode chamber. The catholyte was the minimal medium bubbled with CO2 at a flow rate of 40 mL/min. And the potential of the cathode was applied continuously through the CHI1000C multi-channel electrochemical workstation during the experiment. Cyclic voltammetry (CV) was conducted with a scan rate of 0.05 V/s. Each MES was produced in triplicates. All MES reactors were incubated at 30°C and agitated by magnetic stir bars with a speed of 150 rpm.

Expression, Purification and Analysis of FDH. The transformed strains were cultured in LB medium supplemented with 50 µg/mL kanamycin at 37oC. When the cultures reached an optical density of 0.6~0.8 at 600 nm, IPTG was added to a final concentration of 0.5 mM and FDHs were induced and overexpressed in E. coli BL21(DE3) at 25oC for 12 h. All purification steps were performed at 4 oC. Cells were harvested by centrifugation at 8000 g for 30 min. The centrifuged cell pellet was washed twice with buffer A (50 mM Tris-HCl, 300 mM NaCl, 4 mM β-mercaptoethanol, pH 7.6), resuspended in the buffer A, and disrupted by sonication at 4°C for 5 min (pulsations of 5 s and cooling for 9 s). Then buffer B (500 mM imidazole in Buffer A) was added to the sonicated extract. Cell debris was removed by centrifugation (12000 rpm for 10 min at 4°C). The supernatant was loaded onto a Ni column. Finally, elution buffers with different imidazole concentrations (30, 50, 100, 250, and 500 mM) by regulating the proportions of Buffer A and Buffer B were used to elute the Ni column, and the eluents were collected.

Quantification of PHB. For the quantifcation of PHB, the samples in the MES cultures were firstly centrifuged at 12000

Collected samples from each step and the eluents were analyzed 5


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rpm for 1 min. Then the cell pellet and PHB standard substance (Sigma 363502) were digested into 3-hydroxybutyrate with concentrated H2SO4 at 90oC for 1 h. The digestion dilution was sequentially diluted with deionized water by 50 times and filtered (0.22 µm filter). The pretreated samples were determined by a high performance liquid chromatography system (Waters, e2695) equipped with a 2998 PDA detector (210 nm). An Aminex HPX-87H column was used at 35oC with 4 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min for 30 min. The elution times was ~28 min for PHB.

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We acknowledge the financial support from the National Natural Science Foundation of China (NSFC 21376174, 21621004), the National Basic Research Program of China (“973” Program: 2014CB745100).

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Table 1. Measurements of the catalytic activity of formate dehydrogenases (FDH) originated from different microorganisms.

Specific enzyme

Enzyme

Source

NCBI number

Cs-FDH1

Ceriporiopsis subvermispora

164564765

0.330 ± 0.016

Cs-FDH2

Ceriporiopsis subvermispora

164564767

0.408 ± 0.016

Op-FDH

Ogataea parapolymorpha

25772590

0.160 ± 0.008

Cm-FDH

Candida methylica

1181204

0.184 ± 0.009

ACS Paragon Plus Environment 9

activity (U/mg)

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Figure 1. Schematic of the FDH-assisted microbial electrosynthesis (MES) system to reduce CO2 for the synthesis of poly(3-hydroxybutyrate) (PHB). The design of this MES system involved two feeding mechanisms of both carbon source (CO2) and reducing equivalents (electron). Two modes of CO2 feeding mechanisms were used, i.e., the direct CO2 assimilation (Pathway A, green arrow) and the formate-shuttled CO2 assimilation by the engineered Ralstonia eutropha (Pathway B, green arrow). Two modes of reducing equivalents feeding mechanisms were via the neutral red (NR)-mediated electron transfer to the cell (Pathway I, red arrow), and the formate-mediated electron transfer (Pathway II, red arrow). The NR transferred electrons derived from the cathode to reduce NAD+ for the in vitro NADH regeneration. Formate was then synthesized by the reduction of CO2 catalyzed by the NADH-dependent formate dehydrogenase (FDH) in the cathodic chamber. Formate, as the electron carrier, could subsequently transport into the cells of R. eutropha via Pathway II (the formate-mediated electron transfer mechanism) to participate in the cellular metabolism and the synthesis of PHB. Meanwhile, the reduced NR also directly diffused into R. eutropha to enhance the formation of intracellular reducing equivalents for efficient CO2 bioreduction and PHB biosynthesis via Pathway I (the NR-mediated electron transfer mechanism). Furthermore, the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiscuo) from Synechococcus elongatus PCC7942 was overexpressed in the wild-type R. eutropha H16 to enhance the Calvin-Benson-Bassham (CBB) cycle, which significantly improved the CO2 fixation.


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ACS Catalysis

Figure 2. The selection of electron shuttles in the FDH-assisted MES system. (A) NADH regeneration with different electron shuttles in the cathodic chamber of MES. Abbreviation: NR: neutral red; Rh: rhodium complex; and RF: riboflavin. (B) The color changes in the centrifuged samples. In the MES with 0.05 mM NR, the oxidized state of NR (NRox) accepted electrons from the cathode to form the reduced state of NR (NRred) at the potential of -0.8 V vs. Ag/AgCl. NRred was re-oxidized within the R. eutropha cells to form NRox. The light-yellow broth indicated the formation of NRred, while the red biomass (at the bottom of centrifuge tubes) indicated NRox formed inside R. eutropha.


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Figure 3. Improving the PHB production in MES by optimizing the operational conditions, including the applied potential, and the NR concentration. (A) Cyclic voltammetry curves of the MES in the presence or absence of NR. The scan rate of the CV was 0.05 V/s. The Ag/AgCl electrode was used as the reference electrode, and the carbon cloth as the working electrode. (B) Dependence of the PHB yield in R. eutropha upon the applied cathode potentials. 0.05 mM NR was used as the electron shuttle. (C) Dependence of the PHB yield in R. eutropha upon the NR concentrations. The cathode of the MES was poised at -0.6 V vs. Ag/AgCl. (D) Dependence of cell growth upon the NR concentrations. OD600 was used to monitor the cell growth.


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ACS Catalysis

Figure 4. Construction of the Rubisco-overexpressed R. eutropha strain to enhance the PHB production. (A) The Rubisco gene from Synechococcus elongatus PCC7942 was overexpressed in R. eutropha to facilitate CO2 fixation by enhancing the CBB cycle. (B) The production of PHB by the Rubisco-overexpressed R. eutropha (with the plasmid pHG12B-Rubisco) induced by 0.25% (m/v) L-arabinose in the MES system. 0.05 mM NR was added to the cathodic chamber.


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Figure 5. Improvement of PHB production by FDH-assisted MES system. (A) The PHB yield in WT and Rubisco-overexpressed R. eutropha. The cathode of the MES was poised at -0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR and 4 g/L Cs-FDH2. (B) Enhancement of PHB production upon gradual optimization of the MES process. The yield of PHB was measured at 120 hours of the MES operation. Abbreviations: Control: the WT R. eutropha by MES poised at -0.6 V vs. Ag/AgCl; NR: the WT R. eutropha by MES poised at -0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR; NR+Rubisco: the Rubisco-overexpressed R. eutropha by MES poised at -0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR; NR+FDH: the WT R. eutropha by MES poised at -0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR and 4 g/L Cs-FDH2; and NR+FDH+Rubisco: the Rubisco-overexpressed R. eutropha by MES poised at -0.6 V vs. Ag/AgCl with the addition of 0.05 mM NR and 4 g/L Cs-FDH2.


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Enzyme-Assisted Microbial Electrosynthesis of Poly(3-hydroxybutyrate

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