Insights into cell-free conversion of CO2 to chemicals by a

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Insights into cell-free conversion of CO2 to chemicals by a multienzyme cascade reaction Raushan Singh, Ranjitha Singh, sivakumar Dakshinamurthy, sanath kondaveeti, Taedoo Kim, Jinglin Li, Bong Hyun Sung, Byung-Kwan Cho, Dong Rip Kim, Sun Chang Kim, Vipin Chandra Kalia, Y. -H. Percival Zhang, Huimin Zhao, Yun Chan Kang, and Jung-Kul Lee ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02646 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Insights into cell-free conversion of CO2 to chemicals by a multienzyme cascade reaction Raushan Singh1#, Ranjitha Singh1#, Dakshinamurthy Sivakumar1, Sanath Kondaveeti1, Taedoo Kim1, Jinglin Li1, Bong Hyun Sung2, Byung-Kwan Cho3, Dong Rip Kim4, Sun Chang Kim3, Vipin C. Kalia1, Y-H Percival Zhang5, Huimin Zhao6, Yun Chan Kang 7*, Jung-Kul Lee1* 1Department 2Bioenergy

of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Seoul 05029

and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806

3Department

of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

4School

of Mechanical Engineering, Hanyang University, Seoul 04763, Republic of Korea

5Department

of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA

6Department

of Chemical and Biomolecular Engineering, University of Illinois at UrbanaChampaign, Urbana, IL 61801, USA

7Department

of Materials Science and Engineering, Korea University, Anam-Dong, SeongbukGu, Seoul 02841, Republic of Korea

#

These authors contributed equally to this manuscript.

*Corresponding author. J.-K.L. E-mail: [email protected], Tel: +82-2-450-3505; Fax: +82-2-458-3504 Y.C.K. E-mail: [email protected], Tel: +82-2-3290-3268; Fax: +82-2-928-3584

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ABSTRACT Multi-enzymatic cascade reactions have garnered the attention of many researchers as an approach for converting CO2 into methanol. The cascade reaction used in this study includes the enzymes a formate dehydrogenase (ClFDH), a formaldehyde dehydrogenase (BmFaldDH), and an alcohol dehydrogenase (YADH) from Clostridium ljungdahlii, Burkholderia multivorans, and Saccharomyces cerevisiae, respectively. Because this cascade reaction requires NADH as a cofactor, phosphite dehydrogenase (PTDH) was employed to regenerate the cofactor. The multienzymatic cascade reaction, along with PTDH, yielded 3.28 mM methanol. The key to the success of this cascade reaction was a novel formaldehyde dehydrogenase, BmFaldDH, the enzyme catalyzing reduction of formate to formaldehyde. The methanol yield was further improved by incorporation of 1-ethyl-3-methylimidazolium acetate, resulting in 7.86 mM of methanol. A 500-fold increase in total turnover number was observed for the ClFDHBmFaldDH-YADH cascade system compared to the Candida boidinii FDH-Pseudomonas putida FaldDH-YADH system. We provided detailed insights into the enzymatic reduction of CO2 by determining the thermodynamic parameters (Kd and ΔG) using isothermal titration calorimetry. Furthermore, we demonstrated a novel time-dependent formaldehyde production from CO2. Our results will aid in the understanding and development of a robust multienzyme catalyzed cascade reaction for the reduction of CO2 to value-added chemicals.

KEYWORDS: cascade reaction, formaldehyde, methanol, multienzyme, CO2, FDH, FaldDH


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INTRODUCTION Carbon dioxide (CO2) is a major greenhouse gas which has accumulated steadily over the past few decades1-2. It is generated mainly by fossil fuel combustion, volcanic eruptions, industrial byproducts, and products of respiration3. Increasing atmospheric concentration of CO2 and a constant need for alternative sources of energy have inspired many researchers to investigate different methods to produce methanol from CO24. Recently, reduction of CO2 to yield methanol and other renewable fuels has received considerable attention from the scientific community5-6. There are several routes to achieve CO2 conversion, such as chemical, photochemical, electrochemical, and enzymatic reduction. Of particular interest is enzymatic reduction of CO2, owing to its product selectivity, mild experimental conditions, and environmental friendliness7. However, electrochemical CO2 reduction presents various fundamental and practical challenges, mainly because of its inefficient conversion performance and high cost8. Enzymes are natural catalysts. In living cells, enzymes often work together or in a specific order to catalyze multi-step biochemical reactions, which play a crucial role in the synthesis of natural products9. To mimic microbial multistep reactions, multi-enzyme in vitro systems have been explored for various catalytic reactions where single enzyme catalysis is not effective10. Enzymatic reduction of CO2 is a multi-enzymatic, multistep process which employs three dehydrogenases, namely formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH), acting in a cascade where the product of the first reaction serves as a substrate for the subsequent, downstream enzyme7, 11. In vitro biocatalytic reduction of CO2 to methanol is considered a ‘green’ chemical process, and occurs at ambient temperature and atmospheric pressure12. Cazelles and colleagues have reported the reduction of CO2 to methanol by a polyenzymatic system using FDH from Candida boidinii, FaldDH from Pseudomonas putida, and ADH from Saccharomyces cerevisiae7. Similarly, the enzymatic reduction of CO2 to methanol using three dehydrogenases was achieved by reversing the biological metabolic pathways13-17. However, this multi-enzymatic conversion of CO2 to methanol was found to be quite inefficient, taking several hours to produce a very low concentration of methanol (less than 1 mM). Additionally, the need for high NADH concentrations (up to 100 mM) makes the multi-enzymatic CO2 conversion to methanol more expensive. Here, the low reduction activity of FDH and FaldDH is a major challenge for the reduction of CO2. In order to improve the catalysis efficiency of the multi-enzyme cascade reaction, more active FDH and FaldDH are required. Additionally, each of the three dehydrogenases require reduced nicotinamide adenine dinucleotide (NADH) as a cofactor for their activity. Therefore, in situ regeneration of the cofactor in the multi-enzymatic conversion of CO2 will further improve the productivity. Low solubility of CO2 in aqueous media is one of the possible challenges encountered during design of the enzymatic reduction of CO218. Suitable co-solvents may be introduced to increase the substrate solubility. Ionic liquids (ILs), as an alternative to conventional organic cosolvents, can be used owing to their ‘green’ property19. ILs have been used in various chemical and enzymatic reactions, involving enzymes such as lipases, cellulases, and alcohol dehydrogenases20-22. ILs have also been reported to enhance the stability of various enzymes. Furthermore, Rosen and co-workers reported that the IL 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIM-BF4) lowers the reduction over-potential for CO2 conversion in aqueous media2, 23. They proposed that the effectiveness of this newly observed catalytic reduction relies on the ability of the IL to lower the energy barrier. Wipple et al. (2010) also 3 ACS Paragon Plus Environment

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proposed that the over-potential of CO2 reduction could be lowered by stabilizing intermediate CO2 using a suitable catalyst8. In this study, we constructed a multi-enzymatic, artificial cascade reaction using dehydrogenases from three different organisms, which may not share similar reaction conditions for their respective catalytic activity (Figure 1). We used purified recombinant FDH from Clostridium ljungdahlii (ClFDH), FaldDH from Burkholderia multivorans (BmFaldDH), and ADH from S. cerevisiae (YADH) in the overall enzymatic cascade reaction for the conversion of CO2 to chemicals. To overcome the limitation of reducing cofactor levels during catalytic cycles, an in situ NADH regeneration system was incorporated using phosphite dehydrogenase (PTDH). Cazelles et al. (2013) have reported that PTDH shows highest activity in NADH regeneration7. This work also demonstrates the role of EMIM-Ac in the overall process, by comparing the thermodynamic parameters of the multi-enzymatic artificial cascade reaction in the absence and presence of EMIM-Ac. Transient time analysis for the ClFDH-BmFaldDH-YADH and CbFDHPpFaldDH-YADH cascade system was performed to demonstrate the effect of novel enzymes (ClFDH and BmFaldDH) on the entire cascade reaction.

Figure 1. Schematic illustration of reduction of CO2 into methanol using multi-enzymatic cascade reaction with NADH regeneration by PTDH. CO2, formate, and formaldehyde reductions are catalyzed by ClFDH, BmFaldDH, and YADH, respectively.

MATERIALS AND METHODS Materials. The genes encoding ClFDH, BmFaldDH, and PTDH used in this study were synthesized by GenScript (Piscataway, NJ, USA). Alcohol dehydrogenase (YADH) enzyme (homo-tetramer, 141 kDa) from S. cerevisiae and oxidized and reduced nicotinamide adenine dinucleotide (NAD+, NADH), were purchased from Sigma-Aldrich (St. Louis, MO, USA). For PCR, Ex-Taq DNA polymerase was acquired from Promega (Madison, WI, USA). Restriction enzymes were obtained from New England Biolabs (Ipswich, MA, USA). pET 28(a) and pQE80L expression vector and Ni-NTA Superflow column were purchased from Novagen (Madison, WI, USA) and Qiagen (Hilden, Germany), respectively. Oligonucleotide primers and a plasmid isolation kit were obtained from Bioneer (Daejeon, Korea). Electrophoresis reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA) and all assay chemicals were purchased from Sigma-Aldrich. All reagents were of analytical or biotechnological grade. Screening FDHs and FaldDHs. To identify a suitable FDH and FaldDH capable of catalyzing the reduction reaction, we used a three-step screening approach. The first step was a 4 ACS Paragon Plus Environment

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BLAST-based sequence comparison and systematic sequence analysis screening for evaluating the potential of CO2 or formate-reducing homologues using the EcFDH or PpFaldDH sequences, respectively, as the search driver. Then, based on the phenazine methosulfate (PMS)-nitroblue tetrazolium (NBT) assay24, a library of 20 FDHs (Table S1) and 26 FaldDHs (Table S2) was constructed. Finally, the libraries of 20 FDHs and 26 FaldDHs were analyzed for CO2 or formate reduction, respectively (see Supporting Information for details). Cloning, expression, and purification of ClFDH, BmFaldDH, and PTDH. The Clfdh (UniProt D8GNS3; RefSeq WP_013237369), Bmfalddh (UniProt J4QK49), and Ptdh genes from pUC57 vector were PCR-amplified using specific primers. The amplified fragments of Clfdh and Bmfalddh were digested by NdeI and XhoI, and BamHI and XhoI, respectively, and ligated with pET 28(a) to construct recombinant plasmids pET 28(a)-Clfdh and pET 28(a)-Bmfalddh. pET 28(a) is under the control of a T7 promoter and expresses a hexa-histidine tag fused to the N terminus of the protein of interest. The amplified PTDH gene was cloned into vector pQE-80L. The cloned Clfdh, Bmfalddh, and PTDH genes were confirmed to be free of point mutations by DNA sequencing at Macrogen Inc. (Seoul, Korea). The recombinant plasmids were used to transform competent Escherichia coli BL21 (DE3). Expression and purification of the FDHs were performed as previously described25. The recombinant ClFDH, BmFaldDH, and PTDH were expressed using 0.1, 0.1, and 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C, respectively. Ni-NTA resin (3.4 × 13.5 cm, Qiagen) was used to purify the enzymes ClFDH, BmFaldDH, and PTDH. To obtain pure ClFDH, BmFaldDH, and PTDH enzymes, bound Ni-NTA resin was washed with 5 mL of wash buffer (50 mM NaH2PO4 pH 7.0, 300 mM NaCl, 50 mM imidazole). Purity of the enzymes was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by staining with Coomassie blue R250 (BioShop, Burlington, Canada). Protein quantification and enzyme assay. Protein concentration was estimated by the Bradford protein assay method using bovine serum albumin as a reference protein26. CO2 reduction activity of FDH was measured in sodium phosphate buffer (100 mM; pH 7.0), at 37°C. Reaction mixtures (10 mM NaHCO3, 2 mM NADH, and enzyme) were incubated at 37°C for 15 minutes. Each reaction mixture was placed on ice in a sealed tube and the product was estimated instantaneously following the Lang and Lang method27-28. NaHCO3 was used as a substrate instead of CO2 because the concentration of gaseous CO2 could not be accurately determined29. Therefore, CO2 concentration was represented as that of NaHCO3. The initial velocity of enzyme catalysis for CO2 reduction was analyzed by estimating absorbance of formate at 515 nm. An excess of bicarbonate and NADH were provided to ensure that the assays were initiated far from equilibrium. One unit of reduction activity was defined as the amount of enzyme required to produce 1 mol of formate per minute under standard conditions. For FaldDH, the standard assay was carried out using NADH (0.2 mM), and sodium formate (HCOONa, 5 mM) in sodium phosphate buffer (100 mM; pH 7.0) at 25°C. For the alcohol dehydrogenase (YADH), the standard assay was performed with NADH (0.2 mM), and formaldehyde (HCHO 5 mM) in sodium phosphate buffer (100 mM; pH 7.0) at 25°C. FaldDH and YADH activity was determined by following the decrease in the absorbance of NADH at 340 nm by UV-visible spectroscopy7. Reduction of CO2 into methanol. Reduction of CO2 by cascade reactions was performed using ClFDH, BmFaldDH, and YADH enzymes with and without NADH regeneration. 50 mM sodium phosphite (Na2HPO3) was used as substrate for the recycling of NADH using PTDH in 100 mM phosphate buffer at pH 7. ClFDH, BmFaldDH, and YADH ratios and experimental 5 ACS Paragon Plus Environment

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conditions such as pH, temperature, and substrate concentration were optimized using response surface methodology (RSM)30. The statistical software package Design-Expert (Stat-Ease Inc., Minneapolis, MN, USA) was used for regression analysis of experimental data and to plot response surfaces. One-way analysis of variance (ANOVA) was used to estimate the statistical parameters. The ClFDH-BmFaldDH, BmFaldDH-YADH, CbFDH-PpFaldDH, and PpFaldDHYADH coupled enzyme systems were assayed under standard assay conditions (30ºC, pH 7.0) using stopped-flow spectroscopy by monitoring the decrease in NADH fluorescence or absorbance at 460 or 340 nm, respectively. The transient time was determined as described previously31-32. Methanol was produced from CO2 by purging the gas into a sodium phosphate buffer (100 mM; pH 7.0) for 1 h. The pH was adjusted to 7.0 using NaOH. The reaction mixture was then purged with CO2 at the flow rate of 50 mL/min during the reduction to methanol. The flow rate of the gas was controlled by means of a pressure valve. The reaction solution was continuously stirred at 100 rpm with a magnetic bar and the pressure was maintained below 2 bar. Screening of co-solvents. To identify a suitable co-solvent capable of enhancing the enzymatic conversion of CO2 to methanol by using ClFDH, BmFaldDH, and YADH along with PTDH for NADH regeneration, the optimized ratio of the amounts of ClFDH to BmFaldDH to YADH was employed for NaHCO3 as the starting substrate under optimum reaction conditions. Different types of co-solvents were tested to analyze their effect on methanol production. The initial screening of co-solvents was performed by mixing 100 mM of NaHCO3 with 5% of the co-solvents (v/v), then, they were properly mixed to dissolve the substrate at 30 °C. After 1 h of mixing, the pH was appropriately adjusted to 7 and the reaction was initiated by adding the enzymes and NADH. After 6 h of the reaction, the methanol concentration was determined. Analytical methods. Formate concentration was measured following the method described by Lang and Lang27-28, 33. Samples (100 µL) containing formate were mixed with 0.2 mL of solution A, 10 µL of solution B, and 0.7 mL of 100% acetic anhydride and incubated at 50°C for 2 h with occasional mixing. A red color developed which was measured photometrically at 515 nm. Solution A was prepared by dissolving 0.5 g of citric acid and 10 g of acetamide in 100 mL of isopropanol; solution B was prepared by dissolving 30 g of sodium acetate in 100 mL of water. Sodium formate dissolved in potassium phosphate buffer (100 mM; pH 7.0) was used for standard calibration. For the detection and quantification of methanol, aliquots at various time points were taken and analyzed for methanol content by using an Agilent 7890A gas chromatograph with an Agilent J&W DB-1 non-polar column (60 m × 0.32 mm × 2.0 µm) and a FID detector, with ethyl acetate as the internal standard. A calibration curve was prepared by employing the known concentrations of methanol that ranged from 0.05 to 10.0 mM. To estimate the methanol produced as a result of the enzyme-catalyzed reaction, 1.0 µL of the final reaction solution was used for the GC measurements while the injector temperature was maintained at 200 °C. The concentration of methanol was calculated based on the area corresponding to the characteristic methanol peak observed in the chromatogram.

RESULTS AND DISCUSSION A novel formate reducing FaldDH, BmFaldDH. To date, an FaldDH reducing formate to formaldehyde has not been reported. Recently, Ma and colleagues concluded that the reduction of formate to formaldehyde by PpFaldDH was unfavorable during the multi-enzymatic conversion of CO2 to methanol; although the first reaction, catalyzed by CbFDH, occurs very slowly, it is the second reaction, catalyzed by PpFaldDH, that is the real bottleneck34. Therefore, to overcome this bottleneck in the reduction 6 ACS Paragon Plus Environment

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of CO2 to chemicals by a cascade reaction, we screened a number of FaldDHs and evaluated their formate reduction capabilities. We used bioinformatics tools the National Center for Biotechnology Information, Braunschweig Enzyme Database, and a comprehensive literature review to identify suitable FaldDH candidates. We limited our search to FaldDHs capable of catalyzing reduction of formate and searched for reports of CO2 utilization by microorganisms. A library of 26 putative FaldDHs (Table S2) was constructed based on their formate reduction activities in crude cell lysates of 75 different microorganisms using the PMS-NBT system24. Analysis of the formate-reducing capacity of 26 FaldDHs using the FaldDH assay35 led to the identification of a novel FaldDH from Burkholderia multivorans (BmFaldDH) exhibiting formate reduction activity. Recombinant BmFaldDH proteins were purified (Figure S1) using Ni-NTA affinity column chromatography as described previously36. For formate reduction by BmFaldDH, the optimum pH and temperature were 7.0 and 40°C, respectively (Figure S2). Here, BmFaldDH can reduce formate to formaldehyde without the involvement of other enzymes. No reducing activity was observed for PpFaldDH, a frequently used FaldDH in the cascade reduction of CO2 to methanol in vitro. Similarly, FDHs catalyzing reduction of CO2 to formate were screened from 60 different CO2 assimilating microorganisms using the PMS-NBT system24. The reduction analysis of 20 screened FDHs using the Lang and Lang method27 led to the identification of an FDH from Clostridium ljungdahlii (ClFDH) with the highest CO2 reduction activity. The optimum pH and temperature of ClFDH-catalyzed CO2 reduction were 8.6 and 45°C, respectively (Figure S2). The apparent kcat/Km values of ClFDH and BmFaldDH for CO2 and formate reduction were 183 mM-1s-1 and 0.38 mM-1s-1, respectively. The results presented in Table 1 strongly suggest that ClFDH and BmFaldDH are the most efficient catalysts for the reduction of CO2 to a chemical state in multi-enzymatic cascade reactions. Table 1. Comparison of kinetic parameters of various FDHs and FaldDHs Enzyme

Km, NaHCO3 (mM)

Km, NADH (mM)

kcat/Km (1/mM*s)

Reference

CbFDH

31.3 ± 8.03

0.512 ± 0.186

0.0004

29

CbFDH

30-50a

ND

ND

7

TsFDH

9.23 ± 3.98

0.264 ± 0.076

0.034

29

CcFDH

0.37 ± 0.02

0.050

7.45

25

ClFDH

0.03 ± 0.01

0.003 ± 0.001

183

This study

ClFDH

0.12 ± 0.02a

0.003 ± 0.001

39.2

This study

PpFaldDH

NDb

ND

ND

7

BmFaldDH

1.70 ± 0.20b

0.190 ± 0.13

0.38

This study

[a]

CO2 gas was used as substrate instead of NaHCO3, [b] Sodium formate was used as substrate; “ND” Not detected. Although BmFaldDH and PpFaldDH showed high sequence identity (80%), computational analysis revealed significant differences in the formate orientation in terms of distance and angle 7 ACS Paragon Plus Environment

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with the cofactor NADH (Figure S3). Inductively coupled plasma-optical emission spectrometry (ICP-OES) of BmFaldDH revealed the presence of two zinc atoms per monomer, which are designated as the catalytic and structural metal ions (Table S3). Before hydrogen transfer occurs, the carbonyl oxygen needs to be first activated via the catalytic zinc ion37-38. The average distance between the Zn and carbonyl oxygen should be less than 2.5 Å and the Zn-O-C1 angle should be in the range 142 ± 16° for activation. Since the formate can dock with a delocalized negative charge, the distances between the two oxygens of the formate and the Zn were calculated. The distances Zn-O1 (Zn-O2) for BmFaldDH and PpFaldDH are 1.9 (2.0) and 4.2 (2.1) Å, respectively. Based on the molecular dynamics simulation results (Figure S4), it has been further confirmed that the distance between both the oxygens and Zn in BmFaldDH is very well conserved (1.9 to 2.0 Å), but in PpFaldDH, the distance is highly fluctuating, ranging from 2.1 to 4.2 Å, which offers a significant clue on the difference in the reduction activity of the two enzymes. Similarly, the distances between carbonyl carbon and NADH (C4) are 2.4 and 3.2 Å, respectively (Figure S3D and S3B). To make the hydride transfer effective, the substrate and cofactor angle (C4-H-C1) should be close to 160º37. In BmFaldDH and PpFaldDH, the above angle was observed as 153º and 91º, respectively. A shorter distance between the carbonyl carbon (C1) and NADH (C4) is favorable for hydride transfer39 during the reduction of formate by BmFaldDH. Isothermal titration calorimetry of protein-ligand interactions. Isothermal titration calorimetry (ITC) was performed to evaluate the interaction of BmFaldDH with formate, NADH, and NAD+ (Table 2). The Kd values of formate and NADH to BmFaldDH were 6.20 μM and 0.67 μM, respectively (Figure S5). BmFaldDH showed a stoichiometry (N) of 4. However, the thermodynamic parameters could not be obtained for PpFaldDH because of precipitation of PpFaldDH at high concentrations. The results presented in Table 2 clearly show that BmFaldDH has an affinity for both NADH and NAD+ which implies that BmFaldDH can efficiently catalyze reactions in both directions. In contrast to PpFaldDH, BmFaldDH showed formate reduction to formaldehyde. The ΔG value for BmFaldDH (Table 2) which resulted from BmFaldDH and formate interaction suggests that formate reduction is favored when it is present in excess in the reaction mixture. The ΔG, along with the favorable Kd value for NADH, makes BmFaldDH a more efficient catalyst for enzymatic reduction of formate to formaldehyde. When the ΔG for ClFDH is compared with respect to NAD+ and NADH (Figure S6), it is evident that ClFDH facilitates the reduction of CO2 to formate under controlled experimental conditions (Table 2). Table 2. Thermodynamic parameters determined for FDH and FaldDH.a Enzyme Ligand N Kd (μM) ΔGb (kJ/mol) ClFDH NADH 1.85 ± 0.45 0.21 ± 0.01 -38.67 + NAD 2.10 ± 0.32 0.84 ± 0.03 -35.22 CO2 1.82 ± 0.28 1.92 ± 0.06 -33.15 Formate 2.00 ± 0.33 7.55 ± 0.40 -29.69 CO2 + EMIM-Ac 1.79 ± 0.19 0.44 ± 0.05 -36.85 CbFDH NADH 4.12 ± 0.52 1.04 ± 0.07 -34.67 NAD+ 3.97 ± 0.30 0.07 ± 0.01 -41.47 CO2 3.94 ± 0.51 9.95 ± 0.80 -29.00 Formate 4.21 ± 0.34 0.89 ± 0.07 -35.07 8 ACS Paragon Plus Environment

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BmFaldDH

NADH NAD+ HCHO Formate

3.85 ± 0.22 3.91 ± 0.36 3.98 ± 0.50 3.80 ± 0.46


0.67 ± 0.05 0.37 ± 0.03 3.70 ± 0.24 6.20 ± 0.81

-35.2 -36.7 -75.7 -29.7

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[a]

Ligand binding to proteins was measured by ITC. Titration experiments were performed at 25°C and consisted of twenty 2.0-μL injection volumes and 120-second time intervals between consecutive injections. Construction of the multi-enzymatic cascade reaction for reduction of CO2 to methanol. Overcoming the bottleneck for CO2 conversion to chemicals. Several groups have reported reduction of CO2 into methanol using CbFDH, PpFaldDH, and YADH as cascade enzymes7, 13-15. The biocatalytic reduction of CO2 is thermodynamically unfavorable13, 16, and the multi-enzymatic conversion of CO2 to methanol is quite inefficient owing to the low activities of CbFDH and PpFaldDH. To overcome this limitation, novel ClFDH and BmFaldDH were employed in place of CbFDH and PpFaldDH, respectively. Our system overcomes the bottleneck of the multi-enzymatic cascade reaction by using formate-reducing BmFaldDH. To the best of our knowledge, this is the first report of the enzymatic reduction of CO2 to formaldehyde. ClFDH and BmFaldDH converted CO2 and formate to their products, formate and formaldehyde, respectively, confirming the cascade reaction pathway (Figure S7). We used a statistical RSM technique to optimize the various factors simultaneously (Figure S8). For maximum production of methanol (0.44 mM), the optimum ratios of ClFDH, BmFaldDH, and YADH were 2.5, 2.5, and 50 U, respectively. Optimum pH and temperature for the cascade reaction were found to be 7.0 and 30°C, respectively. Optimum pH was found to be well within the range for all three dehydrogenases. NADH cofactor regeneration. During the complete reduction of one molecule of CO2 into one molecule of methanol, three molecules of NADH have to be spent, making the whole process inefficient without NADH regeneration. The oxidation of NADH leads to the accumulation of NAD+ in the reaction mixture, which can result in product inhibition of CO2 reduction and stimulate the reverse oxidative reaction. The Ki values of ClFDH, BmFaldDH, and YADH for NAD+ were determined to be 0.182 mM, 0.225 mM, and 0.764 mM, respectively (Figure S9). At the end of the cascade reaction consisting of ClFDH, BmFaldDH, and YADH, 1.37 mM of NAD+ was accumulated. Therefore, to maintain the initial amount of NADH in the multi-enzymatic cascade reaction and to drive the reaction toward the formation of methanol, regeneration and recycling of NADH is essential. In this work, PTDH was used for the regeneration of NADH, along with the cascade enzymes (ClFDH, BmFaldDH, and YADH) for methanol production from CO2. PTDH shows optimum activity at pH 6.0–8.0, which is well within the optimized catalytic conditions for the conversion of CO2 into methanol (pH 7.0, 30C) (Figure S10). The optimum initial concentration of NADH was observed to be 50 mM for the production of methanol in the absence of the NADH regeneration system (Figure 2). The conversion of CO2 to methanol showed the highest methanol productivity with 3.5 mg mL-1 PTDH (Figure S11). Incorporation of PTDH lowered the NADH requirement from 50 mM to 1.5 mM for the reduction of CO2 to methanol. In a previous study, 100 mM of NADH was used for enzymatic reduction of CO2 into methanol (0.9 mM; after 48 h) via three enzyme cascade reactions (CbFDH-PpFaldDH-YADH) in the presence of an NADH regeneration system7. In the current study, however, a 66-fold lower NADH concentration was used and the methanol concentration obtained was 3.28 mM.


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A

B

Figure 2. Methanol production as a function of initial NADH amount. (A) Influence of initial NADH concentration on the conversion of CO2 to methanol for ClFDH + BmFaldDH + YADH (■) without PTDH, after 6 h of reaction. Similarly, for CbFDH + PpFaldDH + YADH () without PTDH. Productivity of ClFDH + BmFaldDH + YADH (■) and CbFDH + PpFaldDH + YADH () as a function of initial NADH concentration. All the reactions were carried out at 30°C in phosphate buffer (100 mM, pH 7.0) with NaHCO3 (100 mM). (B) Methanol production by various cascade reaction systems indicating the initial NADH concentrations. 1, 2, 3, and 4 represents CbFDH + PpFaldDH + YADH + 100 mM NADH, CbFDH + PpFaldDH + YADH + 100 mM NADH with PTDH, ClFDH + BmFaldDH + YADH + 50 mM NADH, and ClFDH + BmFaldDH + YADH + 1.5 mM NADH with PTDH, respectively. Conversion of CO2 into formaldehyde and methanol. CO2 was reduced to methanol using the newly constructed multi-enzymatic cascade system (ClFDH, BmFaldDH, and YADH) with NADH regeneration (PTDH) and NaHCO3 as the starting substrate. The time course of methanol production was examined and methanol concentration was estimated by GC (Figure S12A). Methanol reached its highest concentration of 3.28 mM after 6 h. However, the amount of methanol produced by enzymatic reduction of CO2 using CbFDH, PpFaldDH, and YADH without and with NADH regeneration system was only 0.023 mM and 0.26 mM after 6 h, respectively. A

B


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Figure 3. Methanol and formaldehyde production from CO2 by cascade reaction. (A) Time profile of methanol production by multi-enzymatic cascade reactions. (▲) CbFDH + PpFaldDH + YADH with PTDH, (▼) ClFDH + PpFaldDH + YADH with PTDH, (♦) CbFDH + BmFaldDH + YADH with PTDH, (●) ClFDH + BmFaldDH + YADH with PTDH, and (■) ClFDH + BmFaldDH + YADH with PTDH and EMIM-Ac (1%). All reactions were carried out at 30°C in 100 mM phosphate buffer (pH 7.0) with 100 mM NaHCO3. (B) Time profile of formaldehyde production using FDH and FaldDH with NADH regeneration. CbFDH + PpFaldDH with PTDH (▲), ClFDH + BmFaldDH without PTDH (■), and ClFDH + BmFaldDH with PTDH (▲). The reaction mixture consisted of 1.5 mM NADH and 100 mM NaHCO3 in 100 mM phosphate buffer at pH 7 with 2.5 U/mL of ClFDH and 2.5 U/mL BmFaldDH. CbFDH and PpFaldDH were not able to reduce CO2 to formaldehyde within 6 h of the reaction, but ClFDH and BmFaldDH could reduce CO2 to formaldehyde (Figure 3B), resulting in the subsequent efficient reduction of CO2 to methanol. To further identify and quantify the amount of formaldehyde produced, Nash’s reagent-derivatized formaldehyde was analyzed by high-performance liquid chromatography (Figure S12B). These analyses showed a novel timedependent formaldehyde production from CO2. It has been reported that formaldehyde cannot be produced from CO2 or formate by enzymatic action34. Because formaldehyde is an important intermediate used to synthesize a variety of value-added chemicals, this result provides a new platform that can be used to synthesize value-added chemicals from CO2 feedstock. When PpFaldDH was replaced with BmFaldDH, the cascade reaction consisting of CbFDH, BmFaldDH, and YADH could achieve a methanol concentration of 1.69 mM. Here, BmFaldDH overcame the limitation of PpFaldDH for the reduction of formate into formaldehyde and resulted in a 126-fold enhancement in methanol yield from CO2 (Table 3). These results indicate that BmFaldDH plays a crucial role in the reduction of CO2 to methanol. Using ClFDH, BmFaldDH, and YADH coupled with the NADH regeneration system, the highest methanol concentration obtained was 3.28 mM. This result implies that the ClFDH and BmFaldDH multienzymatic system is far superior to existing enzymatic systems composed of CbFDH and PpFaldDH (Figure 3A). Table 3 summarizes the current state of CO2 conversion into methanol using an enzymatic cascade reaction and highlights the superiority of the ClFDH, BmFaldDH, and YADH system. The methanol yield of the ClFDH-BmFaldDH-YADH cascade system was estimated to be 505fold higher than that of the previously reported CbFDH-PpFaldDH-YADH cascade7. We also estimated the productivity and total turnover number (TTN) of CO2 conversion to methanol by the multi-enzymatic cascade system based on the average methanol formation rate per hour and methanol formation per mole of initial NADH. For ClFDH-BmFaldDH-YADH with NADH regeneration, the calculated productivity and TTN were 1.13 mM·h-1 and 4.50 mol·mol-1 of initial NADH, respectively. The enzymatic reduction of CO2 to methanol using CbFDHPpFaldDH-YADH reported by Cazelles et al. exhibited productivity and TTN of 0.014 mM·h-1 and 0.009 mol·mol-1 of initial NADH, respectively7. Recently reported solar-driven methanol production from CO2 by a photocatalyst/biocatalyst integrated system showed productivity of 0.007 mM·h-1 17. Compared to the productivities (0.014 and 0.007 mM·h-1) of the systems described above for CO2 conversion to methanol with NADH regeneration, the productivity of our system was almost 81- and 161-fold higher. Similarly, the ClFDH-BmFaldDH-YADH with NADH regeneration system in the presence of EMIM-Ac (1%) showed more than 500-fold greater TTN than that of the CbFDH-PpFaldDH-YADH with NADH regeneration system. ClFDH-BmFaldDH-YADH with NADH regeneration requires a 66-fold lower cofactor 12 ACS Paragon Plus Environment

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concentration with 81-fold higher methanol productivity compared to that in a previous report7. The lower cofactor requirement and higher productivity makes this cascade system efficient. We also tested CO2 gas as a substrate. Figure 4 shows that 7.86 mM of methanol was produced by the ClFDH-BmFaldDH-YADH cascade reaction in the presence of a NADH regeneration system and EMIM-Ac (1%) when CO2 gas was used as the starting substrate. However, the CbFDH-PpFaldDH-YADH cascade produced only 0.20 mM of methanol. Here, the methanol yield of the ClFDH-BmFaldDH-YADH cascade system in the presence of the NADH regeneration system was estimated to be 1.59×103%, which is 529-fold higher than that of the previously reported CbFDH-PpFaldDH-YADH system with NADH regeneration13. A C B

Figure 4. Methanol production via multi-enzymatic cascade reaction using CO2 gas. (A) Time profile of methanol production by multi-enzymatic cascade reactions. (▲) CbFDH + PpFaldDH + YADH with PTDH, (▼) ClFDH + PpFaldDH + YADH with PTDH, (♦) CbFDH + BmFaldDH + YADH with PTDH, (●) ClFDH + BmFaldDH + YADH with PTDH, and (■) ClFDH + BmFaldDH + YADH with PTDH and EMIM-Ac (1%). (B) Total turnover number as a function of time for different combinations of FDH, FaldDH, and YADH enzymes: (black color) CbFDH + PpFaldDH + YADH with PTDH, (green color) ClFDH + PpFaldDH + YADH with PTDH, (purple color) CbFDH + BmFaldDH + YADH with PTDH, (red color) ClFDH + BmFaldDH + YADH with PTDH, and (blue color) ClFDH + BmFaldDH + YADH with PTDH and EMIM-Ac (1%). (C) Formation of formate and formaldehyde during the reduction of CO2 to methanol catalyzed by ClFDH + BmFaldDH + YADH with PTDH. All reactions were performed at 30 °C in phosphate buffer (100 mM, pH 7.0). CO2 gas was bubbled into the phosphate buffer and the reaction was initiated by adding the respective cascade enzymes.


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Table 3. Enzymatic methanol production by various cascade reaction systems. Methanol Enzymes System (mM)

Productivity (mM·h-1)

Methanol /NADH

Reference

CbFDH + PpFaldDH + YADH + GDH

0.02 ± 0.003

Co-immobilized

0.040

0.046

[40]

CbFDH + PpFaldDH + YADH

0.015 ± 0.002

Immobilized

0.002

0.075

[41]


0.023 ± 0.004

Free enzyme

0.007

0.001

[7 ]

CbFDH + PpFaldDH + YADH + PTDH

0.20 ± 0.03

Free enzyme

0.014

0.009

[ 7]


0.011

Free enzyme

0.007

0.028

[17]


0.10 ± 0.02

Free

0.20

0.010

[13]


0.10 ± 0.01

Immobilization

0.20

0.010

[13]

ClFDH + BmFaldDH + YADH + PTDH

3.28 ± 0.20

Free enzyme

0.55

2.19

This report

0.49

1.97

This report

1.13

4.50

This report

1.31

5.24

This report

ClFDH + BmFaldDH + YADH + PTDHa 2.95 ± 0.34 Free enzyme ClFDH + BmFaldDH + YADH + PTDH 6.75 ± 0.24 Free enzyme + EMIM-Ac ClFDH + BmFaldDH + YADH + PTDH 7.86 ± 0.41 Free enzyme + EMIM-Aca “a” CO2 gas used as substrate; “Productivity” Average methanol formation rate per hour.


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Protein-ligand interactions in the presence of co-solvent. A multi-enzyme cascade reaction is a complex reaction that involves several enzymes and their respective substrates, along with cofactors. Owing to differences in their solubility and behavior in aqueous media, co-solvents are often introduced in biochemical reactions to enhance their efficacy. As shown in Table S4, we screened a wide spectrum of co-solvents to achieve higher methanol concentrations. Although co-solvents have been used in many enzymecatalyzed reactions, this is the first report where the effects of co-solvents were investigated for multi-enzymatic reduction of CO2 into methanol. Among all the screened co-solvents, EMIM-Ac exhibited the highest enhancing effect on the reduction of CO2 to methanol (Table S4). The EMIM-Ac concentration was further optimized (ranging from 0.1 to 10% (v/v)) to achieve maximum methanol production. Methanol production by the cascade reaction was the highest (6.75 mM) at 1.0% EMIM-Ac concentration when NaHCO3 was used as the starting substrate. As discussed above, EMIM-Ac enhanced methanol production from 3.28 mM to 6.75 mM. Here, the optimized concentration of EMIM-Ac (1.0%) was added to the cascade reaction along with NADH regeneration (PTDH). CO2 interacts with electronegative nitrogen and oxygen atoms. This causes the solubility of CO2 to be higher in EMIM-Ac19. The highest methanol concentration was found to be 6.75 mM after 6 h of reaction, as presented in Figure 3A. This indicates that EMIM-Ac plays crucial roles during CO2 reduction in the cascade reaction. To date, aqueous medium was quite popular among researches for enzymatic reduction of CO2. EMIMAc is a green solvent, completely miscible with water at all concentrations and is extremely hygroscopic. The solubility of CO2 is reported to be higher in EMIM-Ac than in water, predominantly due to anion-CO2 interactions9. Effects of EMIM-Ac on enzyme activity was evaluated by performing enzyme assay in the presence of EMIM-Ac (Table S5). The results presented in Table S5 clearly indicate that EMIM-Ac does not increase the activity of ClFDH, BmFaldDH, or YADH. In order to investigate the role of EMIM-Ac in the enhancement of methanol production, ITC was performed to determine the thermodynamics of ClFDH and CO2 interactions in the presence (Figure S13) and absence of EMIM-Ac. The Kd value of ClFDH for CO2 in the presence of EMIM-Ac was 4.4-fold lower than that of ClFDH for CO2 in the absence of EMIMAc (Table 2). The ΔG value of CO2 interactions with ClFDH in the absence of EMIM-Ac was 33.15 kJ mol-1, which is higher than that (-36.85 kJ mol-1) of CO2 and ClFDH interaction in the presence of EMIM-Ac. Here, the lower binding enthalpy (ΔH value) reflects the increased strength of interaction between CO2 and ClFDH in the presence of EMIM-Ac. It has been reported that the presence of EMIM+ cations around CO2 molecules can reduce the reaction barrier for electrons passing into CO2 9-10. Thus, the observed lower Kd and ΔG values of CO2 in presence of EMIM-Ac supports the enhanced methanol production by the cascade reaction. Transient time of multi-enzymatic cascade reaction. Multi-enzyme cascade reactions are well coordinated and possess systematic pathways to reach the final metabolite. During the whole process of CO2 reduction to methanol, the transfer of a reaction intermediate from the ClFDH active site to BmFaldDH and subsequently to YADH is an important factor. To gain insight into the stepwise reduction of CO2 to methanol by the newly constructed cascade reaction (ClFDH, BmFaldDH, and YADH), transient time (τ) was determined for each coupled enzyme step (Figure 5)42-43. τ is an apparent lag phase to reach steady state flux of the intermediate in a coupled reaction32. τ1 is the time required to reach steady state when CO2 is converted to formaldehyde by ClFDH and BmFaldDH via formate. Similarly, τ2 is the time prior to reaching steady state while the formate is being converted to 15 ACS Paragon Plus Environment

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methanol via formaldehyde. Transient time was measured over the course of the reaction using stopped-flow spectroscopy. τ1 for the ClFDH-BmFaldDH coupled reaction was observed to be 19 s (Figure 5C). In comparison, τ2 was found to be 14 s for a BmFaldDH-YADH coupled reaction (Figure 5D). Under the same experimental conditions, the τ1 and τ2 for a commercial enzyme cascade reaction construct (CbFDH, PpFaldDH, and YADH) could not be determined due to the extended lag phase (>60 s). A decrease in transient time indicates a decreased lag phase between the two consecutive reactions catalyzed by two consecutive enzymes and highlights the efficiency of a multi-enzyme (ClFDH, BmFaldDH, and YADH) cascade reaction.

CONCLUSIONS We have demonstrated that the newly constructed multi-enzyme cascade system using a novel formate-reducing BmFaldDH in the presence of EMIM-Ac can overcome the bottleneck (reduction of formate to formaldehyde) of the previously reported cascade systems for enzymatic conversion of CO2 to chemicals. This is the first report where BmFaldDH and EMIM-Ac were incorporated in the cascade reaction for the reduction of CO2 to chemicals. We also demonstrated biocatalytic production of formaldehyde from CO2 or formate for the first time, revealing the novel potential to synthesize value-added chemicals from CO2. Compared to the CbFDHPpFaldDH-YADH system that was used in all previous studies for the conversion of CO2 into methanol, ClFDH-BmFaldDH-YADH exhibited a 243-fold higher TTN. When EMIM-Ac was used in the multi-enzymatic cascade reaction, it further increased methanol production by improved protein-ligand interaction. This added a new dimension to the role of EMIM-Ac in the enzymatic reduction of CO2 to chemicals. Overall, our novel multi-enzyme cascade system is a promising and highly efficient strategy for CO2-to-chemical conversion.


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A

B

C

D

Figure 5. Determination of the transient time (τ) of a coupled enzyme reaction. (A) and (B) show the raw data image of FDH-FaldDH and FaldDH-YADH catalyzed reactions using a stopped-flow spectrophotometer at 25°C and pH 7.0. ClFDH-BmFaldDH and BmFaldDHYADH are shown with blue lines. CbFDH-PpFaldDH and PpFaldDH-YADH are shown with green lines. Black lines show the control reactions. (C) ClFDH-BmFaldDH (■) coupled reaction is shown with blue line. CbFDH-PpFaldDH (●) coupled reaction is shown with green line. (D) BmFaldDH-YADH (▲) coupled reaction is shown with blue line. PpFaldDH-YADH (●) coupled reaction is shown with green line. A linear fit to the product concentration as a function of time at steady state crosses the x-axis at τ. The y-intercept is equal to the negative of the steady state concentration of the cascade reaction intermediate, [I]ss. ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B3011676, 2017R1A4A1014806, 2013M3A6A8073184). This work was supported by the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (20153030091450). This work was supported by 2017 KU Brain Pool fellowship of Konkuk University. 17 ACS Paragon Plus Environment

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CONFLICT OF INTEREST The authors declare no competing financial interest. SUPPORTING INFORMATION Table S1-S5, Figures S1-S13, and detailed experimental methods.

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Insights into cell-free conversion of CO2 to chemicals by a

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