Bifunctional RhIII-complex catalyzed CO2 reduction and NADH regeneration for direct bioelectrochemical synthesis of C3 and C4

Bifunctional RhIII-complex catalyzed CO2 reduction and NADH regeneration for direct bioelectrochemical synthesis of C3 and C4 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bifunctional Rh III -complex catalyzed CO 2 reduction and NADH regeneration for direct bioelectrochemical synthesis of C 3 and C 4 Yajie Wang, Hailong Li, Yizhou Wu, Yuxuan Wang, Kai Zhang, Jin Zhu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4865792/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bioelectrochemical synthesis is emerging as an eco-friendly method for CO 2 fixation. These systems typically rely on electrochemically regenerated NAD(P)H to provide the necessary reducing equivalents for formate dehydrogenase (FDH) to convert CO 2 into formate. However, the efficiency of these systems is currently unsatisfactory due to the unfavorable dynamics of the CO 2 -to-formate conversion by FDH. In this study, we developed a one-pot cooperative bioelectrochemical system featuring a rhodium-based catalyst [Cp*Rh(bpy)Cl] 2+ (Rh III -complex or [Rh III -H 2 O] 2+ ) working cooperatively with enzymatic cascades of acetyl-CoA synthase (ACS), acetaldehyde dehydrogenase (ACDH), alcohol dehydrogenase (ADH), formolase (FLS), and d-fructose-6-phosphate aldolase mutant FSA A129S to convert CO 2 into several C 2+ chemicals. The bifunctional Rh III -complex concurrently catalyzes the reduction of CO 2 to formate at a rate of 15.8 mM/h and NADH regeneration at a rate of 0.24 mM/min. The formation of formate is 83.2 times faster than using one of the best aerobic FDH from Clostridium ljungdahlii ( Cl FDH), resulting in a 3.6 times enhanced methanol production rate of 0.43 mM/h in the bioelectroenzymatic system (Rh III -complex-ACS-ACDH-ADH) compared to that of 0.12 mM/h in tandem enzymatic system ( Cl FDH-ACS-ACDH-ADH). Bifunctional Rh III -complex also works cooperatively with tandem enzymatic cascades to produce dihydroxyacetone (C 3 ) and L-erythrulose (C 4 ) at the yield of 2.63 mM, and 1.93 mM, respectively. This study leveraged the synthetic capabilities of both electrochemical catalysis and enzymatic catalysis, offering an alternative for electroenzymatic CO 2 reduction to yield value-added compounds with enhanced productivity. Physical sciences/Chemistry/Catalysis/Biocatalysis Physical sciences/Chemistry/Catalysis/Electrocatalysis Carbon dioxide electroreduction NADH regeneration enzymatic cascade bioelectrochemical system Figures Figure 1 Figure 2 Figure 3 Figure 4 Main To address the energy crisis and alleviate the rising level of CO 2 in the atmosphere, various CO 2 capture and utilization techniques have been developed. 1 While electrochemical strategies offer high energy efficiency for CO 2 reduction and transformation, their practical application is limited by low selectivity in producing C 2+ chemicals. 2 In contrast, biocatalysis excels in selectively producing C 2+ chemicals but is less efficient in CO 2 reduction due to the high energy input required to break the O = C = O bond (750 kJ mol − 1 ). 3 In this respect, there is a growing interest in developing electroenzymatic hybrid systems that combine the synthetic power of both catalytic disciplines to efficiently produce value-added compounds from CO 2 . 4 – 7 CO 2 reduction processed by oxidoreductases commonly requires a substantial amount of reducing equivalents. Recent advancements in one-pot bioelectrochemical systems have introduced various NAD(P)H-dependent oxidoreductases within the cathode chamber to convert CO 2 into products like carbon monoxide (CO), 3 formate (HCOOH), 8 methanol (CH 3 OH), 9 ethylene (C 2 H 4 ), 2 and other C 2+ chemicals, 2, 6 utilizing reducing equivalents regenerated from electricity through direct or mediated electron transfer. Among them, formate dehydrogenase (FDH)-catalyzed CO 2 -to-formate conversion is thermodynamic unfavorable and rate-limited due to more positive redox potential of NAD + /NADH (− 0.32 V vs NHE) compared to CO 2 /HCOOH (− 0.43 V vs NHE). 10 This rate-limiting step may also restrict the supply of formate for subsequent C 2+ chemical synthesis catalyzed by enzymatic cascades. 6 Conversely, many electrocatalysts have been developed to efficiently produce formate through electrochemical CO 2 reduction reaction (CO 2 RR) with low overpotential and high Faradaic efficiency. 5 , 11 – 13 Additionally, several electrochemical systems have been widely reported for reducing NAD + to 1,4-NADH with nearly 100% selectivity and good enzyme compatability. 14 , 15 However, electrochemical catalysis for the simultaneous reduction of CO 2 and NAD + , and its cooperative operation with enzymatic cascades for C 2+ chemical synthesis, remains relatively uncommon in this field. Thus, we aimed to develop a “one-pot” biocompatible electrochemical system that can efficiently produce formate from CO 2 and regenerate NADH at the same time. This system can work cooperatively with tandem enzymatic cascades to convert CO 2 into value-added C 2+ chemicals with improved production rate and enantioselectivity. In this study, we developed a cooperative one-pot bioelectrochemical system for the electroenzymatic synthesis of value-added compounds from CO 2 (Scheme 1 ). This system utilizes the bifunctional metal complex [Cp*Rh(bpy)Cl] 2+ (Rh III -complex or [Rh III -H 2 O] 2+ ) to concurrently catalyze the electrochemical reduction of CO 2 to formate and the regeneration of NADH from NAD + . The resulting formate is then converted into methanol (C 1 ), dihydroxyacetone (C 3 ), and L-erythrulose (C 4 ) at the rates of 0.43 mM/h, 0.11 mM/h, and 0.08 mM/h, respectively, through the tandem enzymatic cascades involving ACS-ACDH-ADH, ACS-ACDH-FLS, and ACS-ACDH-FLS-FSA A129S (Scheme 2 ). In those cooperative bioelectroenzymatic synthesis, Rh III -complex exhibited a CO 2 reduction rate of 15.8 mM/h, which is 83.2 times faster than the Cl FDH-containing electroenzymatic system (0.19 mM/h). Meanwhile, Rh III -complex maintained the NADH regeneration at a rate of 0.24 mM/min, efficiently driving the enzymatic cascade for the formation of C 1 -C 4 chemicals. By leveraging the synergies between electrochemical catalysis and biocatalysis, this cooperative bioelectrochemical system enables the conversion of CO 2 into value-added chemicals at a higher rate and yield compared to the electroenzymatic and tandem enzymatic systems with FDH. Results and discussion Firstly, we aimed to design a bifunctional electrocatalyst capable of simultaneously reducing CO 2 and NAD + . Inspired by the studies on CO 2 photoreduction catalyzed by Rh III -based photocatalysts, 16 we selected [Cp*Rh(bpy)H 2 O] 2+ (Rh III -complex or [Rh III -H 2 O] 2+ ) for evaluation because of its established efficiency in electrocatalytic NAD + regeneration and good biocompatibility. 15 , 17 To evaluate the electrocatalytic performance of the Rh III -complex for CO 2 reduction under the mild conditions, we conducted reactions with 0.5 mM Rh III -complex in 0.1 M PBS at pH 7.0 using a three-electrode H-cell device (Carbon felt serves as the working electrode, reversible hydrogen electrode (RHE) as the reference electrode, and Pt plate as the counter electrode) with CO 2 gas bubbling ( Figure S1 ). To be noted, Rh III -complex effectively catalyzed the generation of formate from CO 2 at the rate of 5.5 mM/h with an applied potential of -0.29 V RHE , demonstrating superior efficiency compared to the utilization of bicarbonate as a substrate (Fig. 1 A). To optimize the formate yield, we initially tested the effects of the proton exchange membrane (PEM, Nafion 117) and the anion exchange membrane (AEM, PiperION-A20) on CO 2 reduction and found that PEM performed better than AEM (Fig. 1 A). Subsequently, we evaluated the electrochemical CO 2 reduction under various conditions, including different applied potentials, CO 2 gas flow rates, Rh III -complex concentrations, and PBS pH levels ( Figure S2 ). Under the optimal operating conditions of 0.1M PBS at pH 7.0, a CO 2 gas flow rate of 20 ml/min, and an applied voltage of -0.39 V RHE , the formate production rate reached 7.49 mM/h ( Figure S2D ). Additionally, considering that ionic liquids (ILs) are known to decrease the overpotential of CO 2 reduction in aqueous media 18 , 19 and that 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) has shown superior performance in CO 2 reduction within biocatalytic systems, 20 the performance of EMIM-Ac was evaluated. Incorporating 1% (v/v) EMIM-Ac resulted in the production of 19.32 mM formate within the first hour and 189.63 mM after 12 hours of reaction (Fig. 1 B). For comparison, formate production was evaluated using a conventional electroenzymatic system with one of the most efficient aerobic FDHs from Clostridium ljungdahlii ( Cl FDH) as the biocatalyst, Rh III -complex as the NADH regeneration catalyst, and 100 mM bicarbonate as the substrate. 20 Over a 12-hour reaction, only 2.28 mM of formate was generated, which is 83.2 times lower than that produced by our electrochemical system. Efficient NADH regeneration is essential in electroenzymatic synthesis involving multiple NADH-dependent oxidoreductases. To investigate the dual functionality of Rh III -complex, we evaluated the NADH regeneration rate with or without CO 2 bubbling. The Rh III -complex demonstrated the optimal activity for NADH regeneration under the condition most suitable for CO 2 reduction ( Figure S3 ). The presence of EMIM-Ac had little influence on NADH regeneration (Fig. 1 C). 3 mM of NAD + was electrochemically reduced at a rate of 0.24 mM/min, achieving an 80% recovery within 10 min, which was comparable to most of the reported rhodium complex-catalyzed electrochemical NADH regeneration processes. 14 Additionally, in the presence of CO 2 , 0.5 mM Rh III -complex could maintain catalytic efficiency for both NADH regeneration and CO 2 reduction (Fig. 1 D). To better understand the mechanism of bifunctional Rh III -complex in CO 2 and NAD + reduction, cyclic voltammogram (CV) curves were conducted in 0.1 M PBS at pH of 7.0 with or without 1% (v/v) EMIM-Ac. The overall electrochemical behavior can be explained by an EEC (electron transfer, electron transfer, chemical reaction) mechanism ( Eqs. 1–3 ). As shown in Fig. 2 A, the CV curves of CO 2 with or without EMIM-Ac have no significant differences, indicating that EMIM-Ac does not catalyze CO 2 reduction. In the absence of both CO 2 and EMIM-Ac, [Rh III -H 2 O] 2+ undergoes a two-electron reduction process to form [Rh I -H] + hydride ( Eqs. 1 ) at -0.17 V RHE (-4.67 mA/cm 2 ). After adding 1% EMIM-Ac, the reduction peak slightly shifts to -0.16 V RHE with a higher current density (-6.43 mA/cm 2 ). These data suggest that EMIM-Ac, as a cosolvent, does not have a noticeable interference on the process of [Rh I -H] + formation. Comparing the CV curves of Rh III -complex for CO 2 reduction with or without EMIM-Ac (Fig. 2 B), the cathodic current peak increases from − 8.34 to − 9.56 mA/cm 2 , indicating a higher CO 2 reduction efficiency. Additionally, the reduction peak of [Rh I -H] + hydride-catalyzed CO 2 reduction ( Eqs. 2 ) shifts from − 0.25 V RHE (without EMIM-Ac) to − 0.17 V RHE (with EMIM-Ac), suggesting that EMIM-Ac lowered the energy of the [Rh I -H] + -CO 2 intermediate, thereby reducing the initial reduction barrier. Those findings are consistent with the previous studies showing that ionic liquids can decrease the overpotential for CO 2 to CO conversion. 18 The CV curves of Rh III -complex in the presence of NAD + clarify the interplay between [Rh I -H] + and NAD + (Fig. 2 C and D ). The CV curve of NAD + reduction without Rh III -complex shows no significant differences in reduction peak in the presence or absence of EMIM-Ac, indicating that EMIM-Ac does not impact on NAD + reduction (Fig. 2 C). According to the catalytic mechanism reported by Kim et al. , 21 the [Rh III -H 2 O] 2+ was reduced to [Rh I -H] + ( Eqs. 1 ) by the cathode, and then a hydride transfer reaction takes place between [Rh I -H] + and NAD + to achieve NADH regeneration ( Eqs. 3 ). The reduction peak of [Rh III -H 2 O] 2+ to [Rh I -H] + during NADH regeneration is clearly observed at − 0.19 V RHE (Fig. 2 D). Compared with the Rh III -complex mediated NADH regeneration, the direct NAD + reduction requires a more negative potential of − 0.59 V RHE (Fig. 2 C), confirming that NAD + was reduced by [Rh I -H] + via a hydride transfer pathway in our system. 22 The simultaneous reduction of CO 2 and NAD + begins at − 0.21V RHE in the presence of 1% EMIM-Ac, slightly more negative than the potential of Rh III -complex catalyzed CO 2 reduction (− 0.17 V RHE , Fig. 2 A). CV analysis defines the electrochemical window for the mediated CO 2 reduction and NADH regeneration. Thus, a potential of -0.39 V RHE is sufficient for driving the concurrent CO 2 and NAD + reduction. Formaldehyde is a promising feedstock for tandem enzymatic cascade reactions. 23 However, the enzymatic reduction of formate to formaldehyde is thermodynamically unfavorable. We compared the highest catalytic efficiency of two pathways for synthesizing formaldehyde from formate (Figure S5 , Fig. 3 A ) : the formaldehyde dehydrogenase (FaldDH) pathway and the formolase pathway that utilizes acetyl-CoA synthase (ACS) from Escherichia coli and acetaldehyde dehydrogenase (ACDH) from Listeria monocytogenes . 24 By employing the most efficient enzymes under the optimal reaction conditions in both pathways, we achieved a formaldehyde formation rate of 32.87 µM/h and a final concentration of 98.63 µM within 3 hours by using the formolase pathway with phosphite dehydrogenase (PTDH)-catalyzed NADH regeneration ( Figure S6-S11 ). In contrast, only 18.62 µM formaldehyde was obtained after a 3-hour reaction when using FaldDH from Burkholderia multivorans ( Bm FaldDH) 20 as the biocatalyst (Fig. 3 B ) . The low yield could be attributed to significantly unfavorable thermodynamics of FDH pathway (Δ r G′ m = + 51.7 kJ/mol) compared to the formolase pathway (ΔrG′ m = -8.6 kJ/mol) (Fig. 3 A). Given the preference of Bm FaldDH for formaldehyde oxidation over formate reduction, we added alcohol dehydrogenase (ADH, Figure S12-13 ) from Saccharomyces cerevisiae to convert formaldehyde to methanol, driving the equilibrium towards the reduction direction (Scheme 3 ). With excess formate (150 mM) as the substrate, the formolase pathway ( Route 2 ) outperformed the Bm FaldDH pathway ( Route 1 ), reaching a maximum of 2.78 mM methanol within 6 h, which is 3.4-fold higher than that of Route 1 (Fig. 3 C). At a low formate concentration of 10 mM, Route 2 still produced approximately three times more methanol than Route 1 within the first-hour reaction ( Figure S14-15 ). To this end, we selected the formolase pathway for further establishment of cooperative bioelectrochemical systems. Prior to establishing the cooperative bioelectrochemical system for CO 2 reduction, we assessed the effectiveness of Rh III -complex-catalyzed electrochemical NADH regeneration in supporting the formolase pathway for methanol production ( Route 3 ). As shown in Fig. 3 C, the methanol production rate (0.49 mM/h) and the final concentration (2.87 mM) over a 6-hour reaction period of electrochemical NADH regeneration system is slightly superior to those of PTDH-catalyzed NADH regeneration systems. This suggests that the Rh III -complex is biocompatible for enzymatic system with high NAD + reduction efficiency. We finally set up the cooperative bioelectrochemical system by coupling Rh III -complex-catalyzed CO 2 reduction and NADH regeneration with ACS, ACDH, and ADH to synthesize methanol from CO 2 in a “one-pot” manner ( Route 5 ). The cooperative bioelectrochemical system ( Route 5 ) generates 2.59 mM methanol after 6-hour reaction, which is comparable to the enzymatic system (2.65 mM in Route 2 ) using formate as the substrate (Fig. 3 C). This proves that Rh III -complex-catalyzed electrochemical CO 2 reduction is efficient and biocompatible to the enzymatic cascade. In addition, the cooperative bioelectrochemical system ( Route 5 ) produces 3.8 times higher methanol from gaseous CO 2 compared to the tandem enzymatic system (0.68 mM in Route 4 ) as shown in Fig. 3 D. These underscore the pivotal role of the bifunctional Rh III -complex in the cooperative bioelectrochemical system for efficient CO 2 reduction and subsequent synthesis of valuable compounds. Building on the success of the cooperative bioelectrochemical system for efficient methanol production, we aimed to extend the applications of bifunctional Rh III -complex in electroenzymatic synthesis of C 2+ chemicals from CO 2 . Dihydroxyacetone (DHA) and L-erythrulose are key chemicals in cosmetics, pharmaceuticals, and food industries for their water-evaporation prevention, UV protection, and antioxidant properties. 23 While DHA and L-erythrolose have been produced for decades through oxidative microbial fermentation, direct synthesis from CO 2 is rare. 23 , 25 We were pleased to find that an engineered Benzaldehyde lyase (BAL) variant, formolase (FLS), could catalyze the asymmetric umpolung activation of formaldehyde to DHA. 24 , 26 Additionally, extensive development of d-fructose-6-phosphate aldolase’s derivative A129S (FSA A129S ) has enabled efficient aldol addition between formaldehyde and DHA while maintaining stereochemical fidelity. 26 , 27 With those powerful tools in hand, we aimed to build the cooperative bioelectrochemical systems to convert CO 2 into DHA and L-erythrulose. After optimizing the reaction conditions for converting formaldehyde into DHA and L-erythrulose catalyzed by FLS and FSA A129S ( Figure S17-18 ), we coupled Rh III -complex-ACS-ACDH with FLS and FSA A129S in one-pot to create cooperative bioelectrochemical systems for the direct synthesis of DHA and L-erythrulose from CO 2 respectively (Scheme 4 ). Remarkably, using CO 2 as the initial substrate, the cooperative bioelectrochemical system, Route 6 and Route 7 , produced 2.63 mM and 1.93 mM of DHA and L-erythrulose within 24 h, respectively (Fig. 4 ). These results were comparable to the output of the tandem enzymatic system using formate as the starting materials ( Scheme S1 and Figure S19 ). These findings highlight the bifunctional ability of Rh III -complex to efficiently reduce CO 2 to formate while supplying the necessary reducing equivalents (NADH) to drive the subsequent tandem enzymatic cascades for synthesizing longer-chain, higher-value chiral compounds. Conclusion This study introduces several cooperative one-pot bioelectrochemical systems that consist of a bifunctional Rh III -complex and various tandem enzymatic cascades to directly synthesize methanol (C 1 ), dioxyacetone (C 3 ), and L-erythrulose (C 4 ) from CO 2 , achieving high yields of 2.58 mM, 2.63 mM, and 1.93 mM, respectively. The Rh III -complex catalyzes the reduction of CO 2 to formate at a rate of 15.8 mM/h, which is 83.2 times faster than that of FDH, and simultaneously facilitates NADH regeneration at a rate of 0.24 mM/min to provide the reducing equivalent (NADH) for subsequent tandem enzyme cascades. The rapid generation of formate in the cooperative bioelectroenzymatic system enhances methanol production at 0.43 mM/h, which is 3.58 times faster than the rate achieved in a tandem enzymatic system using FDH for CO 2 reduction. This highlights the ability of Rh III -complex to effectively addresses the limitations of low catalytic activity of FDH in both electroenzymatic and tandem enzymatic reactions. The study emphasizes the potential of bioelectrochemical CO 2 reduction as a viable alternative for synthesizing high valuable C 2+ products and may inspire the direct electrosynthesis of value-added compounds from CO 2 . Methods The activity assay and kinetic assay The activity of Cl FDH was monitored by NADH oxidation at 340 nm. CO 2 reduction activity of FDH was measured in 0.1 M PBS (pH 7.0), at room temperature. Reaction mixtures (0.1M NaHCO 3 , 5 mM NADH, and enzyme) were incubated for 1 hour. Each reaction mixture was placed on stirrer in a sealed tube, and the product was estimated instantaneously following the Lang and Lang method. The activity of FaldDH was monitored by NADH oxidation at 340 nm. The standard assay was carried out using NADH (0.25 mM) and formate (HCOONa, 5 mM) in 0.1 M PBS (pH 7.0) at room temperature. The activity of PTDH was monitored by NAD + reduction at 340 nm. The assay was carried out using NAD + (0.5 mM) and sodium phosphite (Na 2 HPO 3 , 50 mM) in 0.1 M PBS (pH 7.0) at room temperature. The activity of ACDH was monitored by NAD + reduction at 340 nm. The mixture of ACDH, 10 mM aldehyde, 0.5 mM NAD + , 0.5 mM CoA, 0.5 mM DTT, 10 µM ZnSO 4 , and 1× PBS was monitored for NADH formation at 340 nm. The activity of coupled ACS-ACDH was monitored by NAD + reduction at 340 nm. ACS and ACDH were combined with an assay mix of 0.25 mM NADH, 0.2 mM CoA, 0.5 mM DTT, 5 mM ATP, 2 mM MgSO 4 , 0.2 mM TPP, 0.1 mg/mL glycerokinase, 25 mM PBS (pH 7.0), and 50 mM formate. The activity of ADH was monitored by NADH oxidation at 340 nm, the assay was performed with NADH (0.2 mM) and formaldehyde (HCHO 5 mM) in 0.1M PBS (pH 7.0) at room temperature. The activity of FLS was monitored by HPLC. The activity of FLS was assessed in a reaction mixture containing FLS, 10 mM formaldehyde and TEA buffer (25 mM, pH 7.0) containing 1 mM MgSO 4 and 0.1 mM TPP. The samples were stirred at 500 rpm for 12 hours. The samples were then heated at 98°C for 3 minutes, centrifuged for 10 minutes at 30 000 x g and the supernatant solution was analyzed by analytical HPLC. The activity of FSA A129S was monitored by HPLC. The activity of FSA A129S was probed in a reaction mixture containing FSA A129S , 10 mM formaldehyde, 10 mM DHA and TEA buffer (25 mM, pH 7.0) containing 1 mM MgSO4 and 0.1 mM TPP. The samples were stirred at 500 rpm for 12 hours. The samples were heated at 98°C for 3 minutes, centrifuged for 10 minutes at 30 000 x g and the supernatant solution was analyzed by analytical HPLC. Lang and Lang method for formate analysis Formate was analyzed according to Lang and Lang method with modification. 20 Briefly, 100 µL of sample containing formate was mixed with 0.2 mL of solution A, 10 µL of solution B, 0.7 mL of 100% acetic anhydride, and incubated at 50°C for 0.5 h with occasional rapid mixing. A red color could thereby be developed and quantified 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 0.1 M PBS (pH 7.0) was used for standard calibration (0–10 mM) ( Figure S21B ). Nash method for formaldehyde analysis Quantitative analysis of formaldehyde concentration was performed using the optimized Nash method. 28 Standard formaldehyde solution (10 to 100 µM) was used for plotting the calibration curve ( Figure S21C ). The reaction mixture was prepared 1:1 (v/v) with Nash's reagent containing 0.05 mM acetic acid, 0.02 M acetylacetone, and 2 M ammonium acetate. The developed yellow color was measured at 412 nm. GC for methanol analysis For the detection and quantification of methanol, aliquots at various time points were taken and analyzed for methanol content by using an Agilent G7129A gas chromatograph with an Agilent J&W DB-1 nonpolar column (60 m × 0.32 mm × 2.0 µm) and an FID detector. A calibration curve was prepared by employing the known concentrations of methanol that ranged from 0.1 to 10 mM ( Figure S21D ). To estimate the methanol produced as a result of the 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 amount of products were quantified from the peak areas and calibration curves. HPLC for dihydroxyacetone and L-erythrulose analysis For the detection and quantification of dihydroxyacetone and L-erythrulose, aliquots at various time points were taken and analyzed for dihydroxyacetone and L-erythrulose content by using a Simazu Liquid Chromatograph with a 300 × 7.8 mm HPX-87H column. Samples (10 µL) were injected and eluted under the following conditions: isocratic solvent system 5 mM aqueous sulfuric acid (H 2 SO 4 ) solution, 30 min run time per sample, flow rate 0.6 mL min − 1 , detection using both refractive index (RI) and ultraviolet (UV) at 192 nm detectors, column temperature 26°C. The amount of products were quantified from the peak areas and calibration curves ( Figure S21E and F ) Production of formate Rh III -complex-catalyzed the reduction of CO 2 to formate: The standard reaction condition was carried out for CO 2 reduction with CO 2 gas flow rate at 20ml/min in 0.1M PBS, pH 7.0, 0.5 mM Rh III -complex at -0.39 V RHE in the presence of 1% (v/v) EMIM-Ac. Cl FDH-catalyzed the reduction of CO 2 to formate: The standard reaction condition was carried out for CO 2 reduction in 0.1M PBS, pH 7.0, 100 µM Cl FDH, 0.1M NaHCO 3 , 0.01% (v/v) antifoam, 3 mM NAD + , 0.5 mM Rh III -complex-complex at -0.39 V RHE for NADH regeneration in the presence of 1% (v/v) EMIM-Ac. NADH regeneration Rh III -complex-catalyzed the reduction of NAD + to NADH: The standard reaction condition was carried out for NAD + reduction in 0.1M PBS, pH 7.0, 3 mM NAD + , 0.5 mM Rh III -complex at -0.39 V RHE . PTDH-catalyzed the reduction of NAD + to NADH: The standard reaction condition was carried out for NAD + reduction in 25 mM PBS, pH 7.0, 3 mM NAD + , and 50 mM sodium phosphite (Na 2 HPO 3 ). Production of formaldehyde Bm FaldDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PB, pH 7.0, 50 mM formate, 100 µM Bm FaldDH, 50 mM NADH. Bm FaldDH-PTDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PB, pH 7.0, 50 mM formate, 100 µM Bm FaldDH, 100 µM PTDH, 3 mM NAD + , and 50 mM sodium phosphite (Na 2 HPO 3 ). ACS-ACDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PBS, pH 7.0, 50 mM formate, 160 µM ACS, 40 µM ACDH, 50 mM NADH, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system (5 mM MgCl 2 , 30 mM creatine phosphate, 5 mM ATP, 0.2 mg/mL creatine phosphokinase, and 1.3 mg/mL bovine serum albumin), 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase. ACS-ACDH-PTDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PBS, pH 7.0, 50 mM formate, 50 mM Na 2 HPO 3 , 160 µM ACS, 40 µM ACDH, 100 µM PTDH, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase. Production of methanol Bm FaldDH-ADH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PB, pH 7.0, 150 mM formate, 100 µM Bm FaldDH, 10 U/ml ADH, and 50 mM NADH. Bm FaldDH-ADH-PTDH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PB, pH 7.0, 150 mM formate, 50 mM Na 2 HPO 3 , 100 µM Bm FaldDH, 10 U/ml ADH, 100 µM PTDH, and 3 mM NAD + . ACS-ACDH-ADH-catalyed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 160 µM ACS, 40 µM ACDH, 10 U/ml ADH, 50 mM NADH, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase. ACS-ACDH-ADH-PTDH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 50 mM Na 2 HPO 3 , 160 µM ACS, 40 µM ACDH, 10 U/ml ADH, 100 µM PTDH, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase. ACS-ACDH-Rh III -complex-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE . FDH-ACS-ACDH-ADH-PTDH-catalyzed the reduction of CO 2 to methanol: The standard reaction condition was carried out for methanol production from CO 2 in 25 mM PBS, pH 7.0, CO 2 gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 50 mM Na 2 HPO 3 , 100 µM Cl FDH, 160 µM ACS, 40 µM ACDH, 10 U/ml ADH, 100 µM PTDH, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase in the presence of 1% EMIM-Ac. Rh III -complex-ACS-ACDH-ADH-catalyzed the reduction of CO 2 to methanol: The standard reaction condition was carried out for methanol production from CO 2 in 25 mM PBS, pH 7.0, CO 2 gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 10 U/ml ADH, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE in the presence or absence of 1% EMIM-Ac. Production of dihydroxyacetone and L-erythrulose ACS-ACDH-FLS- Rh III -complex-catalyzed the conversion of formate to DHA: The standard reaction condition was carried out for DHA production from formate in 25 mM TEA, pH 7.0, 150 mM formate, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 100 µM FLS, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE . Rh III -complex-ACS-ACDH-FLS-catalyzed the conversion of CO 2 to DHA: The standard reaction condition was carried out for DHA production from CO 2 in 25 mM TEA, pH 7.0, CO 2 gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 100 µM FLS, 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE in the presence of 1% EMIM-Ac. ACS-ACDH-FLS-FSA A129S -Rh III -complex-catalyzed the conversion of formate to L-erythrulose: The standard reaction condition was carried out for L-erythrulose production from formate in 25 mM TEA, pH 7.5, 150 mM formate, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 100 µM FLS, 50 µM FSA A129S , 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE . Rh III -complex-ACS-ACDH-FLS-FSA A129S -catalyzed the conversion of CO 2 to L-erythrulose: The standard reaction condition was carried out for L-erythrulose production from CO 2 in 25 mM TEA, pH 7.5, CO 2 gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh III -complex, 160 µM ACS, 40 µM ACDH, 100 µM FLS, 50 µM FSA A129S , 3 mM NAD + , 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO 4 , 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V RHE in the presence of 1% EMIM-Ac. Declarations Acknowledgements This work was financially supported by the following grants: Key project on glucose water hydrogen production: [10311053A022301/002], National Key R&D Program of China (2022YFA0912002, 2022YFA0911900), National Natural Science Foundation of China (22309149 to Y.W.), Special Fund for Synthetic Biology [211000006022301/010], Competitive Research Funding Program (WU2022A006) in Center for Synthetic Biology and Integrated Bioengineering at Westlake University, and Westlake Education Foundation. Author Contributions Yajie Wang and Licheng Sun conceived and organized the study, contributed to drafting the manuscript, and revised it. Hailong Li and Yizhou Wu made equal contributions to designing the experiments, acquiring and analyzing the data, interpreting the results, and writing the manuscript. Yuxuan Wang contributed to data acquisition. Kai Zhang, Jin Zhu, Yuan Ji, and Tao Gu contributed to data analysis. All authors have read and approved the final manuscript. Competing interests C.N. Patent No. 202410990776.6 had been filed. The inventors include Yajie Wang, Licheng Sun, and Hailong Li. The other authors declare no competing interests. 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Supplementary Files GraphicalabstractNatureCommunicationsH.Lietal.docx SupplementaryinformationNatureCommunications.docx Schemes.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4865792","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":348207543,"identity":"fc77ecb7-c1bf-4ec0-b4c9-d04a4919164b","order_by":0,"name":"Yajie Wang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6216-791X","institution":"Westlake University","correspondingAuthor":true,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Wang","suffix":""},{"id":348207544,"identity":"23f2e6bc-9991-44de-b166-9ef3ce9b7c87","order_by":1,"name":"Hailong Li","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Li","suffix":""},{"id":348207545,"identity":"95945ba4-a312-4c6a-ae38-94b62d7c787a","order_by":2,"name":"Yizhou Wu","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Yizhou","middleName":"","lastName":"Wu","suffix":""},{"id":348207546,"identity":"fd7a3045-d11e-40f1-9b68-6c47727ccbb9","order_by":3,"name":"Yuxuan Wang","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Wang","suffix":""},{"id":348207547,"identity":"1f05787b-2647-464e-b848-b08a4b3e8e13","order_by":4,"name":"Kai Zhang","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhang","suffix":""},{"id":348207548,"identity":"ac294411-a6b0-4aef-8f7d-61aab7ebbe91","order_by":5,"name":"Jin Zhu","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Zhu","suffix":""},{"id":348207549,"identity":"47c2d3fa-fe12-4ae9-8d95-98c1d088805a","order_by":6,"name":"Yuan Ji","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Ji","suffix":""},{"id":348207550,"identity":"a0e05c21-9fe7-4411-b632-60b1589fed6e","order_by":7,"name":"Tao Gu","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Gu","suffix":""},{"id":348207551,"identity":"636067b3-2805-487c-92a4-759708d46382","order_by":8,"name":"Weixuan Nie","email":"","orcid":"","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Weixuan","middleName":"","lastName":"Nie","suffix":""},{"id":348207552,"identity":"720dcfdb-a3e2-42fb-a5da-bd230a7f39f6","order_by":9,"name":"Licheng Sun","email":"","orcid":"https://orcid.org/0000-0002-4521-2870","institution":"Westlake University","correspondingAuthor":false,"prefix":"","firstName":"Licheng","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-08-06 05:50:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4865792/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4865792/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63994703,"identity":"300facbd-dbae-4665-8b80-545ff142e2b6","added_by":"auto","created_at":"2024-09-04 16:15:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136785,"visible":true,"origin":"","legend":"\u003cp\u003eBifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed CO\u003csub\u003e2\u003c/sub\u003e reduction to formate and NADH regeneration. Unless specified, the reactions were performed with 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex in 0.1 M PBS at pH of 7.0, a CO\u003csub\u003e2\u003c/sub\u003e gas flow rate of 20 ml/min, and an applied potential of -0.39 V\u003csub\u003eRHE\u003c/sub\u003e. A) Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate at an applied potential of -0.29 V\u003csub\u003eRHE\u003c/sub\u003e with PEM or AEM. (B) The effects of EMIM-Ac on formate production. C) Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed NADH generation in the presence or absence of EMIM-Ac. D) Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed concurrent formate production and NADH regeneration.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/69367bd3720175f0ef007b69.png"},{"id":63994244,"identity":"2cc8df8a-5715-4a8c-900a-35919b28d454","added_by":"auto","created_at":"2024-09-04 16:07:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":135453,"visible":true,"origin":"","legend":"\u003cp\u003eCV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed reduction of CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e. Unless specified, the reactions were performed in 0.1 M PBS at pH of 7.0, with a scan rate of 50 mV/s. A) CV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex and CO\u003csub\u003e2\u003c/sub\u003e with or without 1% EMIM-Ac. B) CV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex for CO\u003csub\u003e2\u003c/sub\u003e reduction with or without 1% EMIM-Ac. C) CV curves of NAD\u003csup\u003e+\u003c/sup\u003e with or without 1% EMIM-Ac. D) CV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex for NAD\u003csup\u003e+\u003c/sup\u003e and CO\u003csub\u003e2\u003c/sub\u003e reduction with 1% EMIM-Ac.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/ab381eb304d1a1051f826400.png"},{"id":63994247,"identity":"fbe13b0e-bf51-4dd3-9171-b180c659b875","added_by":"auto","created_at":"2024-09-04 16:07:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96490,"visible":true,"origin":"","legend":"\u003cp\u003eThe production of formaldehyde and methanol by various tandem enzymatic and bioelectrochemical systems. A) The Gibbis energies of the formaldehyde dehydrogenase (FaldDH) pathway and the formolase pathway. B) The production of formaldehyde by \u003cem\u003eBm\u003c/em\u003eFaldDH or ACS-ACDH with NADH or PTDH-catalyzed NADH regeneration system. C) The production of methanol by enzymatic cascades of \u003cem\u003eBm\u003c/em\u003eFaldDH-ADH and ACS-ACDH-ADH. D) The production of methanol by enzymatic cascades of \u003cem\u003eCl\u003c/em\u003eFDH-\u003cem\u003eBm\u003c/em\u003eFaldDH-ADH-PTDH and cooperative bioelectrochemical system of Rh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH-ADH.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/619a8e714f0c94b188faf3f6.png"},{"id":63994243,"identity":"960e0886-8d4d-4cf1-a161-1f5f821067cc","added_by":"auto","created_at":"2024-09-04 16:07:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54553,"visible":true,"origin":"","legend":"\u003cp\u003eCooperative one-pot bioelectrochemical systems for synthesizing DHA and L-erythrulose from CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/48fce2d5e8d18d41871077e2.png"},{"id":63995272,"identity":"f288dcd9-b14a-4cf8-8762-d4da8abf83a2","added_by":"auto","created_at":"2024-09-04 16:23:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1045093,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/74e79a3a-91ce-4510-b552-100b1b35f9c6.pdf"},{"id":63994248,"identity":"d1d332ee-15a7-4e24-9d17-9d0f2a193624","added_by":"auto","created_at":"2024-09-04 16:07:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163823,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractNatureCommunicationsH.Lietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/0ca124d2ae841246f4c2d62b.docx"},{"id":63994250,"identity":"a4ca40f8-27a7-492d-86a9-38db1f33bdff","added_by":"auto","created_at":"2024-09-04 16:07:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3149514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryinformationNatureCommunications.docx","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/13d86f304c561d9239392b88.docx"},{"id":63994704,"identity":"d7d1cf2e-67ca-4b7c-bf1b-53d6f8a5b8b3","added_by":"auto","created_at":"2024-09-04 16:15:16","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":420774,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes.docx","url":"https://assets-eu.researchsquare.com/files/rs-4865792/v1/f81b8a8c6749d44ec2954302.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eBifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex catalyzed CO\u003csub\u003e2\u003c/sub\u003e reduction and NADH regeneration for direct bioelectrochemical synthesis of C\u003csub\u003e3 \u003c/sub\u003eand C\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e","fulltext":[{"header":"Main","content":"\u003cp\u003eTo address the energy crisis and alleviate the rising level of CO\u003csub\u003e2\u003c/sub\u003e in the atmosphere, various CO\u003csub\u003e2\u003c/sub\u003e capture and utilization techniques have been developed.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e While electrochemical strategies offer high energy efficiency for CO\u003csub\u003e2\u003c/sub\u003e reduction and transformation, their practical application is limited by low selectivity in producing C\u003csub\u003e2+\u003c/sub\u003e chemicals.\u003csup\u003e2\u003c/sup\u003e In contrast, biocatalysis excels in selectively producing C\u003csub\u003e2+\u003c/sub\u003e chemicals but is less efficient in CO\u003csub\u003e2\u003c/sub\u003e reduction due to the high energy input required to break the O\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O bond (750 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003csup\u003e3\u003c/sup\u003e In this respect, there is a growing interest in developing electroenzymatic hybrid systems that combine the synthetic power of both catalytic disciplines to efficiently produce value-added compounds from CO\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e reduction processed by oxidoreductases commonly requires a substantial amount of reducing equivalents. Recent advancements in one-pot bioelectrochemical systems have introduced various NAD(P)H-dependent oxidoreductases within the cathode chamber to convert CO\u003csub\u003e2\u003c/sub\u003e into products like carbon monoxide (CO),\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e formate (HCOOH),\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e methanol (CH\u003csub\u003e3\u003c/sub\u003eOH),\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e ethylene (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e),\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and other C\u003csub\u003e2+\u003c/sub\u003e chemicals,\u003csup\u003e2, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e utilizing reducing equivalents regenerated from electricity through direct or mediated electron transfer. Among them, formate dehydrogenase (FDH)-catalyzed CO\u003csub\u003e2\u003c/sub\u003e-to-formate conversion is thermodynamic unfavorable and rate-limited due to more positive redox potential of NAD\u003csup\u003e+\u003c/sup\u003e/NADH (\u0026minus;\u0026thinsp;0.32 V vs NHE) compared to CO\u003csub\u003e2\u003c/sub\u003e/HCOOH (\u0026minus;\u0026thinsp;0.43 V vs NHE).\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e This rate-limiting step may also restrict the supply of formate for subsequent C\u003csub\u003e2+\u003c/sub\u003e chemical synthesis catalyzed by enzymatic cascades.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eConversely, many electrocatalysts have been developed to efficiently produce formate through electrochemical CO\u003csub\u003e2\u003c/sub\u003e reduction reaction (CO\u003csub\u003e2\u003c/sub\u003eRR) with low overpotential and high Faradaic efficiency.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Additionally, several electrochemical systems have been widely reported for reducing NAD\u003csup\u003e+\u003c/sup\u003e to 1,4-NADH with nearly 100% selectivity and good enzyme compatability.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, electrochemical catalysis for the simultaneous reduction of CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e, and its cooperative operation with enzymatic cascades for C\u003csub\u003e2+\u003c/sub\u003e chemical synthesis, remains relatively uncommon in this field. Thus, we aimed to develop a \u0026ldquo;one-pot\u0026rdquo; biocompatible electrochemical system that can efficiently produce formate from CO\u003csub\u003e2\u003c/sub\u003e and regenerate NADH at the same time. This system can work cooperatively with tandem enzymatic cascades to convert CO\u003csub\u003e2\u003c/sub\u003e into value-added C\u003csub\u003e2+\u003c/sub\u003e chemicals with improved production rate and enantioselectivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we developed a cooperative one-pot bioelectrochemical system for the electroenzymatic synthesis of value-added compounds from CO\u003csub\u003e2\u003c/sub\u003e (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This system utilizes the bifunctional metal complex [Cp*Rh(bpy)Cl]\u003csup\u003e2+\u003c/sup\u003e (Rh\u003csup\u003eIII\u003c/sup\u003e-complex or [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e) to concurrently catalyze the electrochemical reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate and the regeneration of NADH from NAD\u003csup\u003e+\u003c/sup\u003e. The resulting formate is then converted into methanol (C\u003csub\u003e1\u003c/sub\u003e), dihydroxyacetone (C\u003csub\u003e3\u003c/sub\u003e), and L-erythrulose (C\u003csub\u003e4\u003c/sub\u003e) at the rates of 0.43 mM/h, 0.11 mM/h, and 0.08 mM/h, respectively, through the tandem enzymatic cascades involving ACS-ACDH-ADH, ACS-ACDH-FLS, and ACS-ACDH-FLS-FSA\u003csup\u003eA129S\u003c/sup\u003e (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In those cooperative bioelectroenzymatic synthesis, Rh\u003csup\u003eIII\u003c/sup\u003e-complex exhibited a CO\u003csub\u003e2\u003c/sub\u003e reduction rate of 15.8 mM/h, which is 83.2 times faster than the \u003cem\u003eCl\u003c/em\u003eFDH-containing electroenzymatic system (0.19 mM/h). Meanwhile, Rh\u003csup\u003eIII\u003c/sup\u003e-complex maintained the NADH regeneration at a rate of 0.24 mM/min, efficiently driving the enzymatic cascade for the formation of C\u003csub\u003e1\u003c/sub\u003e-C\u003csub\u003e4\u003c/sub\u003e chemicals. By leveraging the synergies between electrochemical catalysis and biocatalysis, this cooperative bioelectrochemical system enables the conversion of CO\u003csub\u003e2\u003c/sub\u003e into value-added chemicals at a higher rate and yield compared to the electroenzymatic and tandem enzymatic systems with FDH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eFirstly, we aimed to design a bifunctional electrocatalyst capable of simultaneously reducing CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e. Inspired by the studies on CO\u003csub\u003e2\u003c/sub\u003e photoreduction catalyzed by Rh\u003csup\u003eIII\u003c/sup\u003e-based photocatalysts,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e we selected [Cp*Rh(bpy)H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e (Rh\u003csup\u003eIII\u003c/sup\u003e-complex or [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e) for evaluation because of its established efficiency in electrocatalytic NAD\u003csup\u003e+\u003c/sup\u003e regeneration and good biocompatibility.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e To evaluate the electrocatalytic performance of the Rh\u003csup\u003eIII\u003c/sup\u003e-complex for CO\u003csub\u003e2\u003c/sub\u003e reduction under the mild conditions, we conducted reactions with 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex in 0.1 M PBS at pH 7.0 using a three-electrode H-cell device (Carbon felt serves as the working electrode, reversible hydrogen electrode (RHE) as the reference electrode, and Pt plate as the counter electrode) with CO\u003csub\u003e2\u003c/sub\u003e gas bubbling (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). To be noted, Rh\u003csup\u003eIII\u003c/sup\u003e-complex effectively catalyzed the generation of formate from CO\u003csub\u003e2\u003c/sub\u003e at the rate of 5.5 mM/h with an applied potential of -0.29 V\u003csub\u003eRHE\u003c/sub\u003e, demonstrating superior efficiency compared to the utilization of bicarbonate as a substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo optimize the formate yield, we initially tested the effects of the proton exchange membrane (PEM, Nafion 117) and the anion exchange membrane (AEM, PiperION-A20) on CO\u003csub\u003e2\u003c/sub\u003e reduction and found that PEM performed better than AEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subsequently, we evaluated the electrochemical CO\u003csub\u003e2\u003c/sub\u003e reduction under various conditions, including different applied potentials, CO\u003csub\u003e2\u003c/sub\u003e gas flow rates, Rh\u003csup\u003eIII\u003c/sup\u003e-complex concentrations, and PBS pH levels (\u003cb\u003eFigure S2\u003c/b\u003e). Under the optimal operating conditions of 0.1M PBS at pH 7.0, a CO\u003csub\u003e2\u003c/sub\u003e gas flow rate of 20 ml/min, and an applied voltage of -0.39 V\u003csub\u003eRHE\u003c/sub\u003e, the formate production rate reached 7.49 mM/h (\u003cb\u003eFigure S2D\u003c/b\u003e). Additionally, considering that ionic liquids (ILs) are known to decrease the overpotential of CO\u003csub\u003e2\u003c/sub\u003e reduction in aqueous media\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and that 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) has shown superior performance in CO\u003csub\u003e2\u003c/sub\u003e reduction within biocatalytic systems,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e the performance of EMIM-Ac was evaluated. Incorporating 1% (v/v) EMIM-Ac resulted in the production of 19.32 mM formate within the first hour and 189.63 mM after 12 hours of reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For comparison, formate production was evaluated using a conventional electroenzymatic system with one of the most efficient aerobic FDHs from \u003cem\u003eClostridium ljungdahlii\u003c/em\u003e (\u003cem\u003eCl\u003c/em\u003eFDH) as the biocatalyst, Rh\u003csup\u003eIII\u003c/sup\u003e-complex as the NADH regeneration catalyst, and 100 mM bicarbonate as the substrate.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Over a 12-hour reaction, only 2.28 mM of formate was generated, which is 83.2 times lower than that produced by our electrochemical system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEfficient NADH regeneration is essential in electroenzymatic synthesis involving multiple NADH-dependent oxidoreductases. To investigate the dual functionality of Rh\u003csup\u003eIII\u003c/sup\u003e-complex, we evaluated the NADH regeneration rate with or without CO\u003csub\u003e2\u003c/sub\u003e bubbling. The Rh\u003csup\u003eIII\u003c/sup\u003e-complex demonstrated the optimal activity for NADH regeneration under the condition most suitable for CO\u003csub\u003e2\u003c/sub\u003e reduction (\u003cb\u003eFigure S3\u003c/b\u003e). The presence of EMIM-Ac had little influence on NADH regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). 3 mM of NAD\u003csup\u003e+\u003c/sup\u003e was electrochemically reduced at a rate of 0.24 mM/min, achieving an 80% recovery within 10 min, which was comparable to most of the reported rhodium complex-catalyzed electrochemical NADH regeneration processes.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Additionally, in the presence of CO\u003csub\u003e2\u003c/sub\u003e, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex could maintain catalytic efficiency for both NADH regeneration and CO\u003csub\u003e2\u003c/sub\u003e reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the mechanism of bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex in CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e reduction, cyclic voltammogram (CV) curves were conducted in 0.1 M PBS at pH of 7.0 with or without 1% (v/v) EMIM-Ac. The overall electrochemical behavior can be explained by an EEC (electron transfer, electron transfer, chemical reaction) mechanism (\u003cb\u003eEqs.\u0026nbsp;1\u0026ndash;3\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the CV curves of CO\u003csub\u003e2\u003c/sub\u003e with or without EMIM-Ac have no significant differences, indicating that EMIM-Ac does not catalyze CO\u003csub\u003e2\u003c/sub\u003e reduction. In the absence of both CO\u003csub\u003e2\u003c/sub\u003e and EMIM-Ac, [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e undergoes a two-electron reduction process to form [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e hydride (\u003cb\u003eEqs.\u0026nbsp;1\u003c/b\u003e) at -0.17 V\u003csub\u003eRHE\u003c/sub\u003e (-4.67 mA/cm\u003csup\u003e2\u003c/sup\u003e). After adding 1% EMIM-Ac, the reduction peak slightly shifts to -0.16 V\u003csub\u003eRHE\u003c/sub\u003e with a higher current density (-6.43 mA/cm\u003csup\u003e2\u003c/sup\u003e). These data suggest that EMIM-Ac, as a cosolvent, does not have a noticeable interference on the process of [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e formation. Comparing the CV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex for CO\u003csub\u003e2\u003c/sub\u003e reduction with or without EMIM-Ac (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), the cathodic current peak increases from \u0026minus;\u0026thinsp;8.34 to \u0026minus;\u0026thinsp;9.56 mA/cm\u003csup\u003e2\u003c/sup\u003e, indicating a higher CO\u003csub\u003e2\u003c/sub\u003e reduction efficiency. Additionally, the reduction peak of [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e hydride-catalyzed CO\u003csub\u003e2\u003c/sub\u003e reduction (\u003cb\u003eEqs.\u0026nbsp;2\u003c/b\u003e) shifts from \u0026minus;\u0026thinsp;0.25 V\u003csub\u003eRHE\u003c/sub\u003e (without EMIM-Ac) to \u0026minus;\u0026thinsp;0.17 V\u003csub\u003eRHE\u003c/sub\u003e (with EMIM-Ac), suggesting that EMIM-Ac lowered the energy of the [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e-CO\u003csub\u003e2\u003c/sub\u003e intermediate, thereby reducing the initial reduction barrier. Those findings are consistent with the previous studies showing that ionic liquids can decrease the overpotential for CO\u003csub\u003e2\u003c/sub\u003e to CO conversion.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe CV curves of Rh\u003csup\u003eIII\u003c/sup\u003e-complex in the presence of NAD\u003csup\u003e+\u003c/sup\u003e clarify the interplay between [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e and NAD\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e). The CV curve of NAD\u003csup\u003e+\u003c/sup\u003e reduction without Rh\u003csup\u003eIII\u003c/sup\u003e-complex shows no significant differences in reduction peak in the presence or absence of EMIM-Ac, indicating that EMIM-Ac does not impact on NAD\u003csup\u003e+\u003c/sup\u003e reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). According to the catalytic mechanism reported by Kim \u003cem\u003eet al.\u003c/em\u003e,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e the [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e was reduced to [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e (\u003cb\u003eEqs.\u0026nbsp;1\u003c/b\u003e) by the cathode, and then a hydride transfer reaction takes place between [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e and NAD\u003csup\u003e+\u003c/sup\u003e to achieve NADH regeneration (\u003cb\u003eEqs.\u0026nbsp;3\u003c/b\u003e). The reduction peak of [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e to [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e during NADH regeneration is clearly observed at \u0026minus;\u0026thinsp;0.19 V\u003csub\u003eRHE\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Compared with the Rh\u003csup\u003eIII\u003c/sup\u003e-complex mediated NADH regeneration, the direct NAD\u003csup\u003e+\u003c/sup\u003e reduction requires a more negative potential of \u0026minus;\u0026thinsp;0.59 V\u003csub\u003eRHE\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), confirming that NAD\u003csup\u003e+\u003c/sup\u003e was reduced by [Rh\u003csup\u003eI\u003c/sup\u003e-H]\u003csup\u003e+\u003c/sup\u003e \u003cem\u003evia\u003c/em\u003e a hydride transfer pathway in our system.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The simultaneous reduction of CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e begins at \u0026minus;\u0026thinsp;0.21V\u003csub\u003eRHE\u003c/sub\u003e in the presence of 1% EMIM-Ac, slightly more negative than the potential of Rh\u003csup\u003eIII\u003c/sup\u003e-complex catalyzed CO\u003csub\u003e2\u003c/sub\u003e reduction (\u0026minus;\u0026thinsp;0.17 V\u003csub\u003eRHE\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). CV analysis defines the electrochemical window for the mediated CO\u003csub\u003e2\u003c/sub\u003e reduction and NADH regeneration. Thus, a potential of -0.39 V\u003csub\u003eRHE\u003c/sub\u003e is sufficient for driving the concurrent CO\u003csub\u003e2\u003c/sub\u003e and NAD\u003csup\u003e+\u003c/sup\u003e reduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFormaldehyde is a promising feedstock for tandem enzymatic cascade reactions.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e However, the enzymatic reduction of formate to formaldehyde is thermodynamically unfavorable. We compared the highest catalytic efficiency of two pathways for synthesizing formaldehyde from formate \u003cb\u003e(Figure S5\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e: the formaldehyde dehydrogenase (FaldDH) pathway and the formolase pathway that utilizes acetyl-CoA synthase (ACS) from \u003cem\u003eEscherichia coli\u003c/em\u003e and acetaldehyde dehydrogenase (ACDH) from \u003cem\u003eListeria monocytogenes\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e By employing the most efficient enzymes under the optimal reaction conditions in both pathways, we achieved a formaldehyde formation rate of 32.87 \u0026micro;M/h and a final concentration of 98.63 \u0026micro;M within 3 hours by using the formolase pathway with phosphite dehydrogenase (PTDH)-catalyzed NADH regeneration (\u003cb\u003eFigure S6-S11\u003c/b\u003e). In contrast, only 18.62 \u0026micro;M formaldehyde was obtained after a 3-hour reaction when using FaldDH from \u003cem\u003eBurkholderia multivorans\u003c/em\u003e (\u003cem\u003eBm\u003c/em\u003eFaldDH) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e as the biocatalyst (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The low yield could be attributed to significantly unfavorable thermodynamics of FDH pathway (Δ\u003csub\u003er\u003c/sub\u003eG\u0026prime;\u003csup\u003em\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;+\u0026thinsp;51.7 kJ/mol) compared to the formolase pathway (ΔrG\u0026prime;\u003csup\u003em\u003c/sup\u003e = -8.6 kJ/mol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eGiven the preference of \u003cem\u003eBm\u003c/em\u003eFaldDH for formaldehyde oxidation over formate reduction, we added alcohol dehydrogenase (ADH, \u003cb\u003eFigure S12-13\u003c/b\u003e) from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e to convert formaldehyde to methanol, driving the equilibrium towards the reduction direction (Scheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). With excess formate (150 mM) as the substrate, the formolase pathway (\u003cb\u003eRoute 2\u003c/b\u003e) outperformed the \u003cem\u003eBm\u003c/em\u003eFaldDH pathway (\u003cb\u003eRoute 1\u003c/b\u003e), reaching a maximum of 2.78 mM methanol within 6 h, which is 3.4-fold higher than that of \u003cb\u003eRoute 1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At a low formate concentration of 10 mM, \u003cb\u003eRoute 2\u003c/b\u003e still produced approximately three times more methanol than \u003cb\u003eRoute 1\u003c/b\u003e within the first-hour reaction (\u003cb\u003eFigure S14-15\u003c/b\u003e). To this end, we selected the formolase pathway for further establishment of cooperative bioelectrochemical systems.\u003c/p\u003e \u003cp\u003ePrior to establishing the cooperative bioelectrochemical system for CO\u003csub\u003e2\u003c/sub\u003e reduction, we assessed the effectiveness of Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed electrochemical NADH regeneration in supporting the formolase pathway for methanol production (\u003cb\u003eRoute 3\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the methanol production rate (0.49 mM/h) and the final concentration (2.87 mM) over a 6-hour reaction period of electrochemical NADH regeneration system is slightly superior to those of PTDH-catalyzed NADH regeneration systems. This suggests that the Rh\u003csup\u003eIII\u003c/sup\u003e-complex is biocompatible for enzymatic system with high NAD\u003csup\u003e+\u003c/sup\u003e reduction efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe finally set up the cooperative bioelectrochemical system by coupling Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed CO\u003csub\u003e2\u003c/sub\u003e reduction and NADH regeneration with ACS, ACDH, and ADH to synthesize methanol from CO\u003csub\u003e2\u003c/sub\u003e in a \u0026ldquo;one-pot\u0026rdquo; manner (\u003cb\u003eRoute 5\u003c/b\u003e). The cooperative bioelectrochemical system (\u003cb\u003eRoute 5\u003c/b\u003e) generates 2.59 mM methanol after 6-hour reaction, which is comparable to the enzymatic system (2.65 mM in \u003cb\u003eRoute 2\u003c/b\u003e) using formate as the substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This proves that Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed electrochemical CO\u003csub\u003e2\u003c/sub\u003e reduction is efficient and biocompatible to the enzymatic cascade. In addition, the cooperative bioelectrochemical system (\u003cb\u003eRoute 5\u003c/b\u003e) produces 3.8 times higher methanol from gaseous CO\u003csub\u003e2\u003c/sub\u003e compared to the tandem enzymatic system (0.68 mM in \u003cb\u003eRoute 4\u003c/b\u003e) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. These underscore the pivotal role of the bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex in the cooperative bioelectrochemical system for efficient CO\u003csub\u003e2\u003c/sub\u003e reduction and subsequent synthesis of valuable compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBuilding on the success of the cooperative bioelectrochemical system for efficient methanol production, we aimed to extend the applications of bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex in electroenzymatic synthesis of C\u003csub\u003e2+\u003c/sub\u003e chemicals from CO\u003csub\u003e2\u003c/sub\u003e. Dihydroxyacetone (DHA) and L-erythrulose are key chemicals in cosmetics, pharmaceuticals, and food industries for their water-evaporation prevention, UV protection, and antioxidant properties.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e While DHA and L-erythrolose have been produced for decades through oxidative microbial fermentation, direct synthesis from CO\u003csub\u003e2\u003c/sub\u003e is rare. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e We were pleased to find that an engineered Benzaldehyde lyase (BAL) variant, formolase (FLS), could catalyze the asymmetric umpolung activation of formaldehyde to DHA.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Additionally, extensive development of d-fructose-6-phosphate aldolase\u0026rsquo;s derivative A129S (FSA\u003csup\u003eA129S\u003c/sup\u003e) has enabled efficient aldol addition between formaldehyde and DHA while maintaining stereochemical fidelity.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e With those powerful tools in hand, we aimed to build the cooperative bioelectrochemical systems to convert CO\u003csub\u003e2\u003c/sub\u003e into DHA and L-erythrulose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter optimizing the reaction conditions for converting formaldehyde into DHA and L-erythrulose catalyzed by FLS and FSA\u003csup\u003eA129S\u003c/sup\u003e (\u003cb\u003eFigure S17-18\u003c/b\u003e), we coupled Rh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH with FLS and FSA\u003csup\u003eA129S\u003c/sup\u003e in one-pot to create cooperative bioelectrochemical systems for the direct synthesis of DHA and L-erythrulose from CO\u003csub\u003e2\u003c/sub\u003e respectively (Scheme \u003cspan refid=\"Sch4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Remarkably, using CO\u003csub\u003e2\u003c/sub\u003e as the initial substrate, the cooperative bioelectrochemical system, \u003cb\u003eRoute 6\u003c/b\u003e and \u003cb\u003eRoute 7\u003c/b\u003e, produced 2.63 mM and 1.93 mM of DHA and L-erythrulose within 24 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results were comparable to the output of the tandem enzymatic system using formate as the starting materials (\u003cb\u003eScheme S1\u003c/b\u003e and \u003cb\u003eFigure S19\u003c/b\u003e). These findings highlight the bifunctional ability of Rh\u003csup\u003eIII\u003c/sup\u003e-complex to efficiently reduce CO\u003csub\u003e2\u003c/sub\u003e to formate while supplying the necessary reducing equivalents (NADH) to drive the subsequent tandem enzymatic cascades for synthesizing longer-chain, higher-value chiral compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study introduces several cooperative one-pot bioelectrochemical systems that consist of a bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex and various tandem enzymatic cascades to directly synthesize methanol (C\u003csub\u003e1\u003c/sub\u003e), dioxyacetone (C\u003csub\u003e3\u003c/sub\u003e), and L-erythrulose (C\u003csub\u003e4\u003c/sub\u003e) from CO\u003csub\u003e2\u003c/sub\u003e, achieving high yields of 2.58 mM, 2.63 mM, and 1.93 mM, respectively. The Rh\u003csup\u003eIII\u003c/sup\u003e-complex catalyzes the reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate at a rate of 15.8 mM/h, which is 83.2 times faster than that of FDH, and simultaneously facilitates NADH regeneration at a rate of 0.24 mM/min to provide the reducing equivalent (NADH) for subsequent tandem enzyme cascades. The rapid generation of formate in the cooperative bioelectroenzymatic system enhances methanol production at 0.43 mM/h, which is 3.58 times faster than the rate achieved in a tandem enzymatic system using FDH for CO\u003csub\u003e2\u003c/sub\u003e reduction. This highlights the ability of Rh\u003csup\u003eIII\u003c/sup\u003e-complex to effectively addresses the limitations of low catalytic activity of FDH in both electroenzymatic and tandem enzymatic reactions. The study emphasizes the potential of bioelectrochemical CO\u003csub\u003e2\u003c/sub\u003e reduction as a viable alternative for synthesizing high valuable C\u003csub\u003e2+\u003c/sub\u003e products and may inspire the direct electrosynthesis of value-added compounds from CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe activity assay and kinetic assay\u003c/h2\u003e \u003cp\u003eThe activity of \u003cem\u003eCl\u003c/em\u003eFDH was monitored by NADH oxidation at 340 nm. CO\u003csub\u003e2\u003c/sub\u003e reduction activity of FDH was measured in 0.1 M PBS (pH 7.0), at room temperature. Reaction mixtures (0.1M NaHCO\u003csub\u003e3\u003c/sub\u003e, 5 mM NADH, and enzyme) were incubated for 1 hour. Each reaction mixture was placed on stirrer in a sealed tube, and the product was estimated instantaneously following the Lang and Lang method.\u003c/p\u003e \u003cp\u003eThe activity of FaldDH was monitored by NADH oxidation at 340 nm. The standard assay was carried out using NADH (0.25 mM) and formate (HCOONa, 5 mM) in 0.1 M PBS (pH 7.0) at room temperature.\u003c/p\u003e \u003cp\u003eThe activity of PTDH was monitored by NAD\u003csup\u003e+\u003c/sup\u003e reduction at 340 nm. The assay was carried out using NAD\u003csup\u003e+\u003c/sup\u003e (0.5 mM) and sodium phosphite (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e, 50 mM) in 0.1 M PBS (pH 7.0) at room temperature.\u003c/p\u003e \u003cp\u003eThe activity of ACDH was monitored by NAD\u003csup\u003e+\u003c/sup\u003e reduction at 340 nm. The mixture of ACDH, 10 mM aldehyde, 0.5 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.5 mM CoA, 0.5 mM DTT, 10 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e, and 1\u0026times; PBS was monitored for NADH formation at 340 nm.\u003c/p\u003e \u003cp\u003eThe activity of coupled ACS-ACDH was monitored by NAD\u003csup\u003e+\u003c/sup\u003e reduction at 340 nm. ACS and ACDH were combined with an assay mix of 0.25 mM NADH, 0.2 mM CoA, 0.5 mM DTT, 5 mM ATP, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, 0.1 mg/mL glycerokinase, 25 mM PBS (pH 7.0), and 50 mM formate.\u003c/p\u003e \u003cp\u003eThe activity of ADH was monitored by NADH oxidation at 340 nm, the assay was performed with NADH (0.2 mM) and formaldehyde (HCHO 5 mM) in 0.1M PBS (pH 7.0) at room temperature.\u003c/p\u003e \u003cp\u003eThe activity of FLS was monitored by HPLC. The activity of FLS was assessed in a reaction mixture containing FLS, 10 mM formaldehyde and TEA buffer (25 mM, pH 7.0) containing 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e and 0.1 mM TPP. The samples were stirred at 500 rpm for 12 hours. The samples were then heated at 98\u0026deg;C for 3 minutes, centrifuged for 10 minutes at 30 000 x g and the supernatant solution was analyzed by analytical HPLC.\u003c/p\u003e \u003cp\u003eThe activity of FSA\u003csup\u003eA129S\u003c/sup\u003e was monitored by HPLC. The activity of FSA\u003csup\u003eA129S\u003c/sup\u003e was probed in a reaction mixture containing FSA\u003csup\u003eA129S\u003c/sup\u003e, 10 mM formaldehyde, 10 mM DHA and TEA buffer (25 mM, pH 7.0) containing 1 mM MgSO4 and 0.1 mM TPP. The samples were stirred at 500 rpm for 12 hours. The samples were heated at 98\u0026deg;C for 3 minutes, centrifuged for 10 minutes at 30 000 x g and the supernatant solution was analyzed by analytical HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLang and Lang method for formate analysis\u003c/h2\u003e \u003cp\u003eFormate was analyzed according to Lang and Lang method with modification.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Briefly, 100 \u0026micro;L of sample containing formate was mixed with 0.2 mL of solution A, 10 \u0026micro;L of solution B, 0.7 mL of 100% acetic anhydride, and incubated at 50\u0026deg;C for 0.5 h with occasional rapid mixing. A red color could thereby be developed and quantified 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 0.1 M PBS (pH 7.0) was used for standard calibration (0\u0026ndash;10 mM) (\u003cb\u003eFigure S21B\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNash method for formaldehyde analysis\u003c/h2\u003e \u003cp\u003eQuantitative analysis of formaldehyde concentration was performed using the optimized Nash method.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Standard formaldehyde solution (10 to 100 \u0026micro;M) was used for plotting the calibration curve (\u003cb\u003eFigure S21C\u003c/b\u003e). The reaction mixture was prepared 1:1 (v/v) with Nash's reagent containing 0.05 mM acetic acid, 0.02 M acetylacetone, and 2 M ammonium acetate. The developed yellow color was measured at 412 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGC for methanol analysis\u003c/h2\u003e \u003cp\u003eFor the detection and quantification of methanol, aliquots at various time points were taken and analyzed for methanol content by using an Agilent G7129A gas chromatograph with an Agilent J\u0026amp;W DB-1 nonpolar column (60 m \u0026times; 0.32 mm \u0026times; 2.0 \u0026micro;m) and an FID detector. A calibration curve was prepared by employing the known concentrations of methanol that ranged from 0.1 to 10 mM (\u003cb\u003eFigure S21D\u003c/b\u003e). To estimate the methanol produced as a result of the reaction, 1.0 \u0026micro;L of the final reaction solution was used for the GC measurements while the injector temperature was maintained at 200\u0026deg;C. The amount of products were quantified from the peak areas and calibration curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHPLC for dihydroxyacetone and L-erythrulose analysis\u003c/h2\u003e \u003cp\u003eFor the detection and quantification of dihydroxyacetone and L-erythrulose, aliquots at various time points were taken and analyzed for dihydroxyacetone and L-erythrulose content by using a Simazu Liquid Chromatograph with a 300 \u0026times; 7.8 mm HPX-87H column. Samples (10 \u0026micro;L) were injected and eluted under the following conditions: isocratic solvent system 5 mM aqueous sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) solution, 30 min run time per sample, flow rate 0.6 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, detection using both refractive index (RI) and ultraviolet (UV) at 192 nm detectors, column temperature 26\u0026deg;C. The amount of products were quantified from the peak areas and calibration curves (\u003cb\u003eFigure S21E and F\u003c/b\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProduction of formate\u003c/h2\u003e \u003cp\u003eRh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed the reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate: The standard reaction condition was carried out for CO\u003csub\u003e2\u003c/sub\u003e reduction with CO\u003csub\u003e2\u003c/sub\u003e gas flow rate at 20ml/min in 0.1M PBS, pH 7.0, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex at -0.39 V\u003csub\u003eRHE\u003c/sub\u003e in the presence of 1% (v/v) EMIM-Ac.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCl\u003c/em\u003eFDH-catalyzed the reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate: The standard reaction condition was carried out for CO\u003csub\u003e2\u003c/sub\u003e reduction in 0.1M PBS, pH 7.0, 100 \u0026micro;M \u003cem\u003eCl\u003c/em\u003eFDH, 0.1M NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.01% (v/v) antifoam, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex-complex at -0.39 V\u003csub\u003eRHE\u003c/sub\u003e for NADH regeneration in the presence of 1% (v/v) EMIM-Ac.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNADH regeneration\u003c/h2\u003e \u003cp\u003eRh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed the reduction of NAD\u003csup\u003e+\u003c/sup\u003e to NADH: The standard reaction condition was carried out for NAD\u003csup\u003e+\u003c/sup\u003e reduction in 0.1M PBS, pH 7.0, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex at -0.39 V\u003csub\u003eRHE\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003ePTDH-catalyzed the reduction of NAD\u003csup\u003e+\u003c/sup\u003e to NADH: The standard reaction condition was carried out for NAD\u003csup\u003e+\u003c/sup\u003e reduction in 25 mM PBS, pH 7.0, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, and 50 mM sodium phosphite (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProduction of formaldehyde\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBm\u003c/em\u003eFaldDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PB, pH 7.0, 50 mM formate, 100 \u0026micro;M \u003cem\u003eBm\u003c/em\u003eFaldDH, 50 mM NADH.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBm\u003c/em\u003eFaldDH-PTDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PB, pH 7.0, 50 mM formate, 100 \u0026micro;M \u003cem\u003eBm\u003c/em\u003eFaldDH, 100 \u0026micro;M PTDH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, and 50 mM sodium phosphite (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eACS-ACDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PBS, pH 7.0, 50 mM formate, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 50 mM NADH, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system (5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 30 mM creatine phosphate, 5 mM ATP, 0.2 mg/mL creatine phosphokinase, and 1.3 mg/mL bovine serum albumin), 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase.\u003c/p\u003e \u003cp\u003eACS-ACDH-PTDH-catalyzed the reduction of formate to formaldehyde: The standard reaction condition was carried out for formate reduction in 25 mM PBS, pH 7.0, 50 mM formate, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 100 \u0026micro;M PTDH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProduction of methanol\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBm\u003c/em\u003eFaldDH-ADH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PB, pH 7.0, 150 mM formate, 100 \u0026micro;M \u003cem\u003eBm\u003c/em\u003eFaldDH, 10 U/ml ADH, and 50 mM NADH.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBm\u003c/em\u003eFaldDH-ADH-PTDH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PB, pH 7.0, 150 mM formate, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e, 100 \u0026micro;M \u003cem\u003eBm\u003c/em\u003eFaldDH, 10 U/ml ADH, 100 \u0026micro;M PTDH, and 3 mM NAD\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eACS-ACDH-ADH-catalyed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 10 U/ml ADH, 50 mM NADH, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase.\u003c/p\u003e \u003cp\u003eACS-ACDH-ADH-PTDH-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 10 U/ml ADH, 100 \u0026micro;M PTDH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase.\u003c/p\u003e \u003cp\u003eACS-ACDH-Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed the reduction of formate to methanol: The standard reaction condition was carried out for methanol production in 25 mM PBS, pH 7.0, 150 mM formate, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFDH-ACS-ACDH-ADH-PTDH-catalyzed the reduction of CO\u003csub\u003e2\u003c/sub\u003e to methanol: The standard reaction condition was carried out for methanol production from CO\u003csub\u003e2\u003c/sub\u003e in 25 mM PBS, pH 7.0, CO\u003csub\u003e2\u003c/sub\u003e gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e3\u003c/sub\u003e, 100 \u0026micro;M \u003cem\u003eCl\u003c/em\u003eFDH, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 10 U/ml ADH, 100 \u0026micro;M PTDH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase in the presence of 1% EMIM-Ac.\u003c/p\u003e \u003cp\u003eRh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH-ADH-catalyzed the reduction of CO\u003csub\u003e2\u003c/sub\u003e to methanol: The standard reaction condition was carried out for methanol production from CO\u003csub\u003e2\u003c/sub\u003e in 25 mM PBS, pH 7.0, CO\u003csub\u003e2\u003c/sub\u003e gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 10 U/ml ADH, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e in the presence or absence of 1% EMIM-Ac.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProduction of dihydroxyacetone and L-erythrulose\u003c/h2\u003e \u003cp\u003eACS-ACDH-FLS- Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed the conversion of formate to DHA: The standard reaction condition was carried out for DHA production from formate in 25 mM TEA, pH 7.0, 150 mM formate, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 100 \u0026micro;M FLS, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eRh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH-FLS-catalyzed the conversion of CO\u003csub\u003e2\u003c/sub\u003e to DHA: The standard reaction condition was carried out for DHA production from CO\u003csub\u003e2\u003c/sub\u003e in 25 mM TEA, pH 7.0, CO\u003csub\u003e2\u003c/sub\u003e gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 100 \u0026micro;M FLS, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e in the presence of 1% EMIM-Ac.\u003c/p\u003e \u003cp\u003eACS-ACDH-FLS-FSA\u003csup\u003eA129S\u003c/sup\u003e-Rh\u003csup\u003eIII\u003c/sup\u003e-complex-catalyzed the conversion of formate to L-erythrulose: The standard reaction condition was carried out for L-erythrulose production from formate in 25 mM TEA, pH 7.5, 150 mM formate, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 100 \u0026micro;M FLS, 50 \u0026micro;M FSA\u003csup\u003eA129S\u003c/sup\u003e, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eRh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH-FLS-FSA\u003csup\u003eA129S\u003c/sup\u003e-catalyzed the conversion of CO\u003csub\u003e2\u003c/sub\u003e to L-erythrulose: The standard reaction condition was carried out for L-erythrulose production from CO\u003csub\u003e2\u003c/sub\u003e in 25 mM TEA, pH 7.5, CO\u003csub\u003e2\u003c/sub\u003e gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 0.5 mM Rh\u003csup\u003eIII\u003c/sup\u003e-complex, 160 \u0026micro;M ACS, 40 \u0026micro;M ACDH, 100 \u0026micro;M FLS, 50 \u0026micro;M FSA\u003csup\u003eA129S\u003c/sup\u003e, 3 mM NAD\u003csup\u003e+\u003c/sup\u003e, 0.2 mM CoA, 0.5 mM DTT, ATP recycle system, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage \u0026minus;\u0026thinsp;0.39 V\u003csub\u003eRHE\u003c/sub\u003e in the presence of 1% EMIM-Ac.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the following grants: Key project on glucose water hydrogen production: [10311053A022301/002], National Key R\u0026amp;D Program of China (2022YFA0912002, 2022YFA0911900), National Natural Science Foundation of China (22309149 to Y.W.), Special Fund for Synthetic Biology [211000006022301/010], Competitive Research Funding Program (WU2022A006) in Center for Synthetic Biology and Integrated Bioengineering at Westlake University, and Westlake Education Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYajie Wang and Licheng Sun conceived and organized the study, contributed to drafting the manuscript, and revised it. Hailong Li and Yizhou Wu made equal contributions to designing the experiments, acquiring and analyzing the data, interpreting the results, and writing the manuscript. Yuxuan Wang contributed to data acquisition. Kai Zhang, Jin Zhu, Yuan Ji, and Tao Gu contributed to data analysis. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.N. Patent No. 202410990776.6 had been filed. The inventors include Yajie Wang, Licheng Sun, and Hailong Li. The other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhong W, Li H, Wang Y (2023) BioDesign Res 5:0021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo YM, Hong XM, Chen ZM, Lv YQ (2023) J Energy Chem 80:140\u0026ndash;162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuan LK, Ji XL, Guo BX, Cai JD, Dong WR, Huang YH, Zhang SJ (2023) Biotechnol Adv 63:1873\u0026ndash;1899\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurst J, Rudnev A, Dutta A, Fu YC, Herranz J, Kaliginedi V, Kuzume A, Permyakova AA, Paratcha Y, Broekmann P, Schmidt TJ (2015) Chimia 69:769\u0026ndash;776\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirdja YY, P\u0026eacute;rez-Gallent E, Figueiredo MC, G\u0026ouml;ttle AJ, Calle-Vallejo F, Koper MTM (2019) Nat Energy 4:732\u0026ndash;745\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu RR, Li F, Cui XY, Li ZH, Ma CL, Jiang HF, Zhang LL, Zhang Y, Zhao TX, Zhang YP, Li Y, Chen H, Zhu ZG (2023) Angewandte Chemie-International Ed 62:e202218387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeelajaroen H, Bakandritsos A, Otyepka M, Zboril R, Sariciftci NS (2020) ACS Appl Mater Interfaces 12:250\u0026ndash;259\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong HY, Ma CL, Liu P, You C, Lin JP, Zhu ZG (2019) J CO\u003csub\u003e2\u003c/sub\u003e Utilization 34:568\u0026ndash;575\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlager S, Dumitru LM, Haberbauer M, Fuchsbauer A, Neugebauer H, Hiemetsberger D, Wagner A, Portenkirchner E, Sariciftci NS (2016) Chemsuschem 9:631\u0026ndash;635\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang JY, Kerr TA, Wang XS, Barlow JM (2020) J Am Chem Soc 142:19438\u0026ndash;19445\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin XQ, Kanan MW (2015) J Am Chem Soc 137:4701\u0026ndash;4708\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKortlever R, Peters I, Koper S, Koper MTM (2015) ACS Catal 5:3916\u0026ndash;3923\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia BQ, Chen Z, Li CJ, Li ZF, Zhou XX, Wang T, Yang WX, Sun LC, Zhang BB (2023) J Am Chem Soc 145:14101\u0026ndash;14111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang ZB, Li JJ, Ji MB, Liu YR, Wang N, Zhang XP, Zhang SJ, Ji XY (2021) Green Chem 23:2362\u0026ndash;2371\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma VK, Hutchison JM, Allgeier AM (2022) Chemsuschem, 15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh D, Takeda H, Fabry DC, Tamaki Y, Ishitani O (2019) ACS Sustain Chem Eng 7:2648\u0026ndash;2657\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YY, Liu J (2024) Curr Opin Electrochem, 46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosen BA, Salehi-Khojin A, Thorson MR, Zhu W, Whipple DT, Kenis PJA, Masel RI (2011) Science 334:643\u0026ndash;644\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDongare S, Zeeshan M, Aydogdu AS, Dikki R, Kurtoglu-\u0026Ouml;ztulum SF, Coskun OK, Mu\u0026ntilde;oz M, Banerjee A, Gautam M, Ross RD, Stanley JS, Brower RS, Muchharla B, Sacci RL, Vel\u0026aacute;zquez JM, Kumar B, Yang JY, Hahn C, Keskin S, Morales-Guio CG, Uzun A, Spurgeon JM, Gurkan B (2024) Chem Soc Rev, 1460\u0026ndash;4744\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh RK, Singh R, Sivakumar D, Kondaveeti S, Kim T, Li JL, Sung BH, Cho BK, Kim DR, Kim SC, Kalia VC, Zhang Y, Zhao HM, Kang YC, Lee JK (2018) ACS Catal 8:11085\u0026ndash;11093\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Chung GY, Kim SH, Vinothkumar G, Yoon SH, Jung KD (2016) Electrochim Acta 210:837\u0026ndash;845\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SH, Ryu GM, Nam DH, Kim JH, Park CB (2014) Chemsuschem 7:3007\u0026ndash;3011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesnions S, Faur\u0026eacute; 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These systems typically rely on electrochemically regenerated NAD(P)H to provide the necessary reducing equivalents for formate dehydrogenase (FDH) to convert CO\u003csub\u003e2\u003c/sub\u003e into formate. However, the efficiency of these systems is currently unsatisfactory due to the unfavorable dynamics of the CO\u003csub\u003e2\u003c/sub\u003e-to-formate conversion by FDH. In this study, we developed a one-pot cooperative bioelectrochemical system featuring a rhodium-based catalyst [Cp*Rh(bpy)Cl]\u003csup\u003e2+\u003c/sup\u003e (Rh\u003csup\u003eIII\u003c/sup\u003e-complex or [Rh\u003csup\u003eIII\u003c/sup\u003e-H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e2+\u003c/sup\u003e) working cooperatively with enzymatic cascades of acetyl-CoA synthase (ACS), acetaldehyde dehydrogenase (ACDH), alcohol dehydrogenase (ADH), formolase (FLS), and d-fructose-6-phosphate aldolase mutant FSA\u003csup\u003eA129S\u003c/sup\u003e to convert CO\u003csub\u003e2\u003c/sub\u003e into several C\u003csub\u003e2+\u003c/sub\u003e chemicals. The bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex concurrently catalyzes the reduction of CO\u003csub\u003e2\u003c/sub\u003e to formate at a rate of 15.8 mM/h and NADH regeneration at a rate of 0.24 mM/min. The formation of formate is 83.2 times faster than using one of the best aerobic FDH from \u003cem\u003eClostridium ljungdahlii\u003c/em\u003e (\u003cem\u003eCl\u003c/em\u003eFDH), resulting in a 3.6 times enhanced methanol production rate of 0.43 mM/h in the bioelectroenzymatic system (Rh\u003csup\u003eIII\u003c/sup\u003e-complex-ACS-ACDH-ADH) compared to that of 0.12 mM/h in tandem enzymatic system (\u003cem\u003eCl\u003c/em\u003eFDH-ACS-ACDH-ADH). Bifunctional Rh\u003csup\u003eIII\u003c/sup\u003e-complex also works cooperatively with tandem enzymatic cascades to produce dihydroxyacetone (C\u003csub\u003e3\u003c/sub\u003e) and L-erythrulose (C\u003csub\u003e4\u003c/sub\u003e) at the yield of 2.63 mM, and 1.93 mM, respectively. This study leveraged the synthetic capabilities of both electrochemical catalysis and enzymatic catalysis, offering an alternative for electroenzymatic CO\u003csub\u003e2\u003c/sub\u003e reduction to yield value-added compounds with enhanced productivity.\u003c/p\u003e","manuscriptTitle":"Bifunctional RhIII-complex catalyzed CO2 reduction and NADH regeneration for direct bioelectrochemical synthesis of C3 and C4","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 16:07:11","doi":"10.21203/rs.3.rs-4865792/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ba6114f-d056-49bb-bb9a-cced8bfecef9","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":37107516,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":37107517,"name":"Physical sciences/Chemistry/Catalysis/Electrocatalysis"}],"tags":[],"updatedAt":"2024-09-04T16:07:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 16:07:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4865792","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4865792","identity":"rs-4865792","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}