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

doi:10.21203/rs.3.rs-4865792/v1

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Bifunctional Rh^III-complex catalyzed CO₂ reduction and NADH regeneration for direct bioelectrochemical synthesis of C₃and C₄

https://doi.org/10.21203/rs.3.rs-4865792/v1

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Bioelectrochemical synthesis is emerging as an eco-friendly method for CO₂ fixation. These systems typically rely on electrochemically regenerated NAD(P)H to provide the necessary reducing equivalents for formate dehydrogenase (FDH) to convert CO₂ into formate. However, the efficiency of these systems is currently unsatisfactory due to the unfavorable dynamics of the CO₂-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-H₂O]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 CO₂ into several C₂₊ chemicals. The bifunctional Rh^III-complex concurrently catalyzes the reduction of CO₂ 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 (ClFDH), 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 (ClFDH-ACS-ACDH-ADH). Bifunctional Rh^III-complex also works cooperatively with tandem enzymatic cascades to produce dihydroxyacetone (C₃) and L-erythrulose (C₄) 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 CO₂ 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

To address the energy crisis and alleviate the rising level of CO₂ in the atmosphere, various CO₂ capture and utilization techniques have been developed.¹ While electrochemical strategies offer high energy efficiency for CO₂ reduction and transformation, their practical application is limited by low selectivity in producing C₂₊ chemicals.² In contrast, biocatalysis excels in selectively producing C₂₊ chemicals but is less efficient in CO₂ 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 CO₂.^4–7

CO₂ 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 CO₂ into products like carbon monoxide (CO),³ formate (HCOOH),⁸ methanol (CH₃OH),⁹ ethylene (C₂H₄),² and other C₂₊ chemicals,^{2, 6} utilizing reducing equivalents regenerated from electricity through direct or mediated electron transfer. Among them, formate dehydrogenase (FDH)-catalyzed CO₂-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 CO₂/HCOOH (− 0.43 V vs NHE).¹⁰ This rate-limiting step may also restrict the supply of formate for subsequent C₂₊ chemical synthesis catalyzed by enzymatic cascades.⁶

Conversely, many electrocatalysts have been developed to efficiently produce formate through electrochemical CO₂ reduction reaction (CO₂RR) 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 CO₂ and NAD⁺, and its cooperative operation with enzymatic cascades for C₂₊ 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 CO₂ and regenerate NADH at the same time. This system can work cooperatively with tandem enzymatic cascades to convert CO₂ into value-added C₂₊ 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 CO₂ (Scheme 1). This system utilizes the bifunctional metal complex [Cp*Rh(bpy)Cl]²⁺ (Rh^III-complex or [Rh^III-H₂O]²⁺) to concurrently catalyze the electrochemical reduction of CO₂ to formate and the regeneration of NADH from NAD⁺. The resulting formate is then converted into methanol (C₁), dihydroxyacetone (C₃), and L-erythrulose (C₄) 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 CO₂ reduction rate of 15.8 mM/h, which is 83.2 times faster than the ClFDH-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 C₁-C₄ chemicals. By leveraging the synergies between electrochemical catalysis and biocatalysis, this cooperative bioelectrochemical system enables the conversion of CO₂ into value-added chemicals at a higher rate and yield compared to the electroenzymatic and tandem enzymatic systems with FDH.

Firstly, we aimed to design a bifunctional electrocatalyst capable of simultaneously reducing CO₂ and NAD⁺. Inspired by the studies on CO₂ photoreduction catalyzed by Rh^III-based photocatalysts,¹⁶ we selected [Cp*Rh(bpy)H₂O]²⁺ (Rh^III-complex or [Rh^III-H₂O]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 CO₂ 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 CO₂ gas bubbling (Figure S1). To be noted, Rh^III-complex effectively catalyzed the generation of formate from CO₂ 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. 1A).

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 CO₂ reduction and found that PEM performed better than AEM (Fig. 1A). Subsequently, we evaluated the electrochemical CO₂ reduction under various conditions, including different applied potentials, CO₂ 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 CO₂ 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 CO₂ reduction in aqueous media^{18, 19} and that 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) has shown superior performance in CO₂ 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. 1B). For comparison, formate production was evaluated using a conventional electroenzymatic system with one of the most efficient aerobic FDHs from Clostridium ljungdahlii (ClFDH) 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 CO₂ bubbling. The Rh^III-complex demonstrated the optimal activity for NADH regeneration under the condition most suitable for CO₂ reduction (Figure S3). The presence of EMIM-Ac had little influence on NADH regeneration (Fig. 1C). 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 CO₂, 0.5 mM Rh^III-complex could maintain catalytic efficiency for both NADH regeneration and CO₂ reduction (Fig. 1D).

To better understand the mechanism of bifunctional Rh^III-complex in CO₂ 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. 2A, the CV curves of CO₂ with or without EMIM-Ac have no significant differences, indicating that EMIM-Ac does not catalyze CO₂ reduction. In the absence of both CO₂ and EMIM-Ac, [Rh^III-H₂O]²⁺ undergoes a two-electron reduction process to form [Rh^I-H]⁺ hydride (Eqs. 1) at -0.17 V_RHE (-4.67 mA/cm²). After adding 1% EMIM-Ac, the reduction peak slightly shifts to -0.16 V_RHE with a higher current density (-6.43 mA/cm²). 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 CO₂ reduction with or without EMIM-Ac (Fig. 2B), the cathodic current peak increases from − 8.34 to − 9.56 mA/cm², indicating a higher CO₂ reduction efficiency. Additionally, the reduction peak of [Rh^I-H]⁺ hydride-catalyzed CO₂ 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]⁺-CO₂ intermediate, thereby reducing the initial reduction barrier. Those findings are consistent with the previous studies showing that ionic liquids can decrease the overpotential for CO₂ 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. 2C 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. 2C). According to the catalytic mechanism reported by Kim et al.,²¹ the [Rh^III-H₂O]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-H₂O]²⁺ to [Rh^I-H]⁺ during NADH regeneration is clearly observed at − 0.19 V_RHE (Fig. 2D). Compared with the Rh^III-complex mediated NADH regeneration, the direct NAD⁺ reduction requires a more negative potential of − 0.59 V_RHE (Fig. 2C), confirming that NAD⁺ was reduced by [Rh^I-H]⁺ via a hydride transfer pathway in our system.²² The simultaneous reduction of CO₂ 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 CO₂ reduction (− 0.17 V_RHE, Fig. 2A). CV analysis defines the electrochemical window for the mediated CO₂ reduction and NADH regeneration. Thus, a potential of -0.39 V_RHE is sufficient for driving the concurrent CO₂ 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. 3A): 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 (BmFaldDH) ²⁰ as the biocatalyst (Fig. 3B). The low yield could be attributed to significantly unfavorable thermodynamics of FDH pathway (Δ_rG′^m = + 51.7 kJ/mol) compared to the formolase pathway (ΔrG′^m = -8.6 kJ/mol) (Fig. 3A).

Given the preference of BmFaldDH 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 BmFaldDH 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. 3C). 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 CO₂ 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. 3C, 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 CO₂ reduction and NADH regeneration with ACS, ACDH, and ADH to synthesize methanol from CO₂ 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. 3C). This proves that Rh^III-complex-catalyzed electrochemical CO₂ 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 CO₂ compared to the tandem enzymatic system (0.68 mM in Route 4) as shown in Fig. 3D. These underscore the pivotal role of the bifunctional Rh^III-complex in the cooperative bioelectrochemical system for efficient CO₂ 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 C₂₊ chemicals from CO₂. 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 CO₂ 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 CO₂ 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 CO₂ respectively (Scheme 4). Remarkably, using CO₂ 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 CO₂ to formate while supplying the necessary reducing equivalents (NADH) to drive the subsequent tandem enzymatic cascades for synthesizing longer-chain, higher-value chiral compounds.

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 (C₁), dioxyacetone (C₃), and L-erythrulose (C₄) from CO₂, achieving high yields of 2.58 mM, 2.63 mM, and 1.93 mM, respectively. The Rh^III-complex catalyzes the reduction of CO₂ 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 CO₂ 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 CO₂ reduction as a viable alternative for synthesizing high valuable C₂₊ products and may inspire the direct electrosynthesis of value-added compounds from CO₂.

The activity assay and kinetic assay

The activity of ClFDH was monitored by NADH oxidation at 340 nm. CO₂ reduction activity of FDH was measured in 0.1 M PBS (pH 7.0), at room temperature. Reaction mixtures (0.1M NaHCO₃, 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 (Na₂HPO₃, 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 ZnSO₄, 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 MgSO₄, 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 MgSO₄ 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 (H₂SO₄) 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 CO₂ to formate: The standard reaction condition was carried out for CO₂ reduction with CO₂ 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.

ClFDH-catalyzed the reduction of CO₂ to formate: The standard reaction condition was carried out for CO₂ reduction in 0.1M PBS, pH 7.0, 100 µM ClFDH, 0.1M NaHCO₃, 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 (Na₂HPO₃).

Production of formaldehyde

BmFaldDH-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 BmFaldDH, 50 mM NADH.

BmFaldDH-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 BmFaldDH, 100 µM PTDH, 3 mM NAD⁺, and 50 mM sodium phosphite (Na₂HPO₃).

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 MgCl₂, 30 mM creatine phosphate, 5 mM ATP, 0.2 mg/mL creatine phosphokinase, and 1.3 mg/mL bovine serum albumin), 2 mM MgSO₄, 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 Na₂HPO₃, 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 MgSO₄, 0.2 mM TPP, and 0.1 mg/mL glycerokinase.

Production of methanol

BmFaldDH-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 BmFaldDH, 10 U/ml ADH, and 50 mM NADH.

BmFaldDH-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 Na₂HPO₃, 100 µM BmFaldDH, 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 MgSO₄, 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 Na₂HPO₃, 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 MgSO₄, 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 MgSO₄, 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 CO₂ to methanol: The standard reaction condition was carried out for methanol production from CO₂ in 25 mM PBS, pH 7.0, CO₂ gas flow rate at 20 ml/min, 0.01% (v/v) antifoam, 50 mM Na₂HPO₃, 100 µM ClFDH, 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 MgSO₄, 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 CO₂ to methanol: The standard reaction condition was carried out for methanol production from CO₂ in 25 mM PBS, pH 7.0, CO₂ 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 MgSO₄, 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 MgSO₄, 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 CO₂ to DHA: The standard reaction condition was carried out for DHA production from CO₂ in 25 mM TEA, pH 7.0, CO₂ 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 MgSO₄, 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 MgSO₄, 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 CO₂ to L-erythrulose: The standard reaction condition was carried out for L-erythrulose production from CO₂ in 25 mM TEA, pH 7.5, CO₂ 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 MgSO₄, 0.2 mM TPP, and 0.1 mg/mL glycerokinase at applied voltage − 0.39 V_RHE in the presence of 1% EMIM-Ac.

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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Bifunctional Rh^III-complex catalyzed CO₂ reduction and NADH regeneration for direct bioelectrochemical synthesis of C₃and C₄

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The activity assay and kinetic assay

Lang and Lang method for formate analysis

Nash method for formaldehyde analysis

GC for methanol analysis

HPLC for dihydroxyacetone and L-erythrulose analysis

Production of formate

NADH regeneration

Production of formaldehyde

Production of methanol

Production of dihydroxyacetone and L-erythrulose

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