Photo-enzyme-membrane for ethylene glycol synthesis

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: major_revision

Abstract
The light-driven enzyme catalytic system composed of NAD(P)H regeneration and NAD(P)H-dependent enzymatic process emerges as a biomanufacturing platform for the synthesis of value-added chemicals, where the photo-responsive materials absorb solar energy to drive mass conversion. Herein, we report a photo-enzyme-membrane (PEM) catalytic system coupled with multi-enzyme cascade for ethylene glycol (EG) synthesis, in which membrane mediates energy transfer and mass conversion. Covalent organic polymer membrane as photo-membrane (PM) affords efficient NADH supply through synergistic intensification of electron transfer and proton transfer, where the bipyridine moiety mediates fast electron transfer from the generation site, and the sulfonic acid moiety facilitates proton transfer by enriching protons. Meanwhile, NADH-dependent enzyme is absorbed on PM followed by coating with a silica layer to form PEM, where the enzyme-bearing silica layer is defined as enzyme-membrane (EM). The enzymatic process is intensified by mitigating the adverse effects of PM on enzyme activity through precise regulation of EM thickness. Further, a dual-channel reactor is constructed for sustainable synthesis of EG with an initial synthesis rate of 2.43 mmol g-1 h-1 by continuous supply of methanol. Our study offers an efficient and durable light-driven enzyme catalytic system for the synthesis of C2 from C1 chemicals.

Introduction
The extreme climate change and environmental problems caused by the increase in carbon dioxide concentration are thought-provoking, which provide new opportunities for natural carbon fixation and artificial carbon conversion technologies1–9. Enzyme or microorganism-based biomanufacturing has a broad application prospect in the synthesis and transformation of substances. In particular, biomanufacturing processes powered by renewable energy such as light energy contribute to the green, low-carbon synthesis of energetic chemicals10,11. Typically, light-driven enzyme catalysis is composed of energy supply module and mass transformation module, where cofactor such as NAD(P)H serves as an essential energy carrier, enabling the enzymatic synthesis of value-added chemicals in vitro12–18. The high-performance of light-driven enzyme catalytic system (LECS) primarily relies on efficient NAD(P)H regeneration and stable enzymatic transformation which is determined by the compatibility between NAD(P)H regeneration and NAD(P)H-dependent enzymatic process. Currently, to improve the efficiency of LECS, researchers focus on the intensification of photo-generated electron transfer for ensuring the supply of energy carrier NAD(P)H through constructing heterojunction photocatalysts or shorting the distance between the photocatalyst and catalyst site19–21. However, the transfer and utilization of protons, which are accompanied by photo-generated electrons, have been overlooked. Two electrons and one proton transfer are needed in NAD(P)H regeneration14. The rapid electron-hole recombination hinders sufficient electron transfer to the active site, which reduces the electron utilization efficiency. The proton transfer governs the binding efficiency between NAD(P)+ and proton, while has an impact on the selectivity of generated NAD(P)H and initial reaction rate of NAD(P)H regeneration. The mismatch in the kinetics of electron transfer and proton transfer processes results in the production of by-products and low catalytic performance22. Thus, new insights and relevant design for NAD(P)H regeneration in LECS to coordinate the electron transfer and proton transfer processes are urgently needed.

Meanwhile, to enhance the NAD(P)H transfer efficiency between photocatalytic and enzymatic process, NAD(P)H-dependent enzyme is usually immobilized on photocatalyst to form integrated catalyst23,24. However, the photocatalytic components exhibit detrimental effects on the activity of NAD(P)H-dependent enzyme, thereby compromising the stability of the LECS. The reactive oxygen species (ROS) generated during photocatalytic process and the metal complex utilized in photocatalytic process attacks the active sites of enzyme, leading to enzyme deactivation25–27. Designing physical entrapment or coating on the surface of photocatalyst may be a feasible approach to eliminating or weakening the effect of ROS and metal complex on enzymes28. The dense coating increases the mass transfer resistance, which is conducive to the quenching of ROS, thereby reducing the negative impact on the secondary structure and activity of enzyme. Therefore, the compatibility between NAD(P)H regeneration and NAD(P)H-dependent enzymatic process is crucial to the stability of LECS.

In our study, we reported a membrane-mediated LECS defined as photo-enzyme-membrane (PEM) catalytic system, which then coupled with multi-enzyme cascade for the sustainable synthesis of ethylene glycol (EG) from methanol. The PEM has features of efficient NADH regeneration through the design of photo-membrane (PM) as well as stable enzymatic process through regulating enzyme-membrane (EM). Specifically, covalent organic polymer membrane as PM was designed to synthesize NADH for enzyme catalysis by coordinating electron transfer and proton transfer. A highest NADH regeneration initial reaction rate of 5.6 mmol g-1 h-1 was obtained through optimizing the ratios between electron transfer-facilitating monomer and proton transfer-facilitating monomer in PM. In addition, NADH-dependent enzyme alcohol dehydrogenase (ADH) was absorbed on PM followed by coating silica layer to form PEM for EG synthesis, where ADH-bearing silica layer was defined as EM. Regulating the thickness of the EM can mitigate ROS-induced enzyme deactivation while preserving the integrity of the enzyme secondary structure. A 503% increase in EG production was obtained under the optimal thickness of EM. Ultimately, PEM, when coupled with alcohol oxidase (AOX), catalase (CAT), and glycolaldehyde synthase (GALS), enables sustainable in vitro synthesis of EG from methanol. (Fig. 1). Owing to synergistic optimization of multi-enzyme cascade and PEM, an EG production of 0.71 mmol L-1 with the initial synthesis rate of 2.43 mmol g-1 h-1 was obtained with the continuous supply of methanol. Our study establishes a versatile platform for mass-energy coupled biocatalysis, enabling the efficient and sustainable synthesis of C2 from C1 chemicals.

Results
In PEM, PM harvests light to regenerate NADH, and EM consumes the NADH for EG production. The regeneration of NADH involves both electron and proton transfer processes, which challenges the design of an efficient PM. To coordinate the electron and proton transfer processes, covalent organic polymers with well-defined and tailored functional entities are employed in the construction of PM. In detail, the aldehyde monomer of 1,3,5-tris-(4-formyl-phenyl) triazine (m-TFPT), and the amino monomer of 2,2′-bipyridine-5,5′-diamine (m-Bpy) and 2,5-diamino-1,4-benzenedisulfonic acid (m-PaHSO3) were connected via imine linkage to construct PM. By adjusting the ratio between m-Bpy and m-PaHSO3, different PM named PM-BS30, PM-BS21, and PM-BS12 were obtained (Fig. 2a). Transmission electron microscopy (TEM) showed that all PM were of capsule structures with a wall thickness of 30-38 nm (Fig. 2a). Fourier transform infrared spectrum (FTIR) of PM detected absorption bands at 810 cm-1, 629 cm-1 and 1685 cm-1, which was assigned to the triazine group of m-TFPT, sulfonate group of m-PaHSO3, and imine linkage. This confirmed that PM was formed by the covalent linking of amino and aldehyde monomers (Supplementary Fig. 1). Nitrogen adsorption-desorption isotherm and corresponding pore size distribution of the PM-BS12, PM-BS21 and PM-BS30 were reflected in Supplementary Fig. 2. PM-BS12, PM-BS21, and PM-BS30 exhibited pore sizes of 3.0 nm (Supplementary Fig. 2a, b); 1.7 nm and 3.0 nm (Supplementary Fig. 2c, d); and 3.0 nm and 5.3 nm (Supplementary Fig. 2e, f), respectively. This pore size distribution enabled NAD+/NADH with a hydrodynamic diameter of ~1.7 nm to shuttle through the pores29. As shown in Supplementary Fig. 3a, elemental mapping images of nitrogen (N) and sulfur (S) element were observed in PM-BS12 and PM-BS21, while there were no S elemental signals in PM-BS30. Supplementary Fig. 3b exhibited the proportion variation between element N and S. The S content increased nearly 100% in PM-BS12 compared to PM-BS21, which was consistent with the proportion between m-Bpy and m-PaHSO3 used for the synthesized PM. X-ray photoelectron spectroscopy (XPS) was also applied to characterize the chemical composition of PM. In N 1s spectra, the peaks of triazine group/imine linkage (n1), bipyridyl group (n2), and N-H bond (n3) were detected (Supplementary Fig. 4a–c). The proportion of n3 was consistent, suggesting that different PM had the same amounts of free amino groups. The proportion of n2 was approximately 1:2:3, indicating that the proportion of bipyridyl group in all PM samples was consistent with the proportion of m-Bpy used for the synthesis of PM. Based on the above analysis, the structure unit of PM was shown in Supplementary Figs. 5–7. Moreover, the conduction band potential of PM was higher than the NADH reduction potential under normalized hydrogen electrode (NHE) at pH 7.0, which can theoretically drive the NADH regeneration reaction (Supplementary Fig. 8a–d).

To evaluate the electron transfer and proton transfer performance, PM was characterized by transient absorption spectroscopy (TAS), time-resolved photoluminescence (TR-PL) spectroscopy and AC impedance tests. As shown in Fig. 2b, τ1 represents the time of photo-generated electron-hole recombination, and τ2 represents the relaxation time of non-electron hole recombination. τ2 got prolonged as the ratio of m-Bpy to m-PaHSO3 increased, suggesting that PM-BS30 with more dipyridyl groups was constructive for electron transfer. TR-PL spectroscopy revealed that the average lifetime of photogenerated electrons in PM-BS30 (374 ps) was much longer than in PM-BS21 (229 ps) and PM-BS12 (145 ps) (Supplementary Fig. 9). However, the decrease in m-PaHSO3 would limit the proton transfer performance of PM. PM-BS12 (170.8 mS cm-1) exhibited better proton conductivity than PM-BS21 (63.4 mS cm-1) and PM-BS30 (11.5 mS cm-1) (Fig. 2c and Supplementary Fig. 10). We deduced that electron transfer capability of PM was enhanced with the increased ratio of m-Bpy to m-PaHSO3, while the proton conductivity would be weakened. Then, density functional theory (DFT) calculation was conducted to analyze the energy-level structure and proton adsorption energy of PM. With the molecular cluster formed by one m-Bpy and two m-TFPT units as the model, the HOMO (-5.26 eV) was mainly derived from m-Bpy, while the LUMO (-3.26 eV) originated from m-TFPT (Fig. 2d and Supplementary Data 1). Moreover, both HOMO and LUMO contain imine bonds which were formed by the linking of m-Bpy and m-TFPT, confirming the existence of a linear combination of orbitals. The electrons from m-Bpy were excited and then may transfer to m-TFPT, which would enhance the electron intensity at the imine bond. For proton transfer, PM-BS12 (-4.757 eV) exhibited a more negative proton adsorption energy compared to PM-BS21 (-4.718 eV) and PM-BS30 (-3.445 eV) (Supplementary Fig. 11). To further prove our designed PM can enrich the protons, three types of PM were first filtered to form membranes on the polyacrylonitrile (PAN) substrate (PM-PAN membrane, Supplementary Fig. 12a–c). Then, the PM-PAN membranes were set at the diffusion cell, where 1:5000 (v/v) hydrochloric acid solution and aqueous solution were separately set at both sides. As shown in Supplementary Fig. 13, the pH value of the aqueous solution was monitored over time. The pH value of PM-BS12-PAN changed slightly compared to PM-BS21-PAN and PM-BS30-PAN, which demonstrated that PM-BS12-PAN could more efficiently adsorb protons. Therefore, the optimal molar ratio of m-Bpy to m-PaHSO3 was determined to be 2:1, resulting in enhanced local intensities of electrons and protons in PM-BS21, facilitating NADH regeneration (Fig. 2e).

As shown in Supplementary Fig. 14a–c, the NADH regeneration catalyzed by PM was performed under different light densities. With the increase of light intensity from 100 to 200 mW/cm2, the NADH regeneration yield was gradually improved. This suggested that light absorption was the rate-determining step when light intensity was between 100 and 200 mW/cm2. With the further increase of light intensity, the NADH yield was only slightly changed. This suggested that under light intensities exceeding 200 mW/cm2, factors other than light became the rate-determining step in the NADH regeneration process. During photocatalytic NADH regeneration, Rh-complex captured two electrons and one proton and then transferred them to NAD+ for the production of NADH. Since both the Rh-complex and the reactant were in sufficient concentration, the electron and proton transfers may be the rate-determining step when light intensity was above 200 mW/cm2. The process of NADH regeneration involves the transfer of two electrons and one proton, where proton transfer determines the binding efficiency of NAD+ and proton. Slower proton transfer would result in the formation of NAD dimers by the binding of the NAD radicals, which ultimately decreases the selectivity of 1,4-NADH. As shown in Fig. 2f, NADH regeneration experiments were carried out at 300 mW/cm2 of light density. Within the first 4 min, owing to better proton transfer, PM-BS12 exhibited a higher NADH regeneration rate and 1,4-NADH selectivity than PM-BS21 and PM-BS30 (Supplementary Fig. 15a and Supplementary Fig. 16). However, at 4–8 min, the NADH regeneration rate by PM-BS21 was improved, benefiting from the coordination of electron and proton transfers (Supplementary Fig. 15b). As for 12 min (Fig. 2f), all PM samples reached the balance of photocatalytic NADH regeneration process, where PM-BS21 exhibited the best NADH regeneration performance (89.5%). The NADH regeneration performance of PM-BS21 surpassed that of most previously reported results (Supplementary Table 1).

Glucose derived from the degradation of biomass can be oxidized to value-added products, such as gluconic acid, and formic acid. Glucose serves as a promising electron donor due to its sustainable origin, economic viability, and value-added chemicals production. To explore a greener route of NADH regeneration, glucose was used as the electron donors in place of triethanolamine (TEOA). With glucose as the electron donor, an NADH regeneration yield of 44.0% was achieved, accompanied by the generation of 3.1 mmol L-1 of gluconic acid, highlighting the potential of using lignocellulose as the electron donor to drive NADH regeneration (Supplementary Figs. 17–18).

To convert glycolaldehyde to EG, ADH can be directly mixed with PM in solution or be immobilized onto PM. However, free Rh-complex used for NADH regeneration and photo-generated ROS would deactivate ADH. As shown in Supplementary Fig. 19a, b, the activity of ADH was decreased to 49.3% and 4.1% after incubation with Rh-complex and photo-generated ROS (Supplementary Note. 1). To minimize the impact of Rh-complex and ROS on enzymatic activity, Rh-complex was loaded on PM to prepare PM-Rh, and then ADH was immobilized onto PM-Rh by forming a silica layer. First, by adding sufficient Rh-complex for PM-Rh synthesis, PM-BS12-Rh0.43, PM-BS21-Rh0.82, and PM-BS30-Rh1.34 were obtained. Inductively coupled plasma optical emission spectroscopy (ICP-OES) test was shown in Supplementary Table 2. Rh-complex was anchored on bipyridine group, while the Rh element loading amounts of PM-BS12-Rh0.43, PM-BS21-Rh0.82, and PM-BS30-Rh1.34 was in a mass ratio of ~1:2:3. Therefore, PM-BS30-Rh1.34 with more anchored Rh-complex exhibited a higher NADH regeneration yield than PM-BS12-Rh0.43 and PM-BS21-Rh0.82 (Supplementary Fig. 20a, c). Furthermore, PM with equal amount of Rh element loading was also prepared. The loading of Rh element was shown in Supplementary Table 3, and the Rh element content in the PM was almost consistent. As shown in Supplementary Fig. 20b, d, PM-BS21-Rh0.50 exhibited the highest NADH regeneration performance with the selectivity of ~90%. Comprehensively considering the NADH regeneration yield and the dosage of Rh-complex, PM-BS21-Rh was used to construct PEM for EG synthesis from GALD.

Subsequently, ADH was immobilized onto PM-BS21-Rh through polyethyleneimine (PEI)-induced silicification to form PEM. In detail, polyethyleneimine (PEI) and ADH were sequentially adsorbed onto PM-BS21-Rh. Then, silica was deposited onto PM-BS21-Rh. The composite layer comprising ADH, and silica layer was named EM. As shown in Supplementary Fig. 21, the loading efficiency of ADH was 85.3-98.4%. Given the amount of PM-BS21-Rh (3 mg) and ADH (1 mg) added initially, one mg PM-BS21-Rh loaded 0.28–0.33 mg ADH could be calculated. The integrated PEM was then constructed, enabling the conversion of GALD to EG through a continuous supply of NADH, among which integrated PEM was compared to the non-integrated system constructed by PM-BS21-Rh and free ADH (Fig. 3a, b). TEM images, mapping images, and XPS spectrum of Si element demonstrated that silica was successfully deposited onto PM (Supplementary Fig. 22a–f and Supplementary Fig. 23a). Confocal laser scanning microscope (CLSM) and XPS spectrum of Rh element showed that ADH and Rh-complex were successfully loaded on EM and PM, respectively (Supplementary Fig. 23b and Supplementary Fig. 24). Due to the loading of Rh-complex into PM-BS21, the activity of ADH was well maintained after incubation with PM-BS21-Rh under both in the dark or light conditions. However, ADH was deactivated after incubation with free Rh-complex under the same condition (Supplementary Fig. 25a, b and Supplementary Note. 1). Moreover, an EM layer of various thicknesses (69–131 nm) was formed by manipulating the concentration of the silica percussor, e.g., sodium silicate (Supplementary Fig. 26). Although silica component in EM was almost optically transparent in the visible light range, the light absorption efficiency of PEM was not constant (Supplementary Fig. 27). EM reduced the light absorption intensity of PEM. The intensity of ROS (Supplementary Fig. 28a), such as hydroxyl radical (•OH) and superoxide radical (•O2-) observed in electron spin resonance spectroscopy (ESR) decreased as the thickness of EM increased. This reduction mitigated the adverse effects of ROS on enzyme activity (Fig. 3c and Supplementary Fig. 28b, c). Moreover, as shown in Fig. 3d and Supplementary Fig. 29a–d, the changes in the secondary structure of the ADH were analyzed. The secondary structure of ADH was almost unchanged under dark condition. In PEM system after illumination, the ratio of α-helix of ADH got decreased, suggesting that ROS generated by light had an adverse effect on the structure of enzymes. As the thickness of the EM increased, the secondary structure of ADH was better protected. For the PEM system, the secondary structure of enzyme was almost unchanged before and after light illumination. The presence of the EM weakened the intensity of ROS, and confined enzyme within a stable space, thus ensuring the stable secondary structure of enzyme. Owing to the protection of EM on ADH, the optimal EG production reached 1.0 mmol L-1 when applying PEM for EG synthesis (Supplementary Fig. 30), representing a 503% enhancement compared to the non-integrated system (Fig. 3e). The improved performance was attributed to the stable enzymatic process provided by the EM construction. However, substrate molecule transfer resistance caused by the excessive thickness of EM reduced the EG production when applying PEM for EG synthesis. Ultimately, EG can still be produced by PEM, when TEOA was replaced by glucose as the electron donor (Supplementary Fig. 31). This established a greener route for EG synthesis.

To acquire value-added products from carbon resources, PEM was coupled with the multi-enzyme cascade comprising AOX, CAT, and GALS to convert methanol to EG (Fig. 4a). First, methanol was converted to formaldehyde and H2O2 by AOX. Due to the adverse effect of H2O2 on the cofactor or enzyme, CAT was added to eliminate H2O2 to ensure sustainable production of formaldehyde. Subsequently, in the presence of GALS, two formaldehyde was condensed into GALD. Finally, GALD was converted to EG by PEM. In the coupled system, the collaboration between AOX, CAT, GALS and PEM ensured the sustainable synthesis of EG. As for the multi-enzyme cascade, we first regulated the concentration and ratio of each enzyme to achieve the optimal production of GALD (Supplementary Note. 2). The optimal formaldehyde production, 8.9 mmol L-1, was achieved when the concentration of AOX and CAT was set to 0.8 and 0.6 g L-1, respectively (Supplementary Fig. 32). Additionally, since GALS was thiamine diphosphate (ThDP)-dependent enzyme30–32, Mg2+ was added to accelerate the enzymatic reactions. Supplementary Fig. 33a, b showed that the optimal concentration of Mg2+ and ThDP for the enzymatic conversion of formaldehyde to GALD were 10.0 mmol L-1 and 1.0 mmol L-1, respectively. 3.7 mmol L-1 of GALD was produced after 24 h of incubation by optimizing the concentration of GALS (Supplementary Fig. 34).

Due to the more negative Gibbs free energy of the formaldehyde hydrogenation reaction (-30.5 kJ mol-1) compared to the GALD hydrogenation reaction (-20.6 kJ mol-1), NADH and ADH preferentially convert formaldehyde to methanol rather than GALD to EG when the multi-enzyme cascade was combined with PEM in a single reactor for one-pot EG synthesis. To address challenges, the multi-enzyme cascade and PEM and PEM were compartmentalized into an enzymatic reactor and a PEM reactor, respectively. Moreover, methanol can disrupt the hydration layer of enzymes, thereby reducing their catalytic activity. To maintain enzyme activity and enhance EG production, a continuous supply of methanol, rather than a high-concentration supply, was employed. Specifically, GALD was produced by multi-enzyme cascade in the enzymatic reactor, while PEM further converted GALD to EG in the PEM reactor. With continuous feeding from the enzymatic reactor to the PEM reactor, 5.1 mmol L-1 of EG at a NAD+ concentration of 10 mmol L-1 was obtained, using TEOA after four light and dark cycles. In particular, 3.1 mmol L-1 EG was obtained in the presence of 2 mmol L-1 NAD+, which exhibits the system’s economic viability. This approach offers a new perspective for expanding chemical production (Supplementary Fig. 35 and Supplementary Fig. 36a, b). Notably, after four light and dark cycles, a moderate amount of EG was generated through a greener route using glucose as the electron donor (Supplementary Fig. 37).

In order to integrate multi-enzyme cascade and PEM, a well-established reactor with dual channels was also constructed for continuous EG synthesis. The enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion channel and PEM filtrated to form a PEM-PAN membrane was put in the photo-enzyme catalytic diversion channel. As shown in Fig. 4b, c, 0.71 mmol L-1 EG production with an initial synthesis rate of 2.43 mmol g-1 h-1 was obtained after ten light and dark cycles. The reactor showed a long-term viability, and the total turnover number (TTN) was calculated as 1668. The EG production was moderate compared to other systems (Supplementary Table 4). The design of the reactor enabled the scalable, integrated enzyme-photo-coupled catalytic conversion of feedstocks into valuable chemicals.

In summary, we explore a PEM coupled with multi-enzyme cascade to produce EG. In our PEM, NADH regeneration, by coordinating electron transfer and proton transfer through optimizing the ratios between electron transfer-facilitating monomer and proton transfer-facilitating monomer in PM, ensures the efficient operation of the PEM. An 89.5% NADH yield with an initial reaction rate of 5.6 mmol g-1 h-1 and TOF of 27.9 h-1 is obtained. The thickness-tunable EM mitigates the adverse effects of ROS on the secondary structure and activity of enzyme, enabling stable operation of PEM. A 503% elevation of the EG production in the integrated PEM is obtained compared with the non-integrated system. Through synergistic optimization of multi-enzyme cascade and PEM, 0.71 mmol L-1 EG with the initial synthesis rate of 2.43 mmol g-1 h-1 is obtained with the continuous addition of methanol in a dual-channel reactor, where the photonic quantum efficiency can reach 0.15%. Our study establishes a light-driven enzymatic conversion platform for the sustainable synthesis of C2 from C1 chemicals under continuous operation.

Methods
Synthesis of PM-BS30, PM-BS21, and PM-BS12
PM was prepared by hard stencil method. Firstly, SiO2 and its surface modification polyethyleneimine (PEI) (200 mg) and dopamine (PDA) (200 mg) named SiPP were prepared by previous methods. Then, SiPP powders (50 mg) and 1,3,5-tris-(4-formyl-phenyl) triazine (TFPT) (39.3 mg, 0.1 mmol) was resolved in tetrahydrofuran (THF). The mixture was then treated with ultrasonic for 10 min, following by 6.0 mol L-1 acetic acid addition. And the mixture was hydrothermal treated in flask at 50 °C, 120 r min-1 for 30 min. Then the resolved m-Bpy (18.6 mg, 0.1 mmol) and m-PaHSO3 (9.4 mg, 0.05 mmol) solution by THF or N,N-Dimethylformamide (DMF) were slowly added into flask. After 48 h of reaction, the solid was obtained by filtration and drying. Finally, 6.0 mol L-1 ammonium bifluoride was used to remove the SiPP to obtain the hollow micro-capsule structure photocatalyst PM-BS21. PM-BS30 and PM-BS12 was synthesized by changing the concentration of m-Bpy (27.9 mg, 0.15 mmol or 9.3 mg, 0.05 mmol) and m-PaHSO3 (0 mg, 0 mmol or 18.8 mg, 0.1 mmol), respectively.

Synthesis of PM-BS21-Rh
PM-Rh was synthesized by the coordination of the bipyridine group in the PM with the pentamethylcyclopentadienylrhodium (III) chloride dimer ((Cp*RhCl2)2). Specifically, 10 mg of PM-BS21 with 4 mL of deionized water was sonicated for 10 minutes, followed by the addition of different mass of (Cp*RhCl2)2 (3.32 mg, 0.005 mmol; 6.64 mg, 0.01 mmol; or 9.96 mg, 0.015 mmol). Then the mixture was stirred for 24 h, following by centrifugation, rinsed with deionized water several times, and freeze-dried. The obtained solid was denoted as PM-BS12-Rhx, PM-BS21-Rhx, and PM-BS30-Rhx, where x referred to the content of Rh element. The Rh element content of PM was characterized by ICP test.

Synthesis of PEMx
3 mg of PM-BS21-Rh was added into 2 mL of deionized water with 2 mL of PEI (10 mg, pH 7.0) for 10 min ultrasonic treatment. After stirring for 30 min, the mixture was washed and centrifuged to get the solid. ADH (1 mg) was added into the obtained solid with 2 mL of deionized water stirring for 10 min. Then 2 mL Na2SiO3 solution (pH 7.0, 2, 5, 10, 20 mmol L-1) was mixed with above solution stirring for 20 min. Solid particles defined as PEMx (x=2, 5, 10 or 20) were obtained after centrifugation, washed and freeze-dried. The loading efficiency of ADH was measured by the Bradford method33. The loading efficiency (%) of ADH was defined by the following equation:

Loading efficiency = (m0 - m) / m0 * 100%

where m0 (mg) was initial dosage of ADH, m (mg) was the dosage of free ADH in the supernatant. The loading amount actually refers to the mass (mg) of ADH loaded onto the one mg carrier (PM-BS21-Rh).

The test for NADH regeneration
NADH regeneration was performed in a 3.1 mL quartz test reactor with a reaction volume of 2 mL, where the reaction solution also contained triethanolamine (TEOA) or glucose, tartaric acid, and lactic acid solution of 1000 mmol L-1, pH 8.0, a serious of PM photocatalysts of 1 g L-1, NAD+ of 1 mmol L-1 and Rh-complex of 0.2 mmol L-1. Simulating sunlight used a 100 W LED lamp, and the light was turned on after dark treatment for 5 min. The absorbance of the reaction solution was measured by UV-Vis spectrophotometer after illumination at 340 nm.

The NADH regeneration production was calculated by Lambert-Beer law. The yield of NADH regeneration (%) was the ratio of the concentration of produced NADH to the substrate NAD+. Ultimately, the enzymatic method was used to detect the selectivity of 1,4-NADH. The absorbance of the reaction at 340 nm would decrease arising by the consumption of NADH when the appropriate amount of propanal and ADH added. The selectivity of 1,4-NADH was demonstrated by the ratio of the change in absorbance to the initial absorbance. The initial reaction rate was defined by the following equation:

The initial reaction rate = (Nt - N0) / (m * t),

in which Nt and N0 meant the molar mass of generated NADH production in certain times, m represented the quality of photocatalyst involved in the reaction and t stand for the time of photocatalytic reaction. The TOF based on the reduction reaction center Rh-complex was defined by the following equation:

TOF = (Ct - C0) / (C * t),

in which Ct - C0 represented the change of NADH production in certain times, C represented Rh-complex of 0.2 mmol L-1 and t represented the time of photocatalytic reaction.

The test for the conversion of GALD to EG in PEM
The EG synthesis catalyzed by PEM was carried out in a 3.1 mL quartz test reactor with a reaction volume of 2 mL, where the reaction solution contained TEOA or glucose solution of 1000 mmol L-1, pH 8.0, PM-Rh+free ADH or PEMx (x=2, 5, 10, 20) photocatalyst of 1.5 g L-1, NAD+ of 10 mmol L-1 and GALD of 10 mmol L-1. A 100 W LED lamp was used as the light source, and the light was turned on for 60 min after dark treatment for 5 min. Then high-performance liquid chromatography (HPLC) was used to detect the synthetic production of EG (the standard curve of EG was seen in Supplementary Fig. 40).

The test for the conversion of methanol to EG in PEM coupled with multi-enzyme cascade
To achieve the synthesis of EG in the system, the reactor consisted of two parts (enzymatic reactor and PEM reactor). The enzymatic reactor contained methanol (50 mmol L-1), AOX (0.8 g L-1), CAT (0.6 g L-1), GALS (2 g L-1), MgSO4 (10 mmol L-1) and ThDP (1 mmol L-1). PEM reactor contained TEOA or glucose solution (1000 mmol L-1, pH 8.0), PEMx (x=2, 5, 10, 20) photocatalyst (1.0 g L-1), and NAD+ (2, 5, and 10 mmol L-1). Samples taken from the enzymatic reactor were added into the PEM reactor every three hours, and then PEM reactor was carried out for EG synthesis for four light and dark cycles. The illumination time of each cycle was 0.33 h, and the dark processing time was 2.67 h. The photocatalytic procedures and detection methods were the same as those described above.

The dual-channel reactor for sustainable EG synthesis
A dual-channel reactor was well-established for EG synthesis. The reactor consisted of five parts: upper fixed plate, protective gasket, quartz stone window, photo-enzyme catalytic diversion channel, multi-enzyme cascade diversion channel, and temperature control water channel cover plate. A 100 ml solution containing methanol (100 mmol L-1), MgSO4 (10 mmol L-1), ThDP (1 mmol L-1), TEOA solution (1000 mmol L-1, pH 8.0), and NAD+ (2 mmol L-1) was continuously pumped into the reactor via a peristaltic pump. The enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion channel and PEM filtrated to form a PEM-PAN membrane (12 mg) was put in the photo-enzyme catalytic diversion channel. The reactor was carried out for EG synthesis for ten light and dark cycles. The illumination time of each cycle was 1 h, and the dark processing time was three hours.

The initial synthesis rate of EG is calculated by the following equation:

The initial synthesis rate = nEG / (mPEM * t),

where nEG refers to the mole mass of generated EG under first one hour light illumination, mPEM refers to mass of PEM, and t refers to light illumination (1 h). The total turnover number (TTN) is calculated by the following equation:

TTN = nproduct / ncatalyst,

where nproduct refers to the molar mass of the production of EG and ncatalyst refers to the molar mass of ADH. The photonic quantum efficiency (AQY) in the system of EG production from methanol is calculated by the following equation:

AQY = (2 * m * NA * h * c) / (S * P * t * λ) * 100%,

where m represents the molar amount of the synthesis EG, NA represents the Avogadro constant, h is Planck constant, c is the light speed, S stands for irradiation area, P represents the intensity of the irradiation light, t is irradiation time, and λ stands for wavelength of the light34.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a novel photo-enzyme-membrane (PEM) catalytic system coupled with a multi-enzyme cascade for the light-driven synthesis of ethylene glycol (EG) from methanol. The PEM integrates a covalent organic polymer-based photo-membrane (PM) for efficient NADH regeneration and a silica-coated enzyme-membrane (EM) to protect enzymatic activity. Key achievements include a high NADH regeneration rate (7.9 mmol gPM⁻¹ h⁻¹), a 566% improvement in EG production via EM thickness optimization, and a sustainable EG synthesis rate of 0.25 mmol gPEM⁻¹ h⁻¹ under continuous methanol feeding. The work demonstrates significant innovation in coordinating electron/proton transfer and enzyme stabilization, offering a promising platform for C1-to-C2+ conversion. The data presented are sufficient, supporting the manuscript’s key findings. Therefore, I recommend acceptance of this manuscript after revision. Some points that may help authors to further enhance the accuracy, clarity, and readability of the paper are listed below.\n1) The exploration of glucose as an alternative electron donor (43.6% NADH yield) aligns with green chemistry principles, however, a lower yield was obtained compared to TEOA (89.5%). A short discussion about the economy is suggested.\n2) In Figure 2c, proton conductivity is applied as an indistinct method to explore the proton transfer in the PM systems, where the relationship between NADH regeneration and proton transfer should be discussed. Meanwhile, please provide the details of the experiments in the Supplementary Informationproton\n3) About the optimization, the authors get a high NADH regeneration rate (7.9 mmol gPM⁻¹ h⁻¹), and a 566% improvement in EG production via EM thickness optimization. However, in such a complex multi-enzyme cascade process for the conversion of methanol to EG, which step is the rate-determining step? The charge transfer or chemical reaction? Is there any direct relationship between the EM thickness optimization and the rate-determining step?\n4) The influence of light intensity on the reaction kinetics should be investigated, which may help to identify the rate-determining step. When light intensity does not affect rate, factors such as surface reactions, mass transfer, low reactant concentration, and saturated active sites may control reaction kinetics. On the contrary, when light intensity affects reaction rate, light absorption or other factors may control reaction kinetics.\n5) Correspondingly, for Table S1, light power density density needs to be provided for comparison.\n6) The photonic quantum efficiency for the optimized system needs to be provided.\n7) In Figure S16, the definition of loading amount should be provided.", "reviewer_2": "The manuscript reported a multi-enzyme cascade system for ethylene glycol synthesis, among which membrane mediates energy transfer and mass conversion. However, the work is more likely a simple combination of reported photo-enzyme-membrane design experiences for NADH regeneration and NADH-dependent enzymatic catalysis, as well as a multi-enzyme cascade path for EG synthesis. The current study suffers from shortcomings in novelty and mechanistic insight, and the evidence was not convincing for supporting the conclusion. I would suggest a rejection by Nature Communications as following concerns:\n1. Compared to other synthetic methods, what are the advantages of this complex cascade system for ethylene glycol production? The EG production performance compared to existing systems is also absent?\n2. The authors claim the highlights for synergistic intensification of electron transfer and proton transfer for NADH regeneration. However, the so-called photo-membrane design based on m-TFPT, m-Bpy, and m-PaHSO3 and corresponding characterizations could not support the above ideas.\n-About electron transfer:\nFig. 2d shows that the electrons can be excited from m-Bpy (mainly contribution to HOMO) and transferred to m-TFPT (mainly contribution to LUMO). This photogenerated electron flow direction is unfavorable to the formation of the Rh (I) transition state and subsequent Rh–H species in PM-Rh catalysts due to the coordination of bpy unite to Cp*Rh.\n-About proton transfer:\nThe authors mention that the sulfonic acid moiety in PM-BS12 and PM-BS 21 facilitates proton transfer by enriching protons. However, the proton conductivity tests in Fig. 2c are not sufficient to evaluate the real proton transfer performance of PM samples in aqueous environment. Moreover, it is also questionable whether the electronegativity of sulfonic acid groups makes it easier to enrich hydrated cations or protons. Besides, the imine bonds in PM are prone to protonation. How to distinguish its contribution to proton enrichment from sulfonic acid groups?\n-About NADH regeneration performance:\nAs depicted in Supplementary Fig. 12, within the first 6 min, there is no difference in the initial NADH regeneration rate between PM-BS12 and PM-BS21. At the 8 min, the NADH yield of PM-BS21 is slightly higher (~ 84%) than that of PM-BS12 (~ 78%). At 12 minutes, all samples exhibit similar NADH yields (~80%). Overall, the differences in the NADH regeneration performance among the PM samples are minimal. The regulation of electron and proton migration processes for the optimization of NADH regeneration performance is not convincing. Besides, the detailed reaction time corresponding to initial NADH regeneration performance in Fig. 2f is not clarified.\n-More importantly, in cascade photoenzymatic catalysis for EG synthesis, the applied samples were PM-Rh. However, the corresponding NADH regeneration performance evaluation and comparison are lacking. Meanwhile, the selectivity of enzymatic active 1,4-NADH is also absent.\n3. In the demonstration of electron transfer, the data from transient absorption (Fig. 2b) and time-resolved photoluminescence (Supplementary Fig. 9) are contradictory. Moreover, the TAS, TRPL, and EIS test and fitting results should be reported with error bars and the authors may find that current data are within experimental error. The authors are expected to provide a proper explanation of their data rather than trying to fit the data to what they want. Moreover, the TAS and TRPL do not seem to match their corresponding illustrations.\n4. It seems that the coupled system comprising a multi-enzyme cascade and a PEM cannot complete the synthesis of ethylene glycol. In theory, the proton exchange membrane only allows protons to pass through. If so, how can the products in the enzymatic reactor cross the membrane to participate in photo-enzymatic reactions in the PEM reactor?\n5. Could the DMPO diffuse through the EM to the PM as hole reagents (e.g., TEOA, glucose)? If it is possible, Fig. 3c just illustrates the total amount of hydroxyl radicals in PEM system, but cannot explain the proportions of hydroxyl radicals in photo-membrane (PM) or enzyme-membrane (EM).\n6. The author claims that “the presence of the EM helped preserve the secondary structure of enzyme under light illumination”. As the SiO₂ component in EM is almost optically transparent in the visible light absorption range of PM samples (Supplementary Fig. 8), it is concerned about some contradiction with above conclusion.\n7. Analyzing proton enrichment in a buffer system seems unreasonable. Can the proton enrichment performance of PM catalysts be analyzed without considering the proton sources?\n8. Rather than analyzing only the optimal samples (PM-BS21), the functional groups and pore structures of PM-BS12, 21, and 30 should all be illustrated.\n9. It is unclear how did authors obtain the energy band structures of PM samples (Supplementary Fig. 8).\n10. Have the authors done repeated experiments for the enzymatic reaction, the photocatalytic reaction, and the coupled EG formation reaction? All the corresponding result curves should be reported with error bars.\n11. Analysis of the CD (Fig. 3d) and FITR spectra (Supplementary Fig. 23) data is not informative.", "reviewer_3": "Jiang et al. explore a photo-enzyme-membrane (PEM) coupled with three enzyme cascade to produce C2 chemical from methanol.\n1. The elemental mapping images for PM-BS30 and PM-BS12 should be supplemented to exhibit the proportion variation between element N and S.\n2. For XPS, imine linkage (n1), bipyridyl group (n2), and triazine group (n3) all are C=N bond, Would the binding energy have such differences and where are the C-N =and N-H bond?\n3. The chemical structures are of mistakes. (-C=N-)\n4. It is not necessarily reactive oxygen species that affect enzyme activity. It could also be strong oxidizing holes or free radicals. Please design experiments or characterizations to verify this. In EPR, •OH were monitored, but why h+ and •O2- not monitored?\n5. How does Niquist evaluate proton conductance ability?\n6. In Fig 3d, why are the secondary structures of ADH analyzed under dark conditions different? And is this enzyme without an α-helix structure?\n7. The author used three enzyme to convert methanol to intermediate and then used photo-enzyme catalysis to obtain C2 product, what about the economy and compatibility of this synthetic route? Can it be scaled up for synthesis?"}, "author_response": "Detailed Response to the Reviewers’ Comments\nReviewer: 1\nComments:\nThis manuscript presents a novel photo-enzyme-membrane (PEM) catalytic system coupled with a\nmulti-enzyme cascade for the light-driven synthesis of ethylene glycol (EG) from methanol. The PEM\nintegrates a covalent organic polymer-based photo-membrane (PM) for efficient NADH regeneration\nand a silica-coated enzyme-membrane (EM) to protect enzymatic activity. Key achievements include a\nhigh NADH regeneration rate (7.9 mmol g -1 h-1), a 566% improvement in EG\nproduction via EM PM\nthickness optimization, and a sustainable EG synthesis rate of 0.25 mmol g -1 h-1 under continuous\nPEM\nmethanol feeding. The work demonstrates significant innovation in coordinating electron/proton\ntransfer and enzyme stabilization, offering a promising platform for C to C conversion. The data\n1 2+\npresented are sufficient, supporting the manuscript’s key findings. Therefore, I recommend acceptance\nof this manuscript after revision. Some points that may help authors to further enhance the accuracy,\nclarity, and readability of the paper are listed below.\nAnswer:\nWe sincerely appreciate the reviewer’s positive remarks and valuable guidance.\nQuestion 1:\nThe exploration of glucose as an alternative electron donor (43.6% NADH yield) aligns with green\nchemistry principles, however, a lower yield was obtained compared to TEOA (89.5%). A short\ndiscussion about the economy is suggested.\nAnswer:\nTEOA, a commonly used electron donor, yields low-value oxidation products. Glucose derived from\nthe degradation of biomass can be oxidized to value-added products, such as gluconic acid and formic\nacid. Despite exhibiting a lower NADH regeneration yield relative to TEOA, glucose serves as a\npromising electron donor due to its sustainable origin, economic viability, and value-added chemical\nproduction.\nThe corresponding information could be found in the Revised Manuscript (page 7, line 5-7).\nMoreover, the NADH regeneration yield could be improved by optimizing the glucose oxidation\nreaction via heterojunction engineering or co-catalyst modification. 1) Designing heterojunction\nphotocatalyst can improve the separation efficiency of hole and electron during photocatalysis, which\nensure efficient oxidation of glucose. 2) Loading co-catalysts such as Au nanoparticles onto\nsemiconducting materials can catalyze the selective oxidation of glucose (Nat. Commun., 2021, 12,\n3375). The co-catalyst eliminates the side reaction of glucose oxidation caused by strong oxidants such\nas •OH, enabling the efficient production of gluconic acid or formic acid (Chem. Soc. Rev., 2020, 49,\n6198).\nTherefore, glucose serve as a sustainable electron donor for NADH regeneration.\nQuestion 2:\nIn Figure 2c, proton conductivity is applied as an indistinct method to explore the proton transfer in\nthe PM systems, where the relationship between NADH regeneration and proton transfer should be\ndiscussed. Meanwhile, please provide the details of the experiments in the Supplementary Information.\nAnswer:\nThe process of NADH regeneration involves the transfer of two electrons and one proton, where\nproton transfer determines the binding efficiency of NAD+ and proton. Slow proton transfer would result\nin the formation of NAD dimers caused by the binding of the NAD radicals, which ultimately decrease\nthe selectivity of 1,4-NADH.\nThe corresponding information could be found in the Revised Manuscript (page 6, line 27-30).\nThe details of the proton conductivity experiment are provided in the Revised Supplementary\nInformation (page 2, line 42-48). “The proton conductivities of PM were measured using a two-\nelectrode AC impedance spectroscopy technique. The perturbation voltage was 15 mV, and the\nfrequency was set at a range of 1×106-1 Hz. The sample (2 mm×10 mm) was placed between Pt\nelectrodes (with a spacing of 0.8 mm). Then, the electrodes were put in a constant temperature and\nhumidity chamber to test the proton conductivity. The proton conductivities (σ, mS cm-1) of PM were\ncalculated by the equation of σ=l/(AR), where l (cm), A (cm2), and R (Ω) represented the PM thickness,\ncontact area, and resistance, respectively.”\nQuestion 3:\nAbout the optimization, the authors get a high NADH regeneration rate (7.9 mmol gPM⁻¹ h⁻¹), and a\n566% improvement in EG production via EM thickness optimization. However, in such a complex\nmulti-enzyme cascade process for the conversion of methanol to EG, which step is the rate-determining\nstep? The charge transfer or chemical reaction? Is there any direct relationship between the EM\nthickness optimization and the rate-determining step?\nAnswer:\nIn the complex system for the conversion of methanol to ethylene glycol (EG), multi-enzyme cascade\nand PEM proceed in a step-by-step manner. Multi-enzyme cascade composed of alcohol oxidase,\ncatalase, and glycolaldehyde synthase enables the accumulation of glycolaldehyde (GALD). As shown\nin Supplementary Fig. 32, the ratio of alcohol oxidase and catalase was regulated to improve the\nproduction of formaldehyde from methanol. However, modulating the Mg2+/ThDP ratio did not\nsignificantly enhance GALD production from formaldehyde, and the reaction system still demanded an\nexcessively high amount of glycolaldehyde synthase. Therefore, the conversion of formaldehyde to\nGALD is regarded as rate-determining step due to the low catalytic activity of glycolaldehyde synthase.\nThe production of GALD can be enhanced by improving the activity of glycolaldehyde synthase through\nenzyme modification.\nPEM enables the conversion of GALD to EG through the coupling of alcohol dehydrogenase (ADH)\nin enzyme-membrane layer (EM) and photocatalytic NADH regeneration system in photo-membrane\nlayer (PM). Reactive oxygen species (ROS) generated during photocatalytic process attack the active\nsite of ADH, leading to enzyme deactivation. In addition, the regeneration and transfer of NADH would\nalso restrict the performance of PEM. In our study, EM is designed to enhance NADH transfer and\nprotect ADH from ROS, while PM is designed to facilitate NADH regeneration by coordinating electron\nand proton transfer process. Controlling the thickness of EM is crucial: an optimal thickness preserves\nthe secondary structure of ADH, while excessive thickness impedes molecular transfer. As shown in\nSupplementary Fig. 30, EG production is improved with the increase in the thickness of EM. Therefore,\nthe compatibility between PM and EM is the rate-determining step for the conversion of GALD to EG.\nAlthough thickness of EM provides a better protection of enzyme, but substrate and NAD+/NADH\nmolecule transfer resistance caused by the excessive thickness of EM become rate-determining step\nreducing the EG production.\nQuestion 4:\nThe influence of light intensity on the reaction kinetics should be investigated, which may help to\nidentify the rate-determining step. When light intensity does not affect rate, factors such as surface\nreactions, mass transfer, low reactant concentration, and saturated active sites may control reaction\nkinetics. On the contrary, when light intensity affects reaction rate, light absorption or other factors may\ncontrol reaction kinetics.\nAnswer:\nAs shown in Fig. R1 (Supplementary Fig. 14), the NADH regeneration catalyzed by PM is\nperformed under different light densities. With the increase of light intensity from 100 to 200 mW/cm2,\nthe NADH regeneration yield is gradually improved. This suggests that under light intensities exceeding\n200 mW/cm2, factors other than light become the rate-determining step in the NADH regeneration\nprocess. With the further increase of light intensity, the NADH yield is only slightly changed. This\nsuggests that under light intensities exceeding 200 mW/cm2, factors other than light become the rate-\ndetermining step in the NADH regeneration process. During photocatalytic NADH regeneration, Rh-\ncomplex captured two electrons and one proton and then transfer them to NAD+ for the production of\nNADH. Since both the Rh-complex and the reactant are in sufficient concentration, the electron and\nproton transfer may be the rate-determining step when light intensity is above 200 mW/cm2.\nFig. R1 (Supplementary Fig. 14). The photocatalytic NADH regeneration yield catalyzed by a) PM-\nBS12, b) PM-BS21, and c) PM-BS30 under different light densities.\nThe corresponding information could be found in the Revised Manuscript (page 6, line 18-27) and\nRevised Supplementary Information (page 18).\nQuestion 5:\nCorrespondingly, for Table S1, light power density needs to be provided for comparison.\nAnswer:\nUpon the reviewer’s request, light power density was added in Table R1 (Supplementary Table 1).\nTable R1 (Supplementary Table 1) Summary of NADH Regeneration.\nLight\npower Electron\nPhotocatalyst Light source Yield of NADH Ref.\ndensity donor\n(mW/cm2)\nPM-BS21 420 nm LED 300 TEOA 89.5%, 10 min This work\nPM-BS21 420 nm LED 300 Glucose 44.0%, 30min This work\nT-COF-2 300W Xe lamp - TEOA 74.0%, 10 min 1\nNKCOF-113 300W Xe lamp - TEOA ~60.0%, 30 min 2\nMIL-125-NH 450 nm LED - TEOA 66.4%, 60 min 3\nTP-COFs/Ti C T 300W Xe lamp 153.3 TEOA 95.0%, 30 min 4\n3 2 x\nDBTS-CMP1 410 nm LED - TEOA 84.0%, 45 min 5\nURh 420 nm LED 50-400 TEOA 78.9%, 16 min 6\nCOF-V2-Rh 410 nm LED - TEOA 85.4%, 40 min 7\nReferences\nconversion for efficient photoenzymatic catalysis. J. Am. Chem. Soc. 142, 5958-5963 (2020).\nreduction of CO . Angew. Chem. Int. Ed. 61, e202200261 (2022).\n2\n2\nphotocatalytic NADH regeneration and gas-liquid-solid three-phase enzymatic CO reduction. Angew.\n2\nreduction enabled by a lawn-like TP-COFs/Ti C T (mxene) photocatalyst. ACS Sustain. Chem. Eng.\n3 2 x\n12, 6881-6893 (2024).\nlight-driven nicotinamide adenine dinucleotide regeneration and photobiocatalytic formaldehyde\nreduction. ACS Catal. 10, 12976-12986 (2020).\nas an electron buffer tank for nicotinamide regeneration. ACS Catal. 10, 2894-2905 (2020).\n2\nmatching of linkage and linker in covalent organic frameworks. J. Am. Chem. Soc. 146, 586-598 (2024).\nQuestion 6:\nThe photonic quantum efficiency for the optimized system needs to be provided.\nAnswer:\nUpon the reviewer’s request, the photonic quantum efficiency in the system of EG production from\nmethanol is calculated to be 0.30%. The quantum efficiency is calculated by the equation of\n(2*m*N *h*c)/(S*P*t*λ)*100%, where m represents the molar amount of the synthesis EG, N\nA A\nrepresents the Avogadro constant, h is Planck constant, c is the light speed, S stands for irradiation area,\nP represents the intensity of the irradiation light, t is irradiation time, and λ stands for wavelength of the\nlight.\nThe corresponding information could be found in the Revised Manuscript (page 14, line 13-18).\nQuestion 7:\nIn Figure S16, the definition of loading amount should be provided.\nAnswer:\nUpon the reviewer’s request, the loading amount was revised to loading efficiency, and Fig. R2\n(Supplementary Fig. 21) was revised. The definition of loading efficiency can be seen in the Revised\nManuscript (page 13, line 6-9). The loading efficiency (%) of ADH was defined by the equation of\n(m -m)/m *100%, where m (mg) was initial dosage of ADH, m (mg) was the dosage of free ADH in\n0 0 0\nthe supernatant. The loading amount actually refers to the weight of ADH (mg) loaded onto the one\nmilligram carrier (PM-BS21-Rh).\n100\n)\n%\n(\ny 80\nc\nn\ne\nc i 60\ni\nf\nf\ne\ng 40\nn\ni\nd\na\n20\no\nL\n0\nx=2 x=5 x=10 x=20\nPEM\nx\nFig. R2 (Supplementary Fig. 21). The loading efficiency of ADH in PEM (x=2, 5, 10, and 20).\nx\nThe corresponding information could be found in the Revised Supplementary Information (page\n25).\nReviewer: 2\nComments:\nThe manuscript reported a multi-enzyme cascade system for ethylene glycol synthesis, among which\nmembrane mediates energy transfer and mass conversion. However, the work is more likely a simple\ncombination of reported photo-enzyme-membrane design experiences for NADH regeneration and\nNADH-dependent enzymatic catalysis, as well as a multi-enzyme cascade path for EG synthesis. The\ncurrent study suffers from shortcomings in novelty and mechanistic insight, and the evidence was not\nconvincing for supporting the conclusion. I would suggest a rejection by Nature Communications as\nfollowing concerns.\nAnswer:\nThank the reviewer for the remarks and valuable guidance. We regret that the innovative aspects of\nour study are not clearly articulated in the original manuscript, leading to the impression that our work\nis a simple combination of existing methods.\nUpon your request, we would like to further highlight our key contributions. In this study, we report\na photo-enzyme-membrane (PEM) catalytic system coupled with multi-enzyme cascade for ethylene\nglycol (EG) synthesis, among which membrane mediates energy transfer and mass conversion. We first\nsimultaneously improve energy supply efficiency and enzyme stability in the system through the design\nand construction of photo-enzyme-membrane (PEM), where PM solves efficient energy supply and EM\nensures stable enzymatic process. The system is not a simple combination of reported photo-enzyme-\nmembrane design experiences for NADH regeneration and NADH-dependent enzymatic catalysis, but\na highly integrated system, where each component addresses one key issue within the system.\nOur study establishes a new system for the efficient and sustainable synthesis of C from C\n2+ 1\nchemicals. The system integrates the advantages of enzymatic catalysis and photocatalytic processes,\ncharacterized by clean energy sources, mild reaction conditions, and designable pathways, which also\ncan expand and guide multi-enzyme catalytic chemical synthesis involving hydrogenation process.\nUpon the reviewer’s remarks, we have meticulously revised and refined the manuscript to provide\nmore robust evidence and enhance overall quality. The supplementary data is provided below. For\ndetailed information, please refer to the Revised Manuscript and Revised Supplementary Information.\nQuestion 1:\nCompared to other synthetic methods, what are the advantages of this complex cascade system for\nethylene glycol production? The EG production performance compared to existing systems is also\nabsent?\nAnswer:\nThere are a series of strategies for the synthesis of C to C chemicals, such as thermal catalysis,\n1 2+\nelectrocatalysis, photocatalysis, and enzyme catalysis, etc. Enzyme catalysis features high efficiency\nand specificity, which can facilitate the directional synthesis of C to C chemicals. Most chemicals\n1 2+\ncan be synthesized by multi-enzyme pathway design with high product selectivity. Compared to thermal\ncatalysis, electrocatalysis, and photocatalysis, milder and safer reaction conditions and higher selectivity\nfor enzyme catalysis have unique advantages. However, cofactors such as NADH are used to supply\nhydrogen source for oxidoreductase. The development of multi-enzyme catalysis is limited by the cost\nof coenzyme. The coupling of cofactor regeneration system driven by clean energy (electro or photo\nenergy) and multi-enzyme catalysis can reduce the costs. In our study, the multi-enzyme catalysis\nachieves the accumulation of intermediate products, and the photo-enzyme catalysis comprising of\nphotocatalytic NADH regeneration and NADH-dependent enzymatic process converts intermediate\nproducts into final products. The multi-enzyme catalysis pathway ensures the directional synthesis of\nethylene glycol. The use of solar energy reduces the cost and ensures the high efficiency and\nsustainability of the system.\nActually, our study proposed a system for EG synthesis through the coupling of PEM and multi-\nenzyme cascade, aiming to enhance NADH regeneration in the system by coordinating the electron and\nproton transfers. Our study provides a case to improve hydrogeneration process in multi-enzyme\ncascade.\nUpon the reviewer’s request, the performance of EG production by existing catalytic systems is\nsummarized in Table R2 (Supplementary Table 2). The EG production of this work is moderate\ncompared to other systems.\nTable R2 (Supplementary Table 2). The performance of EG production by different catalytic\nsystems.\nEG production Production rate\nSystem Substrate Ref.\n(mmol L-1) (mmol g-1 h-1)\nPhoto-enzyme-\nMethanol 2.9 2.6 This work\nmembrane (PEM)\nMulti-enzyme catalysis Methanol 14.5 - 8\nElectro-enzyme\nCO 0.016 - 9\ncatalysis 2\nElectro-enzyme\nCO 0.15 - 9\ncatalysis 2\nMulti-enzyme catalysis Formaldehyde 6.6 - 10\nPhotothermal catalysis Methanol 0.216a 7.2 11\nPhotocatalysis Methanol 0.04 0.46 12\nPhotocatalysis Methanol 48 2.0 13\nPhotocatalysis Methanol 1.3a 11 14\nElectrocatalysis CO - 0.5b 15\n2\na refers the molar amount of the synthesis EG (mmol); b stands for the molar amount of EG synthesized\nper hour (mmol h-1).\nReferences\n1 2 4\nChem. Catalysis 2, 2675-2690 (2022).\n2\nnano-reactor. Adv. Sci. 11, e2407204 (2024).\nethylene glycol via glycolaldehyde. Green Chem. 24, 218-226 (2022).\nphotocatalytic C-C coupling of methanol to ethylene glycol with near-unity selectivity. Angew. Chem.\nInt. Ed. e202510993 (2025).\nethylene glycol. Angew. Chem. Int. Ed. 63, e202318236 (2024).\nH bond activation in methanol. J. Am. Chem. Soc. 147, 22509-22516 (2025).\nNat. Commun. 9, 1181 (2018).\n6, 585-595 (2023).\nQuestion 2:\nThe authors claim the highlights for synergistic intensification of electron transfer and proton transfer\nfor NADH regeneration. However, the so-called photo-membrane design based on m-TFPT, m-Bpy,\nand m-PaHSO and corresponding characterizations could not support the above ideas.\n3\n-About electron transfer:\nFig. 2d shows that the electrons can be excited from m-Bpy (mainly contribution to HOMO) and\ntransferred to m-TFPT (mainly contribution to LUMO). This photogenerated electron flow direction is\nunfavorable to the formation of the Rh (I) transition state and subsequent Rh-H species in PM-Rh\ncatalysts due to the coordination of Bpy unite to Cp*Rh.\nAnswer:\nIn PM, Rh-complex as the activity site is loaded to dipyridyl (Bpy) group. As shown in Fig. R3\n(Supplementary Fig. 20a), PM-BS30 exhibits the best NADH regeneration efficiency owing to the\nhigher loading amount of Rh-complex. Therefore, equal amounts of free Rh-complex are used to\neliminate the NADH regeneration yield differences caused by the amounts of Rh-complex. The electron\ntransfer in the skeleton of PM is enhanced by regulating the monomer type, where the Rh-complex can\neasily accept electrons for the further conversion of NAD+. Then, PM-Rh is used for the construction\nof PEM, where Rh-complex in PM would not deactivate alcohol dehydrogenase.\nAs for the reviewer's questions of “This photogenerated electron flow direction is unfavorable to the\nformation of the Rh (I) transition state and subsequent Rh-H species in PM-Rh catalysts due to the\ncoordination of Bpy unite to Cp*Rh”, PM-Rh is a multilayer polymer and its unit is composed of three\nbuilding blocks, TFPT (A), Bpy-Rh (B), and PaHSO (C). Actually, our PM-Rh provides an electron\n3\ntransfer acceleration zone (Fig. R4), where electron from B is transferred to A in plane, then the B from\nadjacent layer can easier obtain electron to realize NADH regeneration.\nPM-BS21-Rh\n% ) 40 PM-BS30-Rh\n(\nH\nD30\nA\nN\nf20\no\nd\nl\ne10\ni\nY\n0\n0 4 8 12 16\nTime (min)\nFig. R3 (Supplementary Fig. 20a). The NADH regeneration yield catalyzed by PM-BS12,\nPM-BS21, and PM-BS30.\nFig. R4. The schematic of “electron transfer acceleration zone” in multilayer PM-Rh.\nThe corresponding information could be found in the Revised Supplementary Information (page\n24).\n-About proton transfer:\nThe authors mention that the sulfonic acid moiety in PM-BS12 and PM-BS21 facilitates proton\ntransfer by enriching protons. However, the proton conductivity tests in Fig. 2c are not sufficient to\nevaluate the real proton transfer performance of PM samples in aqueous environment. Moreover, it is\nalso questionable whether the electronegativity of sulfonic acid groups makes it easier to enrich hydrated\ncations or protons. Besides, the imine bonds in PM are prone to protonation. How to distinguish its\ncontribution to proton enrichment from sulfonic acid groups?\nAnswer:\nIn our study, the proton conductivity was tested at ~100% humidity to simulate the aqueous\nenvironment. Upon the reviewer’s request, we have conducted more experiments to confirm that our\ndesigned PM can enrich the protons. First, three types of PM were filtered to form membranes on the\npolyacrylonitrile (PAN) substrate (PM-PAN membrane, Fig. R5 (Supplementary Fig. 12)). Then, PM-\nPAN membrane is set at the diffusion cell, where 1:5000 (v/v) hydrochloric acid solution and aqueous\nsolution were separately added to both sides. As shown in Fig. R6 (Supplementary Fig. 13), the pH\nvalue of the aqueous solution is monitored over time. The pH value of PM-BS12-PAN changed slightly\ncompared to PM-BS21-PAN and PM-BS30-PAN, which demonstrated that PM-BS12-PAN can more\nefficiently adsorb protons.\nThe corresponding information could be found in the Revised Manuscript (page 6, line 8-15).\nAs reported (J. Am. Chem. Soc. 2025, 147, 12604; Nat. Mater. 2016, 15, 722), the presence of sulfonic\nacid can enhance proton transfer. In our study, the imine group in PM-BS12, PM-BS-21, and PM-BS30\nis the same, while the ratio of sulfonic acid to bipyridine was different. Therefore, the contribution to\nproton enrichment from imine group can be ignored.\nFig. R5 (Supplementary Fig. 12). The images of three types of PM-PAN membrane: a) PM-BS12-\nPAN, b) PM-BS21-PAN, and PM-BS30-PAN.\n6.8\nPM-BS12-PAN\nPM-BS21-PAN\n6.6\nPM-BS30-PAN\ne\nu6.4\nl\na\nv\nH6.2\np\n6.0\n5.8\n0 5 10 15 20 25 30\nTime (min)\nFig. R6 (Supplementary Fig. 13). The change of pH value by different PM-PAN membrane: PM-\nBS12-PAN, PM-BS21-PAN, and PM-BS30-PAN.\nThe corresponding information could be found in the Revised Supplementary Information (page\n16-17).\n-About NADH regeneration performance:\nAs depicted in Supplementary Fig. 12, within the first 6 min, there is no difference in the initial\nNADH regeneration rate between PM-BS12 and PM-BS21. At the 8 min, the NADH yield of PM-BS21\nis slightly higher (~ 84%) than that of PM-BS12 (~ 78%). At 12 minutes, all samples exhibit similar\nNADH yields (~80%). Overall, the differences in the NADH regeneration performance among the PM\nsamples are minimal. The regulation of electron and proton migration processes for the\noptimization of NADH regeneration performance is not convincing. Besides, the detailed reaction time\ncorresponding to initial NADH regeneration performance in Fig. 2f is not clarified.\nAnswer:\nAs shown in Fig. R7 (Fig. 2f), PM was re-performed for photocatalytic NADH regeneration.\nWithin the first 4 min, owing to the better proton transfer, PM-BS12 exhibits a higher NADH\nregeneration rate than PM-BS21 and PM-BS30 (Fig. R8a (Supplementary Fig. 15a)). However, at 4-8\nmin, the NADH regeneration rate by PM-BS21 is improved, benefiting from the coordination of electron\nand proton transfers (Fig. R8b (Supplementary Fig. 15b)). As for 12 min, all PM samples reach the\nbalance of photocatalytic NADH regeneration, where PM-BS21 exhibits the best NADH regeneration\nperformance (89.5%). Overall, rapid proton transfer contributes to the initial reaction rate (0-4 min)\nand selectivity of 1,4-NADH (Fig. R9 (Supplementary Fig. 16)). As the reaction proceeds, PM-BS21\nwith the best\ncoordination of electron and proton transfers exhibits the highest NADH regeneration yield, suggesting\nthat the regulation of electron and proton transfer is effective in optimizing NADH regeneration.\nThe corresponding information can be found in the Revised Manuscript (page 6, line 30-34 and\npage 7, line 1-4).\nPM-BS21\n) 80 PM-BS30\n%\n(\nH\nA\nN\n40\nf\no\nd\ne l 20\ni\nY\n0\n0 4 8 12 16\nTime (min)\nFig. R7 (Fig. 2f). The NADH regeneration yield catalyzed by PM-BS12, PM-BS21, and PM-BS30.\nFig. R8 (Supplementary Fig. 15). The initial reaction rate and TOF of NADH regeneration catalyzed\nby PM-BS12, PM-BS21, and PM-BS30 under a) 0-4 min and b) 4-8 min.\n100\n80\n)\n%\n(\ny 60\nt\ni\nv\ni\nt\nc 40\ne\nl\ne\nS\n20\nFig. R9 (Supplementary Fig. 16). The selectivity of 1,4-NADH catalyzed by PM-BS12,\nPM-BS21, and PM-BS30.\nThe corresponding information can be found in the Revised Supplementary Information (page 19-\n20).\n-More importantly, in cascade photo enzymatic catalysis for EG synthesis, the applied samples were\nPM-Rh. However, the corresponding NADH regeneration performance evaluation and comparison are\nlacking. Meanwhile, the selectivity of enzymatic active 1,4-NADH is also absent.\nAnswer:\nAs shown in Fig. R10a (Supplementary Fig. 20a), PM-Rh was used for NADH regeneration, where\nRh-complex as the active site for NADH regeneration is anchored to dipyridyl group. PM-BS30-Rh\nwith more anchored Rh-complex exhibited a higher NADH regeneration yield than PM-BS12-Rh and\nPM-BS21-Rh. Enzymatic method was used to detect the selectivity of 1,4-NADH (Fig. R10b\n(Supplementary Fig. 20b)). The absorbance of the reaction at 340 nm would decrease with the\nconsumption of NADH when appropriate amount of propanal and ADH are added. The selectivity of\n1,4-NADH is evaluated by calculating the ratio of the change in absorbance to the initial absorbance.\nThe corresponding information can be found in the Revised Manuscript (page 8, line 17-23).\nFig. R10 (Supplementary Fig. 20). The a) yield and b) selectivity of 1,4-NADH catalyzed by PM-\nBS12, PM-BS21, and PM-BS30.\nThe corresponding information can be found in the Revised Supplementary Information (page 24).\nQuestion 3:\nIn the demonstration of electron transfer, the data from transient absorption (Fig. 2b) and time-\nresolved photoluminescence (Supplementary Fig. 9) are contradictory. Moreover, the TAS, TRPL,\nand EIS test and fitting results should be reported with error bars and the authors may find that current data\nare within experimental error. The authors are expected to provide a proper explanation of their data\nrather than trying to fit the data to what they want. Moreover, the TAS and TRPL do not seem to match\ntheir corresponding illustrations.\nAnswer:\nTime-resolved photoluminescence (TR-PL) demonstrated the average lifetime of photogenerated\nelectrons. τ in transient absorption spectroscopy (TAS) represents the time of photogenerated electron-\n1\nhole recombination, and τ represents the relaxation time of non-electron-hole recombination. As shown\n2\nin Fig. R11 (Fig. 2b), PM-BS30 showed longer τ , suggesting that PM-BS30 with more dipyridyl\n2\ngroups is constructive for electron transfer. Upon to reviewer’s remark, Supplementary Fig. 9 was\nrevised to Fig. R12. PM-BS30 with more dipyridyl group showed longer average lifetime of\nphotogenerated electrons, which demonstrated that more electrons can be transferred to the active site\nfor NADH regeneration. As shown in reported literatures (Angew. Chem. Int. Ed., 2022, e202218688;\nJ. Am. Chem. Soc., 2025, 147, 9891; Nat. Commun., 2024, 15, 3930; Nat. Mater., 2024, 23, 519; Nat.\nChem., 2025, https://doi.org/10.1038/s41557-025-01868-y), TAS, TRPL, and EIS test have not error\nbars.\n0.2\nFitted PM-BS12 PM-BS12\nFitted PM-BS21 PM-BS21\n0.1 Fitted PM-BS30 PM-BS30\n)\nD\n0.0\nO\nm\n(\nA-0.1\nΔ\nτ =9.70 ps, τ =99.97 ps\n1 2\n-0.2 τ =12.22 ps, τ =199.88 ps\n1 2\nτ =15.12 ps, τ =710.72 ps\n1 2\n-0.3\n0 2000 4000 6000 8000\nDelay Time (ps)\nFig. R11 (Fig. 2b). The transient absorption spectra of PM-BS30, PM-BS21, and PM-BS12. The\nwavelength of excitation light is 400 nm.\n)\n.\nu\n. 1\na PM-BS12 (145 ps)\n( PM-BS21 (229 ps)\ny PM-BS30 (374 ps)\nt\ni\ns\nn\ne 0.1\nt\nn\nI\ne\nc\nn\ne\nc0.01\ns\ne\nr\no\nu\nl\nF\n2000 3000 4000\nTime (ps)\nFig. R12 (Supplementary Fig. 9). The fluorescence intensity of PM-BS30, PM-BS21, and PM-BS12\nupon excitation at 365 nm.\nThe corresponding information can be found in the Revised Supplementary Information (page 13).\nQuestion 4:\nIt seems that the coupled system comprising a multi-enzyme cascade and a PEM cannot complete the\nsynthesis of ethylene glycol. In theory, the proton exchange membrane only allows protons to pass\nthrough. If so, how can the products in the enzymatic reactor cross the membrane to participate in photo-\nenzymatic reactions in the PEM reactor?\nAnswer:\nWe recognize that the original schematic in Figure 4b may have introduced ambiguity. Accordingly,\nwe have revised the illustration to improve clarity. Fig. 4b and Supplementary Fig. 35 was revised to\nFig. R13 and Fig. R14 in the Revised Manuscript and Revised Supplementary Information.\nActually, there is no proton exchange membrane in our system. In the enzymatic reactor, methanol is\nconverted to glycolaldehyde catalyzed by the multi-enzyme cascade composed of AOX, CAT, and\nGALS. As-generated glycolaldehyde is then added to the PEM reactor for the production of ethylene\nglycol. Ethylene glycol can be continuously produced through the steady addition of glycolaldehyde.\nFig. R13 (Fig. 4b). Scheme of coupled system comprising a multi-enzyme cascade in the enzymatic\nreactor and a PEM in PEM reactor for the synthesis of EG.\nFig. R14 (Supplementary Fig. 35). Scheme of the system comprising AOX, CAT, GALS and free\nADH for the synthesis of EG, which is denoted as blank.\nThe corresponding information can be found in the Revised Manuscript (page 11, line 17) and\nRevised Supplementary Information (page 39).\nQuestion 5:\nCould the DMPO diffuse through the EM to the PM as hole reagents (e.g., TEOA, glucose)? If it is\npossible, Fig. 3c just illustrates the total amount of hydroxyl radicals in PEM system, but cannot explain\nthe proportions of hydroxyl radicals in photo-membrane (PM) or enzyme-membrane (EM).\nQuestion 6:\nThe author claims that “the presence of the EM helped preserve the secondary structure of enzyme\nunder light illumination”. As the SiO component in EM is almost optically transparent in the visible\n2\nlight absorption range of PM samples (Supplementary Fig. 8), it is concerned about some contradiction\nwith above conclusion.\nAnswer:\nEM is composed of ADH and silica, which cannot absorb light and convert solar energy to radicals.\nAlthough DMPO can diffuse through EM to PM as hole reagents, only the hydroxyl radical signals\ngenerated by PM can be detected by ESR test. Owing to the decrease of the intensity of hydroxyl radicals\nin the presence of EM, the secondary structure and activity of enzyme are well maintained (Fig. R15).\nAs pointed out by the reviewer, it is very difficult to explain the proportions of hydroxyl radicals in PM\nor EM. Our study aims to design EM to protect the secondary structure and maintain enzyme activity.\n“the presence of the EM helped preserve the secondary structure of enzyme under light illumination”\nwas revised to “the presence of the EM weakened the intensity of ROS, and confined enzyme within a\nstable space, thus ensuring the stable secondary structure of enzyme”. Although SiO component in EM\n2\nis almost optically transparent in the visible light range, the efficiency of light absorption is not constant\n(Fig. R16 (Supplementary Fig. 27)). EM reduces the light absorption intensity of PEM. Meanwhile,\nthe intensity of ROS is weakened as the increasing thickness of EM, where the silica layer can quench\nROS (J. Am. Chem. Soc., 2022, 144, 7320). Weakened intensity of ROS and confined space provided\nby EM jointly stabilize the secondary structure of the enzyme.\nThe corresponding information can be found in the Revised Manuscript (page 9, line 8-13 and page\n9, line 19-20).\nFig. R15. The attack of hydroxyl radicals on the enzyme in presence and absence of EM.\n0.35 PEM\n5\n0.30\nPEM\n10\n)\nu .0.25 PEM 20\n.\na\n(0.20\ny\nt\ni\ns0.15\nn\ne\nt0.10\nn 630 nm\nI\n0.05 625 nm\n602 nm\n0.00 600 nm\n400 500 600 700 800\nWavelength (nm)\nFig. R16 (Supplementary Fig. 27). The Visible Diffuse Reflectance Spectroscopy of PEM.\nThe corresponding information can be found in the Revised Supplementary Information (page 31).\nQuestion 7:\nAnalyzing proton enrichment in a buffer system seems unreasonable. Can the proton enrichment\nperformance of PM catalysts be analyzed without considering the proton sources?\nAnswer:\nCofactor and enzymes involved in our PEM coupled with multi-enzyme cascade for EG synthesis are\nsensitive to pH of the reaction solution. Buffer is necessary for enzymatic reaction and photocatalytic\nNADH regeneration. In our previous study (Angew. Chem. Int. Ed. 2025, 64, e202424995; Angew.\nChem. Int. Ed. 2023, 62, e202310238; ACS Catal. 2019, 9, 3913), weakly alkaline buffer is beneficial\nfor NADH regeneration and enzymatic reaction. Therefore, we analyze proton enrichment in a buffer\nsystem. Actually, our previous study (ACS Catal. 2020, 10, 2894) proved that the proton used for NADH\nregeneration is from H O. PM can adsorb proton in the reaction solution and provide a proton-enriched\n2\nregion to enhance proton transfer, thus facilitate NADH regeneration.\nIn addition, PM-PAN is prepared to evaluate the ability of proton absorption in the diffusion cell,\nwhere protons are provided by hydrochloric acid solution. PM-BS12-PAN exhibits the highest proton\nadsorption capability Fig. R6 (Supplementary Fig. 13).\n6.8\nPM-BS12-PAN\nPM-BS21-PAN\n6.6\nPM-BS30-PAN\ne\nu6.4\nl\na\nv\nH6.2\np\n6.0\n5.8\n0 5 10 15 20 25 30\nTime (min)\nFig. R6 (Supplementary Fig. 13). The change of pH value in the use of PM-PAN membrane (PM-\nBS12-PAN, PM-BS21-PAN, and PM-BS30-PAN).\nThe corresponding information can be found in the Revised Supplementary Information (page\n17).\nQuestion 8:\nRather than analyzing only the optimal samples (PM-BS21), the functional groups and pore structures\nof PM-BS12, 21, and 30 should all be illustrated.\nAnswer:\nUpon the reviewer’s guidance, elemental mapping images and the proportion variation of nitrogen\n(N) and sulfur (S) for PM-BS12, PM-BS21, and PM-BS30 are added (Fig. R17 (Supplementary Fig.\n3)). As shown in (Fig. R17 (Supplementary Fig. 3)), elemental mapping images of N and S element\nare observed in PM-BS12 and PM-BS21, while there are no S elemental signals in PM-BS30. Fig. R17\n(Supplementary Fig. 3b) exhibits the proportion variation between element N and S. The S content\nincreases nearly 100% in PM-BS12 compared to PM-BS21, which is consistent with the proportion\nbetween m-Bpy and m-PaHSO used for the synthesized PM.\n(Supplementary Fig. 1) in the Revised Supplementary Information. The absorption bands at 810\ncm-1, 629 cm-1 and 1685 cm-1 are assigned to the triazine group of m-TFPT, sulfonate group of m-\nPaHSO , and imine linkage. This confirms that PM is formed by the covalent linking of amino and\n3\naldehyde monomers.\nNitrogen adsorption-desorption isotherm and corresponding pore size distribution of the PM-BS12\nand PM-BS30 are shown in Fig. R19 (Supplementary Fig. 2). PM-BS12, PM-BS21, and PM-BS30\nexhibit pore sizes of 3.0 nm; 1.7 nm and 3.0 nm; and 3.0 nm and 5.3 nm, respectively. This pore size\ndistribution enables NAD+/NADH with a hydrodynamic diameter of ~1.7 nm to shuttle through the\npores.\nThe corresponding information can be found in the Revised Manuscript (page 5, line 4-13).\nFig. R17 (Supplementary Fig. 3). a) The elemental mapping images and b) the proportion variation of\nN and S for PM-BS12, PM-BS21, and PM-BS30.\nFig. R18 (Supplementary Fig. 1). FTIR spectra of m-TFPT, m-Bpy, m-PaHSO , PM-BS12, PM-BS21\n3\nand PM-BS30.\nFig. R19 (Supplementary Fig. 2). a, c, e) Nitrogen adsorption-desorption isotherm and b, d, f)\ncorresponding pore size distribution of PM-BS21, PM-BS21, and PM-BS30.\nThe corresponding information could be found in the Revised Supplementary Information (page\n5-7).\nQuestion 9:\nIt is unclear how did authors obtain the energy band structures of PM samples (Supplementary Fig.\n8).\nAnswer:\nWe deeply regret that the absence of certain experimental data inadvertently led to uncertainties in\nthe reviewers' understanding of the sample's band structure. As shown in Fig. R20 (Supplementary\nFig. 8) in the Revised Supplementary Information, UV-vis DRS and XPS valence band spectra are\nused to calculate the band structures of PM samples.\nFig. R20 (Supplementary Fig. 8). A) UV-vis diffuse reflectance spectra, (b) Tauc plot spectra and (c)\nXPS valance band spectra of PM. D) Schematic band structure of PM and the redox potential of NAD+\n(-0.32 V) under normalized hydrogen electrode (NHE) at pH 7.0.\nThe corresponding information could be found in the Revised Supplementary Information (page\n12).\nQuestion 10:\nHave the authors done repeated experiments for the enzymatic reaction, the photocatalytic reaction,\nand the coupled EG formation reaction? All the corresponding result curves should be reported with\nerror bars.\nAnswer:\nWe repeated the enzymatic reaction, the photocatalytic reaction, and the coupled reaction for EG\nproduction. Fig. 3e, 4c, and Supplementary Fig. 17-18, 30-34, and 36 were revised to Fig. R21-30.\n)\n1\n-L\nol\n4 50\nm Gluconic acid\nm NADH\n( 40 )\nd 3 %\nci (\na H\nc 30 D\nni\nA\no\nc 2 N\nu\ngl 20\no\nf\no f d\no n 1 10 i e l\nti Y\na\nr\nt\nn\ne 0\nn c 0\no 0 5 10 15 20 25 30 35\nC\nTime (min)\nFig. R21 (Supplementary Fig. 17). The yield of NADH regeneration and the concentration of gluconic\nacid when glucose is used as the electron donor.\n20.0k\n18.0k\n)16.0k\n.\nu\n.\na14.0k\n(\ny\nt12.0k\ni\ns\nn10.0k\ne\nt\nn 8.0k\nI\n6.0k\n4.0k\n2 4 6 8 10\nConcentration of gluconic acid (mmol L-1)\nFig. R22 (Supplementary Fig. 18). The standard curve of gluconic acid by HPLC.\n)1.4 Blank\n- 1 L PM-Rh + free ADH\n1.2 PEM\no l 2 1.0 mmol L-1\nm PEM\n1.0 5\nm PEM\n10\n(\n0.8 PEM\nn 20\no\nt\ni0.6\nc\nu\nd0.4\no\nr\np0.2\nG\nE0.0\n0 10 20 30 40 50 60\nTime (min)\nFig. R23 (Supplementary Fig. 30). The EG production from GALD with PM-Rh + ADH or PEM\nx\n(x=2, 5, 10, 20) under illumination for 60 min, where the test group withoutGALD is defined as the\nblank.\n1.2\n)\n1\n-L\n1.0\nl\no\nm\nm0.8\n(\nn\no0.6 503% increase\ni\nt\nc\nu\n0.4\nd\no\nr\np\n0.2\nG\nE0.0\nNon-integrated system Integrated PEM\nFig. R24 (Fig. 3e). The production of EG by non-integrated system and integrated PEM with TEOA as\nthe electron donor.\n1.2\n)\nPEM\n1 10\n-L\n1.0\nl\no\nm\nm0.8\n(\nn\no0.6\ni\nt\nc\nu\n0.4\nd\no\nr\np\n0.2\nG\nE0.0\nWith glucose With TEOA\nFig. R25 (Supplementary Fig. 31). The EG production from GALD catalyzed by PEM when TEOA\n10\nor glucose is used as the electron donor.\nReaction time\n10 8.9 mmol L-1 1 h\nF 2 h\no\nrm 3 h\na ld 8\ne\nh\ny\nd\ne\np 6\nro\nd\nu\nc\ntio 4\nn\n(m\nm\no 2\nl\nL ) -10 C . o 0 nce 0 n . t 2 ratio 0 n . 4 of AO 0 X .6 (g L 0 -)1 .8 1.0-0.2 0.0 Co 0 n . c 2 entra 0 ti . o 4 n o 0 f C .6 AT ( 0 g . L 8 -1)\nFig. R26 (Supplementary Fig. 32). The conversion of methanol to formaldehyde with different\nconcentration of AOX and CAT after 3 h incubation.\nFig. R27 (Supplementary Fig. 33). The conversion of formaldehyde to GALD with different\nconcentration of MgSO and ThDP after 3 h, 6 h, 12 h, and 24 h incubation.\n4\n5\n)\n1\n-L 1 g L-1 GALS\nl 2 g L-1 GALS 3.7 mmol L-1\no4\nm 3 g L-1 GALS\nm\n(3\nn\no\ni\nt\nc2\nu\nd\no\nr1\np\nD\nL\nA0\nG\n0 5 10 15 20 25\nTime (h)\nFig. R28 (Supplementary Fig. 34). The conversion of methanol to GALD with different concentration\nof GALS after 24 h incubation, where the concentration of AOX and CAT are 0.8 and 0.6 g L-1,\nrespectively.\n)\n- 1 L PEM coupled with multi-enzyme cascade\n4\nl\no Light on Light off\nm\nm\n3\n(\nn\no\nt\ni2\nc\nu\nd\no\nr1\np\nG\nE0\n0 1 2 3 4\nCycle number\nFig. R29 (Fig. 4c). The production of EG by the coupled system with TEOA as the electron donor.\n60\n)\nPEM coupled with multi-enzyme cascade\n1\n-L50 by using glucose as electron donor\no l Light on Light off\nm40\nμ\n(\nn30\no\ni\nt\nc\nu20\nd\no\nr10\np\nG\nE 0\n0 1 2 3 4\nCycle number\nFig. R30 (Supplementary Fig. 36). The EG production in PEM coupled with multi-enzyme cascade\nwith glucose as the electron donor.\nThe corresponding information can be found in the Revised Manuscript (page 10, line 1 and page\n11, line 17) and Revised Supplementary Information (page 21-22, 34-38, and 40).\nQuestion 11:\nAnalysis of the CD (Fig. 3d) and FITR spectra (Supplementary Fig. 23) data is not informative.\nAnswer:\nWe deeply regret that the description of “Moreover, as shown in Fig. 3d and Supplementary Fig. 23,\nthe presence of the EM helped preserve the secondary structure of enzyme under light illumination” in\nthe Original Manuscript is not informative. More comprehensive and clearer description of “As shown\nin Fig. 3d and Supplementary Fig. 29, we reanalyzed the changes in the secondary structure of the\nADH, where the secondary structure of the ADH remained almost unchanged under dark conditions. In\nPEM system after illumination, the ratio of α-helix of ADH was decreased which exhibited that ROS\n2\ngenerated by light had an adverse effect on the structure of enzymes. As the thickness of the EM\nincreased, the secondary structure of ADH was better protected. For the PEM system, the secondary\n20\nstructure of enzyme has barely change under before and after light illumination” was added in the\nRevised Manuscript.\nThe corresponding information can be found in the Revised Manuscript (page 9, line 13-19).\nReviewer: 3\nComments:\nJiang et al. explore a photo-enzyme-membrane (PEM) coupled with three enzyme cascades to\nproduce C chemical from methanol.\nThank the reviewer for the remarks and valuable guidance.\nQuestion 1:\nThe elemental mapping images for PM-BS30 and PM-BS12 should be supplemented to exhibit the\nproportion variation between element N and S.\nAnswer:\nWe added the elemental mapping images of N and S for PM (PM-BS12, PM-BS21, and PM-BS30).\nAs shown in Fig. R17a (Supplementary Fig. 3a), N and S elemental signals are observed for PM-BS12\nand PM-BS21, while there is no S elemental for PM-BS30. Fig. R17b (Supplementary Fig. 3b) exhibits\nthe proportion variation between N and S. The S content increases nearly 100% in PM-BS12 compared\nto PM-BS21, which is consistent with the proportion of between Bipyridine and sulfonic acid\nmonomers used for the synthesis of PM.\nFig. R17 (Supplementary Fig. 3). a) The elemental mapping images and b) the proportion variation of\nN and S for PM-BS12, PM-BS21, and PM-BS30.\nThe corresponding information could be found in the Revised Supplementary Information (page\n7).\nQuestion 2:\nFor XPS, imine linkage (n1), bipyridyl group (n2), and triazine group (n3) all are C=N bond, Would\nthe binding energy have such differences and where are the C-N= and N-H bond?\nAnswer:\nWe are sorry for the mistake in the analysis of XPS data. As was reported (J. Mater. Chem. A, 2025,\n13, 1407; Angew. Chem. Int. Ed., 2025, 64, e202415202; ACS Catal., 2023, 13, 5135), the binding\nenergy of bipyridyl group is higher than triazine group. In our proposed PM, N-H bond is assigned to\nthe free amino group of m-Bpy and m-PaHSO , and there is no C-N= bond in PM. As shown in the N\n3\n1s spectrum of XPS (Supplementary Fig. 4), the peaks of triazine group/imine linkage (n ), bipyridyl\n1\ngroup (n ), and N-H bond (n ) are detected. The proportion of n is consistent, suggesting that different\n2 3 3\nPM has the same amounts of free amino groups. The proportion of n is approximately 1:2:3, indicating\n2\nthat the proportion of bipyridyl group in all PM is consistent with the proportion of bipyridyl monomer\nused for the synthesis of PM.\nThe corresponding information can be found in the Revised Manuscript (page 5, line 13-18).\nQuestion 3:\nThe chemical structures are of mistakes. (-C=N-)\nAnswer:\nAs shown in Fig. R31-35, we have corrected the chemical structures of Fig. 2a, Fig. 2e, and\nSupplementary Fig. 5-7 in the Revised Manuscript and Revised Supplementary Information.\nFig. R31 (Fig. 2a). Schematic preparation of PM.\nFig. R32 (Fig. 2e). NADH regeneration catalyzed by PM.\nFig. R33 (Supplementary Fig. 5). The possible structural units of PM-BS30.\nFig. R34 (Supplementary Fig. 6). The possible structural units of PM-BS21.\nFig. R35 (Supplementary Fig. 7). The possible structural units of PM-BS12.\nThe corresponding information can be found in the Revised Manuscript (page 7, line 13) and\nRevised Supplementary Information (page 9-11).\nQuestion 4:\nIt is not necessarily reactive oxygen species that affect enzyme activity. It could also be strong\noxidizing holes or free radicals. Please design experiments or characterizations to verify this. In EPR,\n•OH were monitored, but why h+ and •O - not monitored?\nAs shown in Supplementary Fig. 22a in the Original Supplementary Information, h+, •OH, and •O -\n2\nare detected by ESR. In this study, TEOA is used as the electron donor to capture h+, eliminating the\ninfluence of h+ on the enzymes. The •OH exhibits a significantly stronger oxidizing capability than •O -,\n2\nmaking it more detrimental to enzymes. •O - originated from the reduction of O , and our experiment\n2 2\nfor EG synthesis is carried out in a sealed reactor. Therefore, •O - generated in photocatalytic reaction\n2\nis comparatively limited. Only the analysis of •OH is shown in Original Manuscript and Original\nSupplementary Information. Upon the reviewer’s remarks, the signal intensity of •O - is also detected\n2\nby ESR. As shown in Fig. R36 (Fig. 3c) and Fig. R37 (Supplementary Fig. 28c), the intensity of •O -\n2\nobserved in ESR decreased as the thickness of EM increased, which also mitigated the adverse effects\nof •O - on enzyme activity.\n2\n4 4\n•OH\n•O -\n2\n1 1 ) 3 31 2 )\n0 0\n1 1\n× ×\n3\n(\nm 2 2 3\n(\nm\nm m\ns / s /\nn n\np i 1 1 p i\nS S\n0 0\nx=2 x=5 x=10 x=20\nPEM (x=2, 5 , 10, and 20)\nx\nFig. R36 (Fig. 3c). The intensity of •O - signals generated by PEM (x=2, 5, 10, 20) after 5 min of\n2 x\nillumination.\nPEM\nx\nx=20\n)\n.\nu\n. a\n(\ny x=10\nt\ni\ns\nn\ne x=5\nt\nn\nI\nx=2\n3460 3480 3500 3520 3540 3560\nField (G)\nFig. R37 (Supplementary Fig. 28c). DMPO-•O - signals of PEM (x=2, 5, 10, 20) after 5 min of\n2 x\nillumination.\nThe corresponding information can be found in the Revised Manuscript (page 10, line 1) and\nRevised Supplementary Information (page 32).\nQuestion 5:\nHow does Nyquist evaluate proton conductance ability?\nAnswer:\nThe Nyquist plot (or impedance spectroscopy) is a common electrochemical technique used to\nevaluate the proton conductance ability of materials (Nat. Energy 2017, 2, 877; Nat. Mater. 2016, 15,\n722). A Nyquist plot displays the imaginary impedance (Z'') vs. real impedance (Z') over a range of\nfrequencies. Our PM samples are pressed into a thin film, and then the AC impedance was tested under\ncertain temperature and humidity conditions. The proton conductivity (σ, mS cm-1) of PM is calculated\nby the equation of σ=l/(AR), where l (cm), A (cm2), and R (Ω) represents the PM film thickness,\ncontact area, and resistance (from the Nyquist plot), respectively.\nThe corresponding information can be found in the Revised Supplementary Information (page 2,\nline 42-48).\nQuestion 6:\nIn Fig 3d, why are the secondary structures of ADH analyzed under dark conditions different? And is\nthis enzyme without an α-helix structure?\nAnswer:\nWe sincerely apologize for the confusion caused by the inaccurate description. As shown in Fig. R38\n(Fig. 3d) and Fig. R39 (Supplementary Fig. 29), we reanalyzed the changes in the secondary structure\nof the ADH. The secondary structure of ADH is almost unchanged under dark condition. In PEM\n2\nsystem after illumination, the ratio of α-helix of ADH gets decreased, suggesting that ROS generated\nby light has an adverse effect on the structure of enzymes. As the thickness of the EM increased, the\nsecondary structure of ADH is better protected. For the PEM system, the secondary structure of\n20\nenzyme is almost unchanged before and after light illumination.\nThe corresponding information can be found in the Revised Manuscript (page 9, line 14-19).\nβ-turn α-helix\nRandom coil β-sheet\n%\n)100\n(\nt\nn\ne 75\nt\nn\no\nc\ne 50\nr\nu\nt\nc\nu\nr 25\nt\nS\n0\nx=2 x=5 x=10 x=20\nPEM (x=2, 5, 10, and 20)\nx\n0 1 2 3 4 5 6 7 8 9 10 11 12\nFig. R38 (Fig. 3d). The secondary structure of ADH in PEM incubated under dark or light conditions\nx\n(with the left column representing PEM incubated under dark condition and the right column\nx\nrepresenting PEM incubated under light condition).\nx\nFig. R39 (Supplementary Fig. 29). Second derivative of the FITR spectra of a) PEM , b) PEM ,\n2 5\nPEM , and d) PEM before and after light illumination.\n10 20\nThe corresponding information can be found in the Revised Manuscript (page 10, line 1) and\nRevised Supplementary Information (page 33).\nQuestion 7:\nThe author used three enzymes to convert methanol to intermediate and then used photo-enzyme\ncatalysis to obtain C2 product, what about the economy and compatibility of this synthetic route? Can\nit be scaled up for synthesis?\nAnswer:\nMost C chemicals can be synthesized via multi-enzyme cascade design with minimal by-product\n2+\nformation. However, cofactors such as NADH are used to supply hydrogen source for oxidoreductase.\nThe development of multi-enzyme cascade is limited by the cost of cofactors. In this study, the multi-\nenzyme cascade achieves the accumulation of intermediate products, and the photo-enzyme catalytic\nsystem composed of photocatalytic NADH regeneration and NADH-dependent enzymatic process\nconverts intermediate products into EG. We initially attempted to achieve photocatalytic NADH\nregeneration using glucose as an electron donor, leveraging the theoretical 100% conversion of biomass-\nderived glucose powered by solar energy to enhance the system's economic viability. In our proposed\nsystem, PEM and multi-enzyme catalysis are spatially separated and do not affect each other. PEM can\nimmediately convert the intermediate products accumulated by multi-enzyme cascade into C products.\n2+\nAs shown in Fig. R40, a tube-in-tube reactor designed by coupling of spatial partitions will be used\nto scale up the synthesis of chemicals in future work, where the inner tube is filled with the multi-\nenzyme cascade catalyst and the outer tube is filled with the PEM catalyst. The PE membrane between\nthe inner tube and the outer tube is used for material exchange. The PE membrane needs to ensure the\nshuttle of intermediate products and block the migration of catalyst particles. The reactor can realize\nefficient and continuous production of C to C chemicals.\n1 2 +\nFig. R40. A tube-in-tube reactor for the synthesis of EG.\nA list of major changes\nIn the Revised Manuscript:\nPage 1, line 31\n“0.25 mmol g -1 h-1” was revised as “2.6 mmol g -1 h-1”.\nPEM PEM\nPage 3, line 13\n“7.9 mmol g -1 h-1” was revised as “5.6 mmol g -1 h-1”.\nPM PM\nPage 3, line 18\n“566%” was revised as “503%”.\nPage 3, line 22\n“an EG production of 3.0 mmol L-1 with the synthesis rate of 0.25 mmol g -1 h-1 was obtained…” was\nPEM\nrevised as “an EG production of 2.9 mmol L-1 with the synthesis rate of 2.6 mmol g -1 h-1 was\nPEM\nobtained…”.\nPage 4, line 23\n“Transmission electron microscopy (TEM) and elemental mapping images showed that all PM were of\ncapsule structures with a wall thickness of 30-38 nm” was revised as “Transmission electron\nmicroscopy (TEM) showed that all PM were of capsule structures with a wall thickness of 30-38 nm\n(Fig. 2a)”.\nPage 5, line 4\n“PM-BS21 had a pore size of 3.0 nm…” was revised as “Nitrogen adsorption-desorption isotherm and\ncorresponding pore size distribution of the PM-BS12 and PM-BS30 were reflected in Supplementary\nFig. 2. PM-BS12, PM-BS21, and PM-BS30 exhibited pore sizes of 3.0 nm; 1.7 nm and 3.0 nm; and 3.0\nnm and 5.3 nm, respectively. This pore size distribution enabled NAD+/NADH with a hydrodynamic\ndiameter of ~1.7 nm to shuttle through the pores”.\nPage 5, line 8\n“As shown in Supplementary Fig. 3a, elemental mapping images of nitrogen (N) and sulfur (S) element\nwere observed in PM-BS12 and PM-BS21, while there were no S elemental signals in PM-BS30.\nSupplementary Fig. 3b exhibited the proportion variation between element N and S. The S content\nincreased nearly 100% in PM-BS12 compared to PM-BS21, which was consistent with the proportion\nbetween m-Bpy and m-PaHSO used for the synthesized PM” was added.\n“X-ray photoelectron spectroscopy (XPS) was applied to characterize the chemical composition of PM.\nIn N 1s spectra, the peaks of imine linkage (n ), bipyridyl group (n ), and triazine group (n ) were\n1 2 3\ndetected, where n has two nitrogen atoms and two amino binding sites while n has three nitrogen atoms\n2 3\nand three aldehyde binding sites. For PM-BS30, n and n accounts for 20.14% and 19.43% of the N 1s\n2 3\nspectra, suggesting that the molar ratio between m-TFPT and m-Bpy is 2:3 (Supplementary Fig. 4a).\nFor PM-BS21, 60.66% of the triazine group were linked to the bipyridyl group through imine linkage,\ncould be inferred under the proportion of n and n being 21.02% and 12.75%. Similarly, the n and n\n2 3 2 3\naccounted for 22.87% and 8.62% as the molar ratio of m-Bpy and m-PaHSO was set at 1:2, about\n3\n37.69% of the triazine ring group was linked to the bipyridyl group through imine linkage\n(Supplementary Fig. 4b, c)” was revised as “X-ray photoelectron spectroscopy (XPS) was also applied\nto characterize the chemical composition of PM. In N 1s spectra, the peaks of triazine group/imine\nlinkage (n ), bipyridyl group (n ), and N-H bond (n ) were detected (Supplementary Fig. 4). The\n1 2 3\nproportion of n was consistent, suggesting that different PM had the same amounts of free amino groups.\nPage 5, line 26\n“τ got prolonged as the ratio of m-Bpy to m-PaHSO increased, suggesting enhanced electron transfer”\n2 3\nwas revised as “τ got prolonged as the ratio of m-Bpy to m-PaHSO increased, suggesting that PM-\n2 3\nBS30 with more dipyridyl groups was constructive for electron transfer”.\nPage 6, line 8\n“To further prove our designed PM can enrich the protons, three types of PM were first filtered to form\nmembranes on the polyacrylonitrile (PAN) substrate (PM-PAN membrane, Supplementary Fig. 12).\nThen, the PM-PAN membranes were set at the diffusion cell, where 1:5000 (v/v) hydrochloric acid\nsolution and aqueous solution were separately set at both sides. As shown in Supplementary Fig. 13, the\npH value of the aqueous solution was monitored over time. The pH value of PM-BS12-PAN changed\nslightly compared to PM-BS21-PAN and PM-BS30-PAN, which demonstrated that PM-BS12-PAN\ncould more efficiently adsorb protons” was added.\nPage 6, line 18\n“PM-BS21 achieved an NADH generation yield of 89.5%, an initial regeneration rate of 7.9 mmol g -\nPM\n1 h-1 and a turnover frequency (TOF) of 39.3 h-1 (Fig. 2f and Supplementary Fig. 12). The NADH\nregeneration performance of PM-BS21 surpassed that of most previously reported results\n(Supplementary Table 1)” was removed.\n“As shown in supplementary Fig. 14, the NADH regeneration catalyzed by PM was performed under\ndifferent light densities. With the increase of light intensity from 100 to 200 mW/cm2, the NADH\nregeneration yield was gradually improved. This suggested that light was the rate-determining step when\nlight intensity was between 100 and 200 mW/cm2. With the further increase of light intensity, the NADH\nyield was only slightly changed. This suggested that under light intensities exceeding 200 mW/cm2,\nfactors other than light became the rate-determining step in the NADH regeneration process. During\nphotocatalytic NADH regeneration, Rh-complex captured two electrons and one proton and then\ntransferred them to NAD+ for the production of NADH. Since both the Rh-complex and the reactant\nwere in sufficient concentration, the electron and proton transfers may be the rate-determining step when\nlight intensity was above 200 mW/cm2. The process of NADH regeneration involves the transfer of two\nelectrons and one proton, where proton transfer determines the binding efficiency of NAD+ and proton.\nSlower proton transfer would result in the formation of NAD dimers by the binding of the NAD radicals,\nwhich ultimately decreases the selectivity of 1,4-NADH. As shown in Fig. 2f, NADH regeneration\nexperiments were carried out at 300 mW/cm2 of light density. Within the first 4 min, owing to better\nproton transfer, PM-BS12 exhibited a higher NADH regeneration rate and 1,4-NADH selectivity than\nPM-BS21 and PM-BS30 (Supplementary Fig. 15a and Supplementary Fig. 16). However, at 4-8 min,\nthe NADH regeneration rate by PM-BS21 was improved, benefiting from the coordination of electron\nand proton transfers (Supplementary Fig. 15b). As for 12 min (Fig. 2f), all PM samples reached the\nbalance of photocatalytic NADH regeneration process, where PM-BS21 exhibited the best NADH\nregeneration performance (89.5%). The NADH regeneration performance of PM-BS21 surpassed that\nof most previously reported results (Supplementary Table 1)” was added.\nPage 7, line 5\n“Glucose derived from the degradation of biomass can be oxidized to value-added products, such as\ngluconic acid, and formic acid. Glucose serves as a promising electron donor due to its sustainable origin,\neconomic viability, and value-added chemicals production” was added.\nPage 7, line 9\n“an NADH regeneration yield of 43.6% was achieved, accompanied by the generation of 3.0 mmol L-1\nof gluconic acid…” was revised as “an NADH regeneration yield of 44.0% was achieved, accompanied\nby the generation of 3.1 mmol L-1 of gluconic acid…”.\nPage 7, line 12\nFig.2 was changed.\nPage 8, line 6\n“Initial reaction rate and TOF that based on the reduction reaction center Rh-complex of NADH\nregeneration in the use of PM-BS30, PM-BS21, and PM-BS12” was revised as “The yield of NADH\nregeneration in the use of PM-BS30, PM-BS21, and PM-BS12. In this section, the NADH mentioned\nin the Figures was enzymatic active 1,4-NADH”.\nPage 8, line 15\n“To minimize the impact of Rh-complex and ROS on enzymatic activity, ADH is immobilized onto PM\nby forming a silica layer. PM-BS21, exhibiting the highest NADH regeneration efficiency, was used.\nRh-complex was loaded on PM-BS21 to prepare PM-Rh” was revised as “To minimize the impact of\nRh-complex and ROS on enzymatic activity, Rh-complex was loaded on PM to prepare PM-Rh, and\nthen ADH was immobilized onto PM-Rh by forming a silica layer. PM-Rh were first performed for\nNADH regeneration, where Rh-complex as the active site of NADH regeneration was anchored on\ndipyridyl group. Therefore, PM-BS30-Rh with more anchored Rh-complex exhibited a higher NADH\nregeneration yield than PM-BS12-Rh and PM-BS21-Rh (Supplementary Fig. 20a). Meanwhile, all PM-\nRh samples showed over 70% selectivity of 1,4-NADH (Supplementary Fig. 20b). Comprehensively\nconsidering the NADH regeneration yield and the dosage of Rh-complex, PM-BS21-Rh was used to\nconstruct PEM for EG synthesis from GALD”.\nPage 9, line 8\n“The intensity of ROS (Supplementary Fig. 22a), such as hydroxyl radical (•OH), observed in electron\nspin resonance spectroscopy (ESR) decreased as the thickness of EM increased. This reduction\nmitigated the adverse effects of ROS on enzyme activity (Fig. 3c and Supplementary Fig. 22b).\nMoreover, as shown in Fig. 3d and Supplementary Fig. 23, the presence of the EM helped preserve the\nsecondary structure of enzyme under light illumination” was removed.\n“Although silica component in EM was almost optically transparent in the visible light range, the light\nabsorption efficiency of PEM was not constant (Supplementary Fig. 27). EM reduced the light\nabsorption intensity of PEM. The intensity of ROS (Supplementary Fig. 28a), such as hydroxyl radical\n(•OH) and superoxide radical (•O -) observed in electron spin resonance spectroscopy (ESR) decreased\n2\nas the thickness of EM increased. This reduction mitigated the adverse effects of ROS on enzyme\nactivity (Fig. 3c and Supplementary Fig. 28b-c). Moreover, as shown in Fig. 3d and Supplementary Fig.\n29, the changes in the secondary structure of the ADH were analyzed. The secondary structure of ADH\nwas almost unchanged under dark condition. In PEM system after illumination, the ratio of α-helix of\nenzymes. As the thickness of the EM increased, the secondary structure of ADH was better protected.\nFor the PEM system, the secondary structure of enzyme was almost unchanged before and after light\n20\nillumination. The presence of the EM weakened the intensity of ROS, and confined enzyme within a\nstable space, thus ensuring the stable secondary structure of enzyme” was added.\nPage 9, line 22\n“566% enhancement…” was revised as “503% enhancement…”.\nPage 10, line 1\nFig. 3 was changed.\nPage 10, line 4\n“The intensity of •OH signals generated by PEM after 5 min of illumination” was revised as “The\nx\nintensity of •OH and •O - signals generated by PEM after 5 min of illumination”.\n2 x\nPage 10, line 20\n“The optimal formaldehyde production, 9.18 mmol L-1…” was revised as “The optimal formaldehyde\nproduction, 8.9 mmol L-1…”.\nPage 10, line 24\n“3.6 mmol L-1 of GALD was produced after 24 hours of incubation by optimizing the concentration of\nGALS” was revised as “3.7 mmol L-1 of GALD was produced after 24 hours of incubation by\noptimizing the concentration of GALS”.\nPage 11, line 12\n“3.0 mmol L-1 of EG was obtained, with a synthesis rate of 0.25 mmol g -1 h-1, using TEOA after four\nPEM\nlight and dark cycles” was revised as “2.9 mmol L-1 of EG was obtained, with a synthesis rate of 2.6\nmmol g -1 h-1, using TEOA after four light and dark cycles”.\nPEM\nPage 11, line 14\n“The EG production was moderate compared to other systems (Supplementary Table 2)” was added.\nPage 11, line 18\nFig. 4 was changed.\nPage 12, line 8\n“An 89.5% NADH yield with an initial reaction rate of 7.9 mmol g -1 h-1 and TOF of 39.3 h-1 is\nPM\nobtained” was revised as “An 89.5% NADH yield with an initial reaction rate of 5.6 mmol g -1 h-1\nPM\nand TOF of 27.9 h-1 is obtained”.\nPage 12, line 11\n“A 566% elevation of the EG production in the integrated PEM is obtained compared with the non-\nintegrated system” was revised as “A 503% elevation of the EG production in the integrated PEM is\nobtained compared with the non-integrated system”.\nPage 12, line 12\n“3.0 mmol L-1 EG with the synthesis rate of 0.25 mmol g -1 h-1 is obtained with the continuous\nPEM\naddition of methanol” was revised as “2.9 mmol L-1 EG with the synthesis rate of 2.6 mmol g -1 h-1\nPEM\nis obtained with the continuous addition of methanol, where the photonic quantum efficiency can reach\n0.30%”.\nPage 13, line 9\n“The loading amount actually refers to the mass (mg) of ADH loaded onto the one mg carrier (PM-\nBS21-Rh)” was added.\nPage 13, line 19\n“Ultimately, the enzymatic method was used to detect the selectivity of NADH regeneration” was\nrevised as “Ultimately, the enzymatic method was used to detect the selectivity of 1,4-NADH”.\nPage 13, line 21\n“The selectivity of NADH was demonstrated by the ratio of the change in absorbance to the initial\nabsorbance” was revised as “The selectivity of 1,4-NADH was demonstrated by the ratio of the change\nin absorbance to the initial absorbance”.\nPage 14, line 11\n“The synthesis rate of EG is calculated by m /(m *t), where m refers to the mole mass of generated\nEG PEM EG\nEG under each 20 min light illumination, m refers to mass of PEM, and t refers to light illumination\nPEM\n(20 min). The photonic quantum efficiency in the system of EG production from methanol is calculated\nby the equation of (2*m*N *h*c)/(S*P*t*λ)*100%, where m represents the molar amount of the\nA\nsynthesis EG, N represents the Avogadro constant, h is Planck constant, c is the light speed, S stands\nA\nfor irradiation area, P represents the intensity of the irradiation light, t is irradiation time, and λ stands\nfor wavelength of the light” was added.\nIn the supporting information:\nPage 2, line 39\n“Polyacrylonitrile (PAN, pore size is about 30-40 nm) flat sheets were selected as support and supplied\nby Shandong Megavision Membrane Engineering and Technology Co., Ltd. (Shandong, China)” was\nadded.\nPage 2, line 42\n“The test of proton conductivity. The proton conductivities of PM were measured using a two-\nelectrode AC impedance spectroscopy technique. The perturbation voltage was 15 mV, and the\nfrequency was set at a range of 1×106-1 Hz. The sample (2 mm×10 mm) was placed between Pt\nelectrodes (with a spacing of 0.8 mm). Then, the electrodes were put in a constant temperature and\nhumidity chamber to test the proton conductivity. The proton conductivities (σ, mS cm-1) of PM were\ncalculated by the equation of σ=l/(AR), where l (cm), A (cm2), and R (Ω) represented the PM thickness,\ncontact area, and resistance, respectively” was added.\nPage 5-7, 9-13, 15, 25, 29, and 32-40\nSupplementary Fig. 1-3, 5-9, 11, 21, 25, and 28-36 was changed.\nPage 16-22, 24, and 31\nSupplementary Fig. 12-18, 20, and 27 was added.\nPage 44\nSupplementary Table 1 was changed.\nPage 45\nSupplementary Table 2 was added.\nDetailed Response to the Reviewers’ Comments\nReviewer: 1\nComments:\nMy concerns have been addressed, and the revised manuscript is recommended for publication.\nI have carefully reviewed the authors' revisions and their point-by-point replies to the previous concerns\nraised by Reviewer 3. In my opinion, the authors have adequately addressed the majority of the issues,\nand the revisions have significantly improved the clarity and scientific rigor of the manuscript.\nThere is only one minor point that still requires attention: the structure in Fig. R35 (Supplementary Fig.\n7) appears to contain a persistent inaccuracy. I would kindly ask the authors to make the necessary\ncorrections to this figure before the manuscript proceeds further.\nAnswer:\nWe sincerely appreciate the reviewer’s positive remarks and valuable guidance. We are sorry that\nthere is an inaccuracy about Supplementary Fig. 7. Upon the reviewer’s request, the Supplementary\nFig. 7 in the Supplementary Information was revised and demonstrated as Fig. R1 (Revised\nSupplementary Information) below. We have also meticulously checked the entire text and have\nidentified and resolved the following issues.\nFig. R1 (Supplementary Fig. 7 in the Revised Supplementary Information). The possible structural\nunits of PM-BS12.\n“This suggested that light was the rate-determining step when light intensity was between 100 and\n200 mW/cm2” was revised to “This suggested that light absorption was the rate-determining step when\nlight intensity was between 100 and 200 mW/cm2” (page 6, line 20 in the Revised Manuscript).\nA mistake in the Supplementary Fig. 11 has been revised to Fig. R2.\nFig. R2 (Supplementary Fig. 11 in the Revised Supplementary Information). The proton adsorption\nenergy of PM-BS30, PM-BS21, and PM-BS12 calculated by DFT.\n“The pronton adsorption…” was revised as “The proton adsorption…” (page 15, line 2 in the\nRevised Supplementary Information).\n“A 100 W LED lamp was used as the light, the light was turned on for 60 min after dark treatment\nfor 5 min” was revised as “A 100 W LED lamp was used as the light source, and the light was turned\non for 60 min after dark treatment for 5 min” (page 14, line 23 in the Revised Manuscript).\n“Second derivative of the FITR spectra of …” was revised as “Second derivative of the FTIR spectra\nof …” (page 33, line 2 in the Revised Supplementary Information).\nReviewer: 2\nComments:\nThe manuscript describes a photo-enzyme-membrane (PEM) system coupled with multi-enzyme\ncascades for ethylene glycol (EG) synthesis. Despite revisions, the study lacks transformative novelty,\nexhibits unresolved contradictions in key data, and fails to convincingly demonstrate the system's\npractical advantage. Therefore, I do not support the publication of this work in the prestigious Nature\nCommunications.\nAnswer:\nMany thanks for the reviewer’s valuable guidance. We are regretful that we may fail to clearly convey\nour innovative points. In this regard, we would like to re-clarify the significance of our study. Multi-\nenzyme catalysis is a typical biomanufacturing process, which can achieve the selective transformation\nof raw materials into target products. In general, oxidoreductases that account for over 25% of industrial\nenzymes are usually involved in the process of multi-enzyme catalysis. Catalytic reactions enabled by\noxidoreductases strongly rely on the energy supply of cofactor (e.g., NADH) and stable enzymatic\nprocess to realize the chemicals synthesis. Addressing the issues of insufficient energy supply and\nunstable enzymatic processes in oxidoreductase catalysis is thus of great importance for multi-enzyme\ncatalysis process.\nIn our study, a photo-enzyme-membrane (PEM) platform with efficient NADH regeneration and\nstable enzymatic process is constructed. Covalent organic polymer membrane as photo-membrane (PM)\naffords efficient NADH supply through synergistic intensification of electron transfer and proton\ntransfer. Meanwhile, the construction of enzyme-membrane (EM) mitigates the adverse effects of the\nPM on enzyme activity. Then, the synthesis of EG from methanol catalyzed by multi-enzymes of alcohol\noxidase (AOX), catalase (CAT), glycolaldehyde synthase (GALS), and alcohol dehydrogenase (ADH,\nthe key oxidoreductase) is just chosen as a representative case to demonstrate the superiority of our\nPEM platform. As reported in previous literatures (Nat. Commun., 2025, 16, 10756; ACS Catal., 2024,\n14, 4665; Nat. Commun., 2025, 16, 8079), many multi-enzymatic pathways require cofactors, including\nthe synthesis of lactic acid from methanol, the synthesis of non-canonical amino acids from glycerol,\netc., which further highlights the importance and universality of our PEM.\nAs for the reviewer’s doubts about the unresolved key contradictions, we have carefully revised and\nrefined the manuscript to provide more solid evidence and improve the overall quality. The detailed\npoint-by-point response is provided below. The corresponding information can be found in the Revised\nManuscript and Revised Supplementary Information.\nQuestion 1:\nThe PEM design, featuring a capsule-like structure for light harvesting, cofactor regeneration, and\nenzyme stabilization, closely mirrors the authors' prior work on thylakoid-mimetic or capsule-based\nphotoenzymatic systems. While incremental optimization (e.g., monomer ratios in PM) is presented,\nthe fundamental architecture and operational principles (e.g., silica encapsulation for enzyme protection)\nare not sufficiently distinct from earlier publications to constitute a conceptual breakthrough.\nAnswer:\nWe agree to the reviewer’s viewpoint that the capsule-like structure of PEM and our previous work\nare both designed inspired by the structure of thylakoids in natural photosynthesis. However, the\nphotocatalyst materials, the scientific problem, and the core innovations are fundamentally distinct.\nFirstly, the titania capsule in our previous work was prepared through a calcium carbonate template\nmethod, where the capsule size was 2.5-3.5 μm. In our current work, a covalent organic polymer capsule\nwas prepared through a silica template method. The capsule size was about 400~500 nm, which was\nsmaller than the titania capsule in our previous work. It was difficult to realize precise tunability in\nmolecular scale for the titania capsule. By contrast, the structure of covalent organic polymer capsules\nin molecular scale can be regulated by changing the monomers. The structural versatility enabled the\nmodulation of the photoelectrochemical properties, thereby optimizing the photocatalytic NADH\nregeneration efficiency.\nIn addition, the previous work focused on molecule transfer between photocatalysis and enzyme\ncatalysis. NAD+/NADH molecules was shuttled through the titania capsule wall, which finally realized\nthe conversion of propanal to propanol. In our current work, the design of PM focused on enhancing\nelectron and proton transfers in photocatalytic NADH regeneration, where the bipyridine moiety\nmediated fast electron transfer from the birth site, and the sulfonic acid moiety facilitated proton transfer\nby enriching protons.\nFinally, in our previous work and other literatures, researchers focused on enhancing electron transfer\nin photocatalytic NADH regeneration and molecule transfer in enzyme-photo-coupled catalysis (J. Am.\nChem. Soc., 2022, 144, 4268; ACS Catal., 2021, 11, 476; Angew. Chem. Int. Ed., 2022, 61,\ne202200261; J. Am. Chem. Soc. 2020, 142, 5958-5963). Most researchers ignore the importance of\nproton transfer in photocatalytic NADH regeneration. Our current work primarily emphasizes the\ncoordination between electron transfer and proton transfer in photocatalytic NADH regeneration.\nBesides, few studies have harnessed photocatalysts with enhanced electron and proton transfer,\nintegrated with multi-enzyme catalysis, to drive the synthesis of final products from raw materials.\nTherefore, our current work is not a simple combination, but an advancement in the field of artificial\nphotosynthesis.\nQuestion 2:\nThe proton diffusion experiment (Supplementary Fig. 13) uses a hydrochloric acid gradient to\ndemonstrate proton adsorption by sulfonic-acid-functionalized PMs. However, this setup does not\nreplicate actual catalytic conditions, where buffered solutions (pH 8.0) regulate proton activity. The\nclaimed \"proton enrichment\" effect lacks validation under reaction-relevant pH dynamics, undermining\nits proposed role in enhancing NADH regeneration kinetics.\nAnswer:\nUpon the reviewer’s request, we would like to explain the reason for using a hydrochloric acid\ngradient to demonstrate proton adsorption by sulfonic-acid-functionalized PMs. In acidic solutions with\nhigh proton concentrations, the variation of proton adsorption by PMs is easier to observe. This\ncondition indeed deviates from the actual reaction conditions. The actual reaction system is conducted\nin a PBS buffer to maintain a stable pH value. Based on the reviewer’s valuable guidance, PM-PAN\nmembrane is set at the diffusion cell, where TEOA/PBS buffer (pH 8.0) solution and aqueous solution\nwere separately added to both sides. As shown in Fig. R3, the pH value of the solution is monitored\nover time. The overall pH value of the solution did not change significantly.\nIn our work, the diffusion cell experiment demonstrates that PM exposure to a high-concentration\nproton source induces a measurable change in proton concentration. Owing to the stable pH value\nprovided by PBS buffer in the actual catalytic conditions, it is hard to directly monitor local proton\nadsorption in real time.\nConsequently, the proton transfer facilitated by our PM is inferred indirectly through two primary\nlines of evidence: (i) the measured increase in the proton conductivity of PMs (Fig. R4 (Fig. 2c in the\nRevised Manuscript)), and (ii) DFT analysis of proton adsorption energy, which provides insight into\nthe thermodynamic driving force for proton adsorption (Fig. R2 (Supplementary Fig. 11 in the\nRevised Supplementary Information)).\n8.40\nPM-BS12\nPM-BS21\n8.35 PM-BS30\ne\nu\nal\n8.30\nv\nH\np\n8.25\n8.20\n0 5 10 15 20 25 30\nTime (min)\nFig. R3. The change of pH value by different PM-PAN membrane: PM-BS12-PAN, PM-BS21-PAN,\nand PM-BS30-PAN.\n1\n)200\n-m\nc\nS 160\nm\n(\ny\nt120\nvi\nti\nc\nu\nd 80\nn\no\nc\nn\no 40\nt\no\nP r 0\nPM-BS12 PM-BS21 PM-BS30\nFig. R4 (Fig. 2c in the Revised Manuscript). The proton conductivity of PM-BS30, PM-BS21,\nand PM-BS12 under 40oC, and 93% humidity.\nFig. R2 (Supplementary Fig. 11 in the Revised Supplementary Information). The proton adsorption\nenergy of PM-BS30, PM-BS21, and PM-BS12 calculated by DFT.\nQuestion 3:\nThe revised Fig. 2f shows an abrupt change in NADH regeneration yields compared to earlier\nversions, particularly for PM-BS21. This adjustment appears to force-fit the proton enrichment\nhypothesis.\nAnswer:\nIndeed, as the reviewer pointed out, the NADH regeneration yields have been changed compared to\nthe earlier version. In the current version, we replaced the monomers required in the PM synthesis\nprocess and repeated the experiment three times, obtaining the average results shown in the Fig. R5\n(Fig. 2f in the Revised Manuscript). We infer that the change in performance can be attributed to\nvariations in the monomeric composition of the synthesized samples.\nThe original data can be found in Fig. R6 and the file of source data.\nPM-BS21\n80\n% ) PM-BS30\n(\nH 60\nD\nA\nN\nf\n40\no\nd\nel 20\ni\nY\n0\n0 4 8 12 16\nTime (min)\nFig. R5 (Fig. 2f in the Revised Manuscript). The NADH regeneration yield catalyzed by PM-BS12,\nPM-BS21, and PM-BS30.\nFig. R6. The original data of NADH regeneration yield catalyzed by PM-BS12, PM-BS21, and PM-\nBS30.\nQuestion 4:\nAfter Rh-complex immobilization, PM-BS30-Rh shows higher NADH yield than PM-BS21-Rh or\nPM-BS12-Rh (Supplementary Fig. 20a). The authors attribute this to \"more anchored Rh-complex\" in\nPM-BS30 but provide no quantitative evidence (e.g., ICP-MS for Rh loading). Without equal Rh-\nloading comparisons, the claim remains speculative. Moreover, PM-BS30's superiority contradicts the\nproposed proton/electron balance hypothesis, revealing a fundamental flaw in the PM design rationale.\nAnswer:\nWe are sorry for the missing of ICP-based quantification for the Rh loading. Based on the reviewer’s\nvaluable guidance, we first optimized the preparation process of PM-Rh. The PM and Rh-complex were\nfully mixed through ultrasonication, so as to effectively improve the Rh-complex loading. First, by\nadding sufficient Rh-complex for PM-Rh synthesis, PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-\n0.43 0.82\nRh were obtained. ICP test was shown in Table R1(Supplementary Table 2 in the Revised\n1.34\nSupplementary Information). Rh-complex was anchored on bipyridine group, the Rh element loading\namounts of PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh was in a mass ratio of ~1:2:3. As\n0.43 0.82 1.34\nshown in Fig. R7 (Supplementary Fig. 20a in the Revised Supplementary Information), PM-BS30-\nRh showed the highest NADH regeneration yield owing to the presence of more catalytic active site\n1.34\nRh-complex. Then, PMs with the equal amount of Rh element loading was prepared. The loading of Rh\nelement was shown in Table R2 (Supplementary Table 3 in the Revised Supplementary\nInformation), and the Rh element content in the PM was almost consistent. As shown in Fig. R8\n(Supplementary Fig. 20b in the Revised Supplementary Information), PM-BS21-Rh exhibited\n0.50\nthe highest NADH regeneration performance. It demonstrated that enhancing electron and proton\ntransfer can realize efficient NADH regeneration under equal Rh element loading amount. The tendency\nwas consistent with the performance of NADH regeneration using equal amount of free Rh-complex\n(Fig. R5 (Fig. 2f in the Revised Manuscript)), which demonstrated the feasibility of improving NADH\nregeneration efficiency through coordinating electron and proton transfers.\nTable R1 (Supplementary Table 2 in the Revised Supplementary Information). The ICP data of\nPM-Rh.\nThe content of\nThe Rh content\nPhotocatalyst Rh (ppm)/2 mg\nin the PM (%)\nPM\nPM-BS12-Rh 0.3420 0.4275\nPM-BS21-Rh 0.6529 0.8161\nPM-BS30-Rh 1.0690 1.3363\n0.43\nPM-BS21-Rh\n) 80 0.82\n% PM-BS30-Rh\n( 1.34\nH 60\nD\nA\nN\nf\n40\no\nd\nel 20\ni\nY\n0\n0 5 10 15 20\nTime (min)\nFig. R7 (Supplementary Fig. 20a in the Revised Supplementary Information). The NADH\nregeneration yield catalyzed by PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh .\n0.43 0.82 1.34\nTable R2 (Supplementary Table 3 in the Revised Supplementary Information). The ICP data of\nPM-Rh.\nThe content of\nThe Rh content\nPhotocatalyst Rh (ppm)/2 mg\nin the PM (%)\nPM\nPM-BS12-Rh 0.3420 0.4275\nPM-BS21-Rh 0.3973 0.4966\nPM-BS30-Rh 0.3904 0.4880\n0.43\nPM-BS21-Rh\n%\n)60\nPM-BS30-Rh\n0.50\n( 0.49\nH\nD\nA40\nN\nf\no\nd 20\nel\nY\n0\n0 5 10 15 20 25\nTime (min)\nFig. R8 (Supplementary Fig. 20b in the Revised Supplementary Information). The NADH\nregeneration yield catalyzed by PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh .\n0.43 0.50 0.49\nQuestion 5:\nAll PM-Rh samples show only ~80% 1,4-NADH selectivity (Supplementary Fig. 20b), an\ninefficiency problematic for enzymatic applications. Furthermore, the multi-enzyme system uses 10\nmM NAD⁺ but produces only ~2.9 mM EG (Fig. 4c). Given the high cost of NAD+ cofactors, the\nsystem's economic viability is questionable.\nAnswer:\nThank the reviewer for the valuable guidance. The enzymatic method was used to detect the\nselectivity of 1,4-NADH. The absorbance of the reaction at 340 nm would decrease arising by the\nconsumption of NADH when the appropriate amount of propanal and ADH added. The selectivity of\n1,4-NADH was demonstrated by the ratio of the change in absorbance to the initial absorbance.\nThe initial reaction rate was defined by the equation of (N-N )/(m*t), in which N and N meant the molar\nt 0 0 t\nmass of generated NADH production in certain time, m represented the quality of photocatalyst involved\nin the reaction and t stand for the time of photocatalytic reaction. We extended the reaction time between\nthe addition of propanal/ADH and the generated NADH to 5 minutes. As shown in the Fig, R9\n(Supplementary Fig. 20c, d in the Revised Supplementary Information), the selectivity has\nincreased by approximately 5% compared to the original level, but it still does not reach 100%.\nPrevious studies on immobilized Rh photocatalysts have largely overlooked the issue of selectivity (J. Am.\nChem. Soc., 2024, 146, 586; Angew. Chem. Int. Ed., 2022, 61, e202206283). In contrast, our experimental\nresults indicate that a fraction of electrons and protons can bypass the Rh active sites, directly reducing\nNAD⁺ to form various by-products, including NAD⁺ dimers, 1,2-NADH, and 1,6-NADH. This diversion\nfacilitates non-productive side reactions, giving rise to NADH-derived byproducts and thus\ncompromising overall selectivity.\nFig. R9 (Supplementary Fig. 20c, d in the Revised Supplementary Information). The selectivity of\nNADH catalyzed by a) PM-BS12 , PM-BS21 , PM-BS30 and b) PM-BS12 , PM-BS21 ,\n0.43 0.82 1.34 0.43 0.50\nPM-BS30 .\n0.49\nAs for the PEM coupled with multi-enzyme cascade, the low catalytic activity of GALS is the rate-\nlimiting step in the EG synthesis process. The key to increasing the output of EG lies in enhancing the\nactivity of GALS. We further purified GALS and obtained a more concentrated enzyme solution. As\nshown in Fig. R10 (Supplementary Fig. 36b in the Revised Supplementary Information), different\nconcentration of NAD+ (2, 5, and 10 mmol L-1) was used for the EG synthesis under higher\nconcentration of GALS (3 g L-1). Finally, a 5.1 mmol L-1 EG is obtained with the continuous addition\nof methanol. In particular, 3.1 mmol L-1 EG is obtained in the presence of 2 mmol L-1 NAD+,\nwhich exhibits the system's economic viability.\n10\n) PEM & multi-enzyme cascade (2 mmol L-1 NAD+)\n- 1 L PEM & multi-enzyme cascade (5 mmol L-1 NAD+)\nol 8 PEM & multi-enzyme cascade (10 mmol L-1 NAD+)\nm Blank\nm Light on Light off\n6\n(\nn\no\nc\nti\n4\nu\nd\no\nr 2\np\nG\nE\n0\n0 1 2 3 4\nCycle number\nFig. R10 (Supplementary Fig. 36b in the Revised Supplementary Information). The production of\nEG by the coupled system with TEOA as electron donor under different concentration of NAD+ (2, 5,\nand 10 mmol L-1). Each cycle consists of 0.33 hour of light followed by 2.67 hours of darkness.\nQuestion 6:\nThe PEM solely catalyzes the final steps, namely NADH regeneration and the transformation of\nglycolaldehyde to EG, while the conversion of methanol to glycolaldehyde occurs in a separate reactor\nvia free enzymes (AOX/CAT/GALS). The authors' work thus focuses narrowly on NADH-dependent\nEG reduction, which is a well-established reaction, rather than an integrated platform for overall\nsynthesis.\nAnswer:\nThank the reviewer for the valuable guidance. Alcohol dehydrogenase (ADH) and NADH catalyze\nthe reduction of formaldehyde to methanol, which is unfavorable for the conversion of glycolaldehyde\nto EG. Therefore, we adopted a strategy of separating the multi-enzymatic cascade and PEM.\nThis approach enables continuous synthesis of EG while minimizing undesirable side reactions. Upon the\nreviewer’s request, a well-established reactor with dual-channel was constructed for continuous EG\nsynthesis, where the enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion\nchannel and PEM filtrated to form a PEM-PAN membrane was put in the photo-enzyme catalytic\ndiversion channel (Fig. R11 (Fig. 4c in the Revised Manuscript)). As shown in Fig. R12 (Fig. 4b, c\nin the Revised Manuscript), 0.71 mmol L-1 EG production with an initial generate rate of 2.43 mmol\ng -1 h-1 was obtained after ten light-dark cycles. This is a preliminary attempt, and the product yield\nPEM\ncan be further improved by optimizing the structure of the set-up and conditions of the coupled reaction.\nThe design of the reactor enables the scalable, integrated enzyme-photo coupled catalytic conversion of\nfeedstocks into valuable chemicals.\nFig. R11 (Fig. 4c in the Revised Manuscript). The images of a) PEM-PAN membrane and b) the dual-\nchannel reactor for EG synthesis.\nFig. R12 (Fig. 4b, c in the Revised Manuscript). a) The schematic of the dual-channel reactor for EG\nsynthesis. b) The production of EG by the dual-channel reactor with TEOA as electron donor. Here, the\nsystem containing PM-PAN membrane was set as blank, where PE adsorbed on PAN to form PE-PAN\nmembrane through vacuum filtration. Each cycle consists of one hour of light followed by three hours\nof darkness.\nQuestion 7:\nThe stability of alcohol dehydrogenase (ADH) is tested over only 1.32 hours (Fig. 4c), a timeframe\ntoo short to evaluate long-term viability. Without demonstrating ADH stability under prolonged\noperation or TTN evaluation, the EM's protective efficacy remains unsubstantiated.\nAnswer:\nBased on the reviewer’s valuable guidance, the long-term stability of immobilized ADH was tested\nunder ten light-dark cycle (ten hours illumination time). As shown in Fig. R13 (Fig. 4c in the Revised\nManuscript), with the protection of ADH by EM, the EG production catalyzed by PEM had continued\nto increase during the ten light-dark cycle.\nThe total turnover number (TTN) was also be calculated. TTN referred to n /n , which was\nproduct catalyst\ncalculated as 1668. n referred to the molar mass of the production of EG and n referred to the\nproduct catalyst\nmolar mass of ADH.\n1.0\n) EG synthesis\n1\n-L\nLight on Light off\nol\n0.8\nm\nm\n0.6\n(\nn\no\nti\n4\nu\nd\no\nr0.2\np\nG\nE0.0\n0 1 2 3 4 5 6 7 8 9 10\nCycle number\nFig. R13 (Fig. 4c in the Revised Manuscript). The production of EG by the dual-channel reactor under\nthe ten light-dark cycle, where each cycle consists of one hour of light followed by three hours of\ndarkness.\nA list of major changes\nIn the Revised Manuscript:\nPage 1, line 30\n“Further, PEM is coupled with alcohol oxidase, catalase, and glycolaldehyde synthase for sustainable\nsynthesis of EG with a rate of 2.6 mmol g -1 h-1 by continuous supply of methanol” was revised as\nPEM\n“Further, a dual-channel reactor is constructed for sustainable synthesis of EG with an initial synthesis\nrate of 2.43 mmol g -1 h-1 by continuous supply of methanol”.\nPEM\nPage 3, line 22\n“an EG production of 2.9 mmol L-1 with the synthesis rate of 2.6 g -1 h-1 was obtained with the\nPEM\ncontinuous supply of methanol” was revised as “an EG production of 0.71 mmol L-1 with the initial\nsynthesis rate of 2.43 mmol g -1 h-1 was obtained with the continuous supply of methanol”.\nPEM\nPage 6, line 20\n“This suggested that light was…” was revised as “This suggested that light absorption was…”.\nPage 8, line 18\n“PM-Rh were first performed for NADH regeneration, where Rh-complex as the active site of NADH\nregeneration was anchored on dipyridyl group. Therefore, PM-BS30-Rh with more anchored Rh-\ncomplex exhibited a higher NADH regeneration yield than PM-BS12-Rh and PM-BS21-Rh\n(Supplementary Fig. 20a). Meanwhile, all PM-Rh samples showed over 70% selectivity of 1,4-NADH\n(Supplementary Fig. 20b)” was revised as “First, by adding sufficient Rh-complex for PM-Rh synthesis,\nPM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh were obtained. Inductively coupled plasma\n0.43 0.82 1.34\noptical emission spectroscopy (ICP-OES) test was shown in Supplementary Table 2. Rh-complex was\nanchored on bipyridine group, while the Rh element loading amounts of PM-BS12-Rh , PM-BS21-\n0.43\nRh , and PM-BS30-Rh was in a mass ratio of ~1:2:3. Therefore, PM-BS30-Rh with more\n0.82 1.34 1.34\nanchored Rh-complex exhibited a higher NADH regeneration yield than PM-BS12-Rh and PM-\n0.43\nBS21-Rh (Supplementary Fig. 20a, c). Furthermore, PM with equal amount of Rh element loading\n0.82\nwas also prepared. The loading of Rh element was shown in Supplementary Table 3, and the Rh element\ncontent in the PM was almost consistent. As shown in Supplementary Fig. 20b, d, PM-BS21-Rh\n0.50\nexhibited the highest NADH regeneration performance with the selectivity of ~90%”.\nPage 11, line 12\n“With continuous feeding from the enzymatic reactor to the PEM reactor, 2.9 mmol L-1 of EG was\nobtained, with a synthesis rate of 2.6 mmol g -1 h-1, using TEOA after four light and dark cycles. The\nPEM\nEG production was moderate compared to other systems (Supplementary Table 2)” was revised as\n“With continuous feeding from the enzymatic reactor to the PEM reactor, 5.1 mmol L-1 of EG at a NAD+\nconcentration of 10 mmol L-1 was obtained, using TEOA after four light and dark cycles. In particular,\n3.1 mmol L-1 EG was obtained in the presence of 2 mmol L-1 NAD+, which exhibits the system's\neconomic viability”.\nPage 11, line 19\n“In order to integrate multi-enzyme cascade and PEM, a well-established reactor with dual channels\nwas also constructed for continuous EG synthesis. The enzymes (AOX/CAT/GALS) were put in the\nmulti-enzyme cascade diversion channel and PEM filtrated to form a PEM-PAN membrane was put in\nthe photo-enzyme catalytic diversion channel. As shown in Fig. 4b, c, 0.71 mmol L-1 EG production\nwith an initial synthesis rate of 2.43 mmol g -1 h-1 was obtained after ten light-dark cycles. The reactor\nPEM\nshowed a long-term viability, and the total turnover number (TTN) was calculated as 1668. The EG\nproduction was moderate compared to other systems (Supplementary Table 4). The design of the reactor\nenabled the scalable, integrated enzyme-photo coupled catalytic conversion of feedstocks into valuable\nchemicals” was added.\nPage 12, line 1\nFig. 4 was changed.\nPage 12, line 3\n“b, The coupled system comprising a multi-enzyme cascade in the enzymatic reactor and a PEM in\nPEM reactor for the production of EG. In this system, the generated GALD from the enzymatic reactor\ndiffuses into the PEM reactor, where it was converted to EG. c, The production of EG by the coupled\nsystem with TEOA as electron donor. Here, the multi-enzyme cascade coupled with PM and free ADH\nand was used as blank” was revised as “b, The dual-channel reactor comprising a multi-enzyme cascade\ndiversion channel and a photo-enzyme catalytic diversion channel for the production of EG. In this\nreactor, methanol is continuously pumped into the reactor via a peristaltic pump. The generated GALD\nfrom the multi-enzyme cascade diversion channel diffuses into the photo-enzyme catalytic diversion\nchannel, where it was converted to EG. c, The production of EG by the dual-channel reactor with TEOA\nas electron donor, where each cycle consists of one hour of light followed by three hours of darkness.\nHere, the system containing PM-PAN membrane was set as blank, where PE adsorbed on PAN to form\nPE-PAN membrane through vacuum filtration. The insert images were the schematic of PEM-PAN\nmembrane and the dual-channel reactor”.\nPage 12, line 20\n“Through synergistic optimization of multi-enzyme cascade and PEM, 2.9 mmol L-1 EG with the\nsynthesis rate of 2.6 mmol g -1 h-1 is obtained with the continuous addition of methanol, where the\nPEM\nphotonic quantum efficiency can reach 0.30%” was revised as “Through synergistic optimization of\nmulti-enzyme cascade and PEM, 0.71 mmol L-1 EG with the initial synthesis rate of 2.43 mmol g -1\nPEM\nh-1 is obtained with the continuous addition of methanol in a dual-channel reactor, where the photonic\nquantum efficiency can reach 0.15%”.\nPage 13, line 19\n“Specifically, 10 mg of PM-BS21 was added into 2 mL of deionized water, followed by the addition of\n6.64 mg of (Cp*RhCl ) . Then the mixture was stirred for 24 h, following by centrifugation, rinsed with\n2 2\ndeionized water several times, and freeze-dried. The obtained solid was denoted as PM-BS21-Rh” was\nrevised as “Specifically, 10 mg of PM-BS21 with 4 mL of deionized water was sonicated for 10 minutes,\nfollowed by the addition of different mass of (Cp*RhCl ) . Then the mixture was stirred for 24 h,\n2 2\nfollowing by centrifugation, rinsed with deionized water several times, and freeze-dried. The obtained\nsolid was denoted as PM-BS12-Rh , PM-BS21-Rh , and PM-BS30Rh , where x referred to the content\nx x x\nof Rh element. The Rh element content of PM was characterized by ICP test”.\nPage 14, line 23\n“A 100 W LED lamp was used as the light, the light was turned on for 60 min after dark treatment for\n5 min” was revised as “A 100 W LED lamp was used as the light source, and the light was turned on\nfor 60 min after dark treatment for 5 min”.\nPage 14, line 32\n“NAD+ (10 mmol L-1)” was revised as “NAD+ (2, 5, and 10 mmol L-1)”.\nPage 15, line 3\n“A dual-channel reactor was well-established for EG synthesis. The reactor consisted of five parts: upper\nfixed plate, protective gasket, quartz stone window, photo-enzyme catalytic diversion channel, multi-\nenzyme cascade diversion channel, and temperature control water channel cover plate. A 100 ml solution\ncontaining methanol, MgSO , ThDP, TEOA/PBS, and NAD+ was continuously pumped into the reactor\n4\nvia a peristaltic pump. The enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion\nchannel and PEM filtrated to form a PEM-PAN membrane was put in the photo-enzyme catalytic\ndiversion channel. The reactor was carried out for EG synthesis for ten light and dark cycles. The\nillumination time of each cycle was one hour, and the dark processing time was three hours.\nThe initial synthesis rate of EG is calculated by n /(m *t), where n refers to the mole mass of\nEG PEM EG\ngenerated EG under first one hour light illumination, m refers to mass of PEM, and t refers to light\nPEM\nillumination (one hour). The total turnover number (TTN) is calculated by n /n , where n\nproduct catalyst product\nrefers to the molar mass of the production of EG and n refers to the molar mass of ADH. The\ncatalyst\nphotonic quantum efficiency in the system of EG production from methanol is calculated by the equation\nof (2*m*N *h*c)/(S*P*t*λ)*100%, where m represents the molar amount of the synthesis EG, N\nA A\nrepresents the Avogadro constant, h is Planck constant, c is the light speed, S stands for irradiation area,\nP represents the intensity of the irradiation light, t is irradiation time, and λ stands for wavelength of the\nlight35” was added.\nIn the supporting information:\nPage 3, line 6\n“The data of inductively coupled plasma optical emission spectroscopy (ICP-OES) was performed on\niCAP7400 of Thermo, China” was added.\nPage 11\nSupplementary Fig. 7 was changed.\nPage 15\nSupplementary Fig. 11 was changed.\nPage 15, line 2\n“The pronton adsorption…” was revised as “The proton adsorption…”.\nPage 24\nSupplementary Fig. 20 was changed.\nPage 33, line 2\n“Second derivative of the FITR spectra of …” was revised as “Second derivative of the FTIR spectra\nof …”.\nPage 40\nSupplementary Fig. 36 was added.\nPage 46\nSupplementary Table 2 was added.\nPage 47\nSupplementary Table 3 was added.\nPage 48\nSupplementary Table 4 was changed.\nResponse to comments\nReviewer #2: The reviewer is willing to recommend the revised manuscript to be\npublished in Nat. Commun.\nResponse: Thank the reviewer for the highly positive remarks and support of\npublication of this work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My concerns have been addressed, and the revised manuscript is recommended for publication.\nI have carefully reviewed the authors' revisions and their point-by-point replies to the previous concerns raised by Reviewer 3. In my opinion, the authors have adequately addressed the majority of the issues, and the revisions have significantly improved the clarity and scientific rigor of the manuscript.\nThere is only one minor point that still requires attention: the structure in Fig. R35 (Supplementary Fig. 7) appears to contain a persistent inaccuracy. I would kindly ask the authors to make the necessary corrections to this figure before the manuscript proceeds further.", "reviewer_2": "The manuscript describes a photo-enzyme-membrane (PEM) system coupled with multi-enzyme cascades for ethylene glycol (EG) synthesis. Despite revisions, the study lacks transformative novelty, exhibits unresolved contradictions in key data, and fails to convincingly demonstrate the system's practical advantage. Therefore, I do not support the publication of this work in the prestigious Nature Communications.\n1. The PEM design, featuring a capsule-like structure for light harvesting, cofactor regeneration, and enzyme stabilization, closely mirrors the authors' prior work on thylakoid-mimetic or capsule-based photoenzymatic systems. While incremental optimization (e.g., monomer ratios in PM) is presented, the fundamental architecture and operational principles (e.g., silica encapsulation for enzyme protection) are not sufficiently distinct from earlier publications to constitute a conceptual breakthrough.\n2. The proton diffusion experiment (Supplementary Fig. 13) uses a hydrochloric acid gradient to demonstrate proton adsorption by sulfonic-acid-functionalized PMs. However, this setup does not replicate actual catalytic conditions, where buffered solutions (pH 8.0) regulate proton activity. The claimed \"proton enrichment\" effect lacks validation under reaction-relevant pH dynamics, undermining its proposed role in enhancing NADH regeneration kinetics.\n3. The revised Fig. 2f shows an abrupt change in NADH regeneration yields compared to earlier versions, particularly for PM-BS21. This adjustment appears to force-fit the proton enrichment hypothesis.\n4. After Rh-complex immobilization, PM-BS30-Rh shows higher NADH yield than PM-BS21-Rh or PM-BS12-Rh (Supplementary Fig. 20a). The authors attribute this to \"more anchored Rh-complex\" in PM-BS30 but provide no quantitative evidence (e.g., ICP-MS for Rh loading). Without equal Rh-loading comparisons, the claim remains speculative. Moreover, PM-BS30's superiority contradicts the proposed proton/electron balance hypothesis, revealing a fundamental flaw in the PM design rationale.\n5. All PM-Rh samples show only ~80% 1,4-NADH selectivity (Supplementary Fig. 20b), an inefficiency problematic for enzymatic applications. Furthermore, the multi-enzyme system uses 10 mM NAD⁺ but produces only ~2.9 mM EG (Fig. 4c). Given the high cost of NAD+ cofactors, the system's economic viability is questionable.\n6. The PEM solely catalyzes the final steps, namely NADH regeneration and the transformation of glycolaldehyde to EG, while the conversion of methanol to glycolaldehyde occurs in a separate reactor via free enzymes (AOX/CAT/GALS). The authors' work thus focuses narrowly on NADH-dependent EG reduction, which is a well-established reaction, rather than an integrated platform for overall synthesis.\n7. The stability of alcohol dehydrogenase (ADH) is tested over only 1.32 hours (Fig. 4c), a timeframe too short to evaluate long-term viability. Without demonstrating ADH stability under prolonged operation or TTN evaluation, the EM's protective efficacy remains unsubstantiated."}, "author_response": "Detailed Response to the Reviewers’ Comments\nReviewer: 1\nComments:\nMy concerns have been addressed, and the revised manuscript is recommended for publication.\nI have carefully reviewed the authors' revisions and their point-by-point replies to the previous concerns\nraised by Reviewer 3. In my opinion, the authors have adequately addressed the majority of the issues,\nand the revisions have significantly improved the clarity and scientific rigor of the manuscript.\nThere is only one minor point that still requires attention: the structure in Fig. R35 (Supplementary Fig.\n7) appears to contain a persistent inaccuracy. I would kindly ask the authors to make the necessary\ncorrections to this figure before the manuscript proceeds further.\nAnswer:\nWe sincerely appreciate the reviewer’s positive remarks and valuable guidance. We are sorry that\nthere is an inaccuracy about Supplementary Fig. 7. Upon the reviewer’s request, the Supplementary\nFig. 7 in the Supplementary Information was revised and demonstrated as Fig. R1 (Revised\nSupplementary Information) below. We have also meticulously checked the entire text and have\nidentified and resolved the following issues.\nFig. R1 (Supplementary Fig. 7 in the Revised Supplementary Information). The possible structural\nunits of PM-BS12.\n“This suggested that light was the rate-determining step when light intensity was between 100 and\n200 mW/cm2” was revised to “This suggested that light absorption was the rate-determining step when\nlight intensity was between 100 and 200 mW/cm2” (page 6, line 20 in the Revised Manuscript).\nA mistake in the Supplementary Fig. 11 has been revised to Fig. R2.\nFig. R2 (Supplementary Fig. 11 in the Revised Supplementary Information). The proton adsorption\nenergy of PM-BS30, PM-BS21, and PM-BS12 calculated by DFT.\n“The pronton adsorption…” was revised as “The proton adsorption…” (page 15, line 2 in the\nRevised Supplementary Information).\n“A 100 W LED lamp was used as the light, the light was turned on for 60 min after dark treatment\nfor 5 min” was revised as “A 100 W LED lamp was used as the light source, and the light was turned\non for 60 min after dark treatment for 5 min” (page 14, line 23 in the Revised Manuscript).\n“Second derivative of the FITR spectra of …” was revised as “Second derivative of the FTIR spectra\nof …” (page 33, line 2 in the Revised Supplementary Information).\nReviewer: 2\nComments:\nThe manuscript describes a photo-enzyme-membrane (PEM) system coupled with multi-enzyme\ncascades for ethylene glycol (EG) synthesis. Despite revisions, the study lacks transformative novelty,\nexhibits unresolved contradictions in key data, and fails to convincingly demonstrate the system's\npractical advantage. Therefore, I do not support the publication of this work in the prestigious Nature\nCommunications.\nAnswer:\nMany thanks for the reviewer’s valuable guidance. We are regretful that we may fail to clearly convey\nour innovative points. In this regard, we would like to re-clarify the significance of our study. Multi-\nenzyme catalysis is a typical biomanufacturing process, which can achieve the selective transformation\nof raw materials into target products. In general, oxidoreductases that account for over 25% of industrial\nenzymes are usually involved in the process of multi-enzyme catalysis. Catalytic reactions enabled by\noxidoreductases strongly rely on the energy supply of cofactor (e.g., NADH) and stable enzymatic\nprocess to realize the chemicals synthesis. Addressing the issues of insufficient energy supply and\nunstable enzymatic processes in oxidoreductase catalysis is thus of great importance for multi-enzyme\ncatalysis process.\nIn our study, a photo-enzyme-membrane (PEM) platform with efficient NADH regeneration and\nstable enzymatic process is constructed. Covalent organic polymer membrane as photo-membrane (PM)\naffords efficient NADH supply through synergistic intensification of electron transfer and proton\ntransfer. Meanwhile, the construction of enzyme-membrane (EM) mitigates the adverse effects of the\nPM on enzyme activity. Then, the synthesis of EG from methanol catalyzed by multi-enzymes of alcohol\noxidase (AOX), catalase (CAT), glycolaldehyde synthase (GALS), and alcohol dehydrogenase (ADH,\nthe key oxidoreductase) is just chosen as a representative case to demonstrate the superiority of our\nPEM platform. As reported in previous literatures (Nat. Commun., 2025, 16, 10756; ACS Catal., 2024,\n14, 4665; Nat. Commun., 2025, 16, 8079), many multi-enzymatic pathways require cofactors, including\nthe synthesis of lactic acid from methanol, the synthesis of non-canonical amino acids from glycerol,\netc., which further highlights the importance and universality of our PEM.\nAs for the reviewer’s doubts about the unresolved key contradictions, we have carefully revised and\nrefined the manuscript to provide more solid evidence and improve the overall quality. The detailed\npoint-by-point response is provided below. The corresponding information can be found in the Revised\nManuscript and Revised Supplementary Information.\nQuestion 1:\nThe PEM design, featuring a capsule-like structure for light harvesting, cofactor regeneration, and\nenzyme stabilization, closely mirrors the authors' prior work on thylakoid-mimetic or capsule-based\nphotoenzymatic systems. While incremental optimization (e.g., monomer ratios in PM) is presented,\nthe fundamental architecture and operational principles (e.g., silica encapsulation for enzyme protection)\nare not sufficiently distinct from earlier publications to constitute a conceptual breakthrough.\nAnswer:\nWe agree to the reviewer’s viewpoint that the capsule-like structure of PEM and our previous work\nare both designed inspired by the structure of thylakoids in natural photosynthesis. However, the\nphotocatalyst materials, the scientific problem, and the core innovations are fundamentally distinct.\nFirstly, the titania capsule in our previous work was prepared through a calcium carbonate template\nmethod, where the capsule size was 2.5-3.5 μm. In our current work, a covalent organic polymer capsule\nwas prepared through a silica template method. The capsule size was about 400~500 nm, which was\nsmaller than the titania capsule in our previous work. It was difficult to realize precise tunability in\nmolecular scale for the titania capsule. By contrast, the structure of covalent organic polymer capsules\nin molecular scale can be regulated by changing the monomers. The structural versatility enabled the\nmodulation of the photoelectrochemical properties, thereby optimizing the photocatalytic NADH\nregeneration efficiency.\nIn addition, the previous work focused on molecule transfer between photocatalysis and enzyme\ncatalysis. NAD+/NADH molecules was shuttled through the titania capsule wall, which finally realized\nthe conversion of propanal to propanol. In our current work, the design of PM focused on enhancing\nelectron and proton transfers in photocatalytic NADH regeneration, where the bipyridine moiety\nmediated fast electron transfer from the birth site, and the sulfonic acid moiety facilitated proton transfer\nby enriching protons.\nFinally, in our previous work and other literatures, researchers focused on enhancing electron transfer\nin photocatalytic NADH regeneration and molecule transfer in enzyme-photo-coupled catalysis (J. Am.\nChem. Soc., 2022, 144, 4268; ACS Catal., 2021, 11, 476; Angew. Chem. Int. Ed., 2022, 61,\ne202200261; J. Am. Chem. Soc. 2020, 142, 5958-5963). Most researchers ignore the importance of\nproton transfer in photocatalytic NADH regeneration. Our current work primarily emphasizes the\ncoordination between electron transfer and proton transfer in photocatalytic NADH regeneration.\nBesides, few studies have harnessed photocatalysts with enhanced electron and proton transfer,\nintegrated with multi-enzyme catalysis, to drive the synthesis of final products from raw materials.\nTherefore, our current work is not a simple combination, but an advancement in the field of artificial\nphotosynthesis.\nQuestion 2:\nThe proton diffusion experiment (Supplementary Fig. 13) uses a hydrochloric acid gradient to\ndemonstrate proton adsorption by sulfonic-acid-functionalized PMs. However, this setup does not\nreplicate actual catalytic conditions, where buffered solutions (pH 8.0) regulate proton activity. The\nclaimed \"proton enrichment\" effect lacks validation under reaction-relevant pH dynamics, undermining\nits proposed role in enhancing NADH regeneration kinetics.\nAnswer:\nUpon the reviewer’s request, we would like to explain the reason for using a hydrochloric acid\ngradient to demonstrate proton adsorption by sulfonic-acid-functionalized PMs. In acidic solutions with\nhigh proton concentrations, the variation of proton adsorption by PMs is easier to observe. This\ncondition indeed deviates from the actual reaction conditions. The actual reaction system is conducted\nin a PBS buffer to maintain a stable pH value. Based on the reviewer’s valuable guidance, PM-PAN\nmembrane is set at the diffusion cell, where TEOA/PBS buffer (pH 8.0) solution and aqueous solution\nwere separately added to both sides. As shown in Fig. R3, the pH value of the solution is monitored\nover time. The overall pH value of the solution did not change significantly.\nIn our work, the diffusion cell experiment demonstrates that PM exposure to a high-concentration\nproton source induces a measurable change in proton concentration. Owing to the stable pH value\nprovided by PBS buffer in the actual catalytic conditions, it is hard to directly monitor local proton\nadsorption in real time.\nConsequently, the proton transfer facilitated by our PM is inferred indirectly through two primary\nlines of evidence: (i) the measured increase in the proton conductivity of PMs (Fig. R4 (Fig. 2c in the\nRevised Manuscript)), and (ii) DFT analysis of proton adsorption energy, which provides insight into\nthe thermodynamic driving force for proton adsorption (Fig. R2 (Supplementary Fig. 11 in the\nRevised Supplementary Information)).\n8.40\nPM-BS12\nPM-BS21\n8.35 PM-BS30\ne\nu\nal\n8.30\nv\nH\np\n8.25\n8.20\n0 5 10 15 20 25 30\nTime (min)\nFig. R3. The change of pH value by different PM-PAN membrane: PM-BS12-PAN, PM-BS21-PAN,\nand PM-BS30-PAN.\n1\n)200\n-m\nc\nS 160\nm\n(\ny\nt120\nvi\nti\nc\nu\nd 80\nn\no\nc\nn\no 40\nt\no\nP r 0\nPM-BS12 PM-BS21 PM-BS30\nFig. R4 (Fig. 2c in the Revised Manuscript). The proton conductivity of PM-BS30, PM-BS21,\nand PM-BS12 under 40oC, and 93% humidity.\nFig. R2 (Supplementary Fig. 11 in the Revised Supplementary Information). The proton adsorption\nenergy of PM-BS30, PM-BS21, and PM-BS12 calculated by DFT.\nQuestion 3:\nThe revised Fig. 2f shows an abrupt change in NADH regeneration yields compared to earlier\nversions, particularly for PM-BS21. This adjustment appears to force-fit the proton enrichment\nhypothesis.\nAnswer:\nIndeed, as the reviewer pointed out, the NADH regeneration yields have been changed compared to\nthe earlier version. In the current version, we replaced the monomers required in the PM synthesis\nprocess and repeated the experiment three times, obtaining the average results shown in the Fig. R5\n(Fig. 2f in the Revised Manuscript). We infer that the change in performance can be attributed to\nvariations in the monomeric composition of the synthesized samples.\nThe original data can be found in Fig. R6 and the file of source data.\nPM-BS21\n80\n% ) PM-BS30\n(\nH 60\nD\nA\nN\nf\n40\no\nd\nel 20\ni\nY\n0\n0 4 8 12 16\nTime (min)\nFig. R5 (Fig. 2f in the Revised Manuscript). The NADH regeneration yield catalyzed by PM-BS12,\nPM-BS21, and PM-BS30.\nFig. R6. The original data of NADH regeneration yield catalyzed by PM-BS12, PM-BS21, and PM-\nBS30.\nQuestion 4:\nAfter Rh-complex immobilization, PM-BS30-Rh shows higher NADH yield than PM-BS21-Rh or\nPM-BS12-Rh (Supplementary Fig. 20a). The authors attribute this to \"more anchored Rh-complex\" in\nPM-BS30 but provide no quantitative evidence (e.g., ICP-MS for Rh loading). Without equal Rh-\nloading comparisons, the claim remains speculative. Moreover, PM-BS30's superiority contradicts the\nproposed proton/electron balance hypothesis, revealing a fundamental flaw in the PM design rationale.\nAnswer:\nWe are sorry for the missing of ICP-based quantification for the Rh loading. Based on the reviewer’s\nvaluable guidance, we first optimized the preparation process of PM-Rh. The PM and Rh-complex were\nfully mixed through ultrasonication, so as to effectively improve the Rh-complex loading. First, by\nadding sufficient Rh-complex for PM-Rh synthesis, PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-\n0.43 0.82\nRh were obtained. ICP test was shown in Table R1(Supplementary Table 2 in the Revised\n1.34\nSupplementary Information). Rh-complex was anchored on bipyridine group, the Rh element loading\namounts of PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh was in a mass ratio of ~1:2:3. As\n0.43 0.82 1.34\nshown in Fig. R7 (Supplementary Fig. 20a in the Revised Supplementary Information), PM-BS30-\nRh showed the highest NADH regeneration yield owing to the presence of more catalytic active site\n1.34\nRh-complex. Then, PMs with the equal amount of Rh element loading was prepared. The loading of Rh\nelement was shown in Table R2 (Supplementary Table 3 in the Revised Supplementary\nInformation), and the Rh element content in the PM was almost consistent. As shown in Fig. R8\n(Supplementary Fig. 20b in the Revised Supplementary Information), PM-BS21-Rh exhibited\n0.50\nthe highest NADH regeneration performance. It demonstrated that enhancing electron and proton\ntransfer can realize efficient NADH regeneration under equal Rh element loading amount. The tendency\nwas consistent with the performance of NADH regeneration using equal amount of free Rh-complex\n(Fig. R5 (Fig. 2f in the Revised Manuscript)), which demonstrated the feasibility of improving NADH\nregeneration efficiency through coordinating electron and proton transfers.\nTable R1 (Supplementary Table 2 in the Revised Supplementary Information). The ICP data of\nPM-Rh.\nThe content of\nThe Rh content\nPhotocatalyst Rh (ppm)/2 mg\nin the PM (%)\nPM\nPM-BS12-Rh 0.3420 0.4275\nPM-BS21-Rh 0.6529 0.8161\nPM-BS30-Rh 1.0690 1.3363\n0.43\nPM-BS21-Rh\n) 80 0.82\n% PM-BS30-Rh\n( 1.34\nH 60\nD\nA\nN\nf\n40\no\nd\nel 20\ni\nY\n0\n0 5 10 15 20\nTime (min)\nFig. R7 (Supplementary Fig. 20a in the Revised Supplementary Information). The NADH\nregeneration yield catalyzed by PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh .\n0.43 0.82 1.34\nTable R2 (Supplementary Table 3 in the Revised Supplementary Information). The ICP data of\nPM-Rh.\nThe content of\nThe Rh content\nPhotocatalyst Rh (ppm)/2 mg\nin the PM (%)\nPM\nPM-BS12-Rh 0.3420 0.4275\nPM-BS21-Rh 0.3973 0.4966\nPM-BS30-Rh 0.3904 0.4880\n0.43\nPM-BS21-Rh\n%\n)60\nPM-BS30-Rh\n0.50\n( 0.49\nH\nD\nA40\nN\nf\no\nd 20\nel\nY\n0\n0 5 10 15 20 25\nTime (min)\nFig. R8 (Supplementary Fig. 20b in the Revised Supplementary Information). The NADH\nregeneration yield catalyzed by PM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh .\n0.43 0.50 0.49\nQuestion 5:\nAll PM-Rh samples show only ~80% 1,4-NADH selectivity (Supplementary Fig. 20b), an\ninefficiency problematic for enzymatic applications. Furthermore, the multi-enzyme system uses 10\nmM NAD⁺ but produces only ~2.9 mM EG (Fig. 4c). Given the high cost of NAD+ cofactors, the\nsystem's economic viability is questionable.\nAnswer:\nThank the reviewer for the valuable guidance. The enzymatic method was used to detect the\nselectivity of 1,4-NADH. The absorbance of the reaction at 340 nm would decrease arising by the\nconsumption of NADH when the appropriate amount of propanal and ADH added. The selectivity of\n1,4-NADH was demonstrated by the ratio of the change in absorbance to the initial absorbance.\nThe initial reaction rate was defined by the equation of (N-N )/(m*t), in which N and N meant the molar\nt 0 0 t\nmass of generated NADH production in certain time, m represented the quality of photocatalyst involved\nin the reaction and t stand for the time of photocatalytic reaction. We extended the reaction time between\nthe addition of propanal/ADH and the generated NADH to 5 minutes. As shown in the Fig, R9\n(Supplementary Fig. 20c, d in the Revised Supplementary Information), the selectivity has\nincreased by approximately 5% compared to the original level, but it still does not reach 100%.\nPrevious studies on immobilized Rh photocatalysts have largely overlooked the issue of selectivity (J. Am.\nChem. Soc., 2024, 146, 586; Angew. Chem. Int. Ed., 2022, 61, e202206283). In contrast, our experimental\nresults indicate that a fraction of electrons and protons can bypass the Rh active sites, directly reducing\nNAD⁺ to form various by-products, including NAD⁺ dimers, 1,2-NADH, and 1,6-NADH. This diversion\nfacilitates non-productive side reactions, giving rise to NADH-derived byproducts and thus\ncompromising overall selectivity.\nFig. R9 (Supplementary Fig. 20c, d in the Revised Supplementary Information). The selectivity of\nNADH catalyzed by a) PM-BS12 , PM-BS21 , PM-BS30 and b) PM-BS12 , PM-BS21 ,\n0.43 0.82 1.34 0.43 0.50\nPM-BS30 .\n0.49\nAs for the PEM coupled with multi-enzyme cascade, the low catalytic activity of GALS is the rate-\nlimiting step in the EG synthesis process. The key to increasing the output of EG lies in enhancing the\nactivity of GALS. We further purified GALS and obtained a more concentrated enzyme solution. As\nshown in Fig. R10 (Supplementary Fig. 36b in the Revised Supplementary Information), different\nconcentration of NAD+ (2, 5, and 10 mmol L-1) was used for the EG synthesis under higher\nconcentration of GALS (3 g L-1). Finally, a 5.1 mmol L-1 EG is obtained with the continuous addition\nof methanol. In particular, 3.1 mmol L-1 EG is obtained in the presence of 2 mmol L-1 NAD+,\nwhich exhibits the system's economic viability.\n10\n) PEM & multi-enzyme cascade (2 mmol L-1 NAD+)\n- 1 L PEM & multi-enzyme cascade (5 mmol L-1 NAD+)\nol 8 PEM & multi-enzyme cascade (10 mmol L-1 NAD+)\nm Blank\nm Light on Light off\n6\n(\nn\no\nc\nti\n4\nu\nd\no\nr 2\np\nG\nE\n0\n0 1 2 3 4\nCycle number\nFig. R10 (Supplementary Fig. 36b in the Revised Supplementary Information). The production of\nEG by the coupled system with TEOA as electron donor under different concentration of NAD+ (2, 5,\nand 10 mmol L-1). Each cycle consists of 0.33 hour of light followed by 2.67 hours of darkness.\nQuestion 6:\nThe PEM solely catalyzes the final steps, namely NADH regeneration and the transformation of\nglycolaldehyde to EG, while the conversion of methanol to glycolaldehyde occurs in a separate reactor\nvia free enzymes (AOX/CAT/GALS). The authors' work thus focuses narrowly on NADH-dependent\nEG reduction, which is a well-established reaction, rather than an integrated platform for overall\nsynthesis.\nAnswer:\nThank the reviewer for the valuable guidance. Alcohol dehydrogenase (ADH) and NADH catalyze\nthe reduction of formaldehyde to methanol, which is unfavorable for the conversion of glycolaldehyde\nto EG. Therefore, we adopted a strategy of separating the multi-enzymatic cascade and PEM.\nThis approach enables continuous synthesis of EG while minimizing undesirable side reactions. Upon the\nreviewer’s request, a well-established reactor with dual-channel was constructed for continuous EG\nsynthesis, where the enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion\nchannel and PEM filtrated to form a PEM-PAN membrane was put in the photo-enzyme catalytic\ndiversion channel (Fig. R11 (Fig. 4c in the Revised Manuscript)). As shown in Fig. R12 (Fig. 4b, c\nin the Revised Manuscript), 0.71 mmol L-1 EG production with an initial generate rate of 2.43 mmol\ng -1 h-1 was obtained after ten light-dark cycles. This is a preliminary attempt, and the product yield\nPEM\ncan be further improved by optimizing the structure of the set-up and conditions of the coupled reaction.\nThe design of the reactor enables the scalable, integrated enzyme-photo coupled catalytic conversion of\nfeedstocks into valuable chemicals.\nFig. R11 (Fig. 4c in the Revised Manuscript). The images of a) PEM-PAN membrane and b) the dual-\nchannel reactor for EG synthesis.\nFig. R12 (Fig. 4b, c in the Revised Manuscript). a) The schematic of the dual-channel reactor for EG\nsynthesis. b) The production of EG by the dual-channel reactor with TEOA as electron donor. Here, the\nsystem containing PM-PAN membrane was set as blank, where PE adsorbed on PAN to form PE-PAN\nmembrane through vacuum filtration. Each cycle consists of one hour of light followed by three hours\nof darkness.\nQuestion 7:\nThe stability of alcohol dehydrogenase (ADH) is tested over only 1.32 hours (Fig. 4c), a timeframe\ntoo short to evaluate long-term viability. Without demonstrating ADH stability under prolonged\noperation or TTN evaluation, the EM's protective efficacy remains unsubstantiated.\nAnswer:\nBased on the reviewer’s valuable guidance, the long-term stability of immobilized ADH was tested\nunder ten light-dark cycle (ten hours illumination time). As shown in Fig. R13 (Fig. 4c in the Revised\nManuscript), with the protection of ADH by EM, the EG production catalyzed by PEM had continued\nto increase during the ten light-dark cycle.\nThe total turnover number (TTN) was also be calculated. TTN referred to n /n , which was\nproduct catalyst\ncalculated as 1668. n referred to the molar mass of the production of EG and n referred to the\nproduct catalyst\nmolar mass of ADH.\n1.0\n) EG synthesis\n1\n-L\nLight on Light off\nol\n0.8\nm\nm\n0.6\n(\nn\no\nti\n4\nu\nd\no\nr0.2\np\nG\nE0.0\n0 1 2 3 4 5 6 7 8 9 10\nCycle number\nFig. R13 (Fig. 4c in the Revised Manuscript). The production of EG by the dual-channel reactor under\nthe ten light-dark cycle, where each cycle consists of one hour of light followed by three hours of\ndarkness.\nA list of major changes\nIn the Revised Manuscript:\nPage 1, line 30\n“Further, PEM is coupled with alcohol oxidase, catalase, and glycolaldehyde synthase for sustainable\nsynthesis of EG with a rate of 2.6 mmol g -1 h-1 by continuous supply of methanol” was revised as\nPEM\n“Further, a dual-channel reactor is constructed for sustainable synthesis of EG with an initial synthesis\nrate of 2.43 mmol g -1 h-1 by continuous supply of methanol”.\nPEM\nPage 3, line 22\n“an EG production of 2.9 mmol L-1 with the synthesis rate of 2.6 g -1 h-1 was obtained with the\nPEM\ncontinuous supply of methanol” was revised as “an EG production of 0.71 mmol L-1 with the initial\nsynthesis rate of 2.43 mmol g -1 h-1 was obtained with the continuous supply of methanol”.\nPEM\nPage 6, line 20\n“This suggested that light was…” was revised as “This suggested that light absorption was…”.\nPage 8, line 18\n“PM-Rh were first performed for NADH regeneration, where Rh-complex as the active site of NADH\nregeneration was anchored on dipyridyl group. Therefore, PM-BS30-Rh with more anchored Rh-\ncomplex exhibited a higher NADH regeneration yield than PM-BS12-Rh and PM-BS21-Rh\n(Supplementary Fig. 20a). Meanwhile, all PM-Rh samples showed over 70% selectivity of 1,4-NADH\n(Supplementary Fig. 20b)” was revised as “First, by adding sufficient Rh-complex for PM-Rh synthesis,\nPM-BS12-Rh , PM-BS21-Rh , and PM-BS30-Rh were obtained. Inductively coupled plasma\n0.43 0.82 1.34\noptical emission spectroscopy (ICP-OES) test was shown in Supplementary Table 2. Rh-complex was\nanchored on bipyridine group, while the Rh element loading amounts of PM-BS12-Rh , PM-BS21-\n0.43\nRh , and PM-BS30-Rh was in a mass ratio of ~1:2:3. Therefore, PM-BS30-Rh with more\n0.82 1.34 1.34\nanchored Rh-complex exhibited a higher NADH regeneration yield than PM-BS12-Rh and PM-\n0.43\nBS21-Rh (Supplementary Fig. 20a, c). Furthermore, PM with equal amount of Rh element loading\n0.82\nwas also prepared. The loading of Rh element was shown in Supplementary Table 3, and the Rh element\ncontent in the PM was almost consistent. As shown in Supplementary Fig. 20b, d, PM-BS21-Rh\n0.50\nexhibited the highest NADH regeneration performance with the selectivity of ~90%”.\nPage 11, line 12\n“With continuous feeding from the enzymatic reactor to the PEM reactor, 2.9 mmol L-1 of EG was\nobtained, with a synthesis rate of 2.6 mmol g -1 h-1, using TEOA after four light and dark cycles. The\nPEM\nEG production was moderate compared to other systems (Supplementary Table 2)” was revised as\n“With continuous feeding from the enzymatic reactor to the PEM reactor, 5.1 mmol L-1 of EG at a NAD+\nconcentration of 10 mmol L-1 was obtained, using TEOA after four light and dark cycles. In particular,\n3.1 mmol L-1 EG was obtained in the presence of 2 mmol L-1 NAD+, which exhibits the system's\neconomic viability”.\nPage 11, line 19\n“In order to integrate multi-enzyme cascade and PEM, a well-established reactor with dual channels\nwas also constructed for continuous EG synthesis. The enzymes (AOX/CAT/GALS) were put in the\nmulti-enzyme cascade diversion channel and PEM filtrated to form a PEM-PAN membrane was put in\nthe photo-enzyme catalytic diversion channel. As shown in Fig. 4b, c, 0.71 mmol L-1 EG production\nwith an initial synthesis rate of 2.43 mmol g -1 h-1 was obtained after ten light-dark cycles. The reactor\nPEM\nshowed a long-term viability, and the total turnover number (TTN) was calculated as 1668. The EG\nproduction was moderate compared to other systems (Supplementary Table 4). The design of the reactor\nenabled the scalable, integrated enzyme-photo coupled catalytic conversion of feedstocks into valuable\nchemicals” was added.\nPage 12, line 1\nFig. 4 was changed.\nPage 12, line 3\n“b, The coupled system comprising a multi-enzyme cascade in the enzymatic reactor and a PEM in\nPEM reactor for the production of EG. In this system, the generated GALD from the enzymatic reactor\ndiffuses into the PEM reactor, where it was converted to EG. c, The production of EG by the coupled\nsystem with TEOA as electron donor. Here, the multi-enzyme cascade coupled with PM and free ADH\nand was used as blank” was revised as “b, The dual-channel reactor comprising a multi-enzyme cascade\ndiversion channel and a photo-enzyme catalytic diversion channel for the production of EG. In this\nreactor, methanol is continuously pumped into the reactor via a peristaltic pump. The generated GALD\nfrom the multi-enzyme cascade diversion channel diffuses into the photo-enzyme catalytic diversion\nchannel, where it was converted to EG. c, The production of EG by the dual-channel reactor with TEOA\nas electron donor, where each cycle consists of one hour of light followed by three hours of darkness.\nHere, the system containing PM-PAN membrane was set as blank, where PE adsorbed on PAN to form\nPE-PAN membrane through vacuum filtration. The insert images were the schematic of PEM-PAN\nmembrane and the dual-channel reactor”.\nPage 12, line 20\n“Through synergistic optimization of multi-enzyme cascade and PEM, 2.9 mmol L-1 EG with the\nsynthesis rate of 2.6 mmol g -1 h-1 is obtained with the continuous addition of methanol, where the\nPEM\nphotonic quantum efficiency can reach 0.30%” was revised as “Through synergistic optimization of\nmulti-enzyme cascade and PEM, 0.71 mmol L-1 EG with the initial synthesis rate of 2.43 mmol g -1\nPEM\nh-1 is obtained with the continuous addition of methanol in a dual-channel reactor, where the photonic\nquantum efficiency can reach 0.15%”.\nPage 13, line 19\n“Specifically, 10 mg of PM-BS21 was added into 2 mL of deionized water, followed by the addition of\n6.64 mg of (Cp*RhCl ) . Then the mixture was stirred for 24 h, following by centrifugation, rinsed with\n2 2\ndeionized water several times, and freeze-dried. The obtained solid was denoted as PM-BS21-Rh” was\nrevised as “Specifically, 10 mg of PM-BS21 with 4 mL of deionized water was sonicated for 10 minutes,\nfollowed by the addition of different mass of (Cp*RhCl ) . Then the mixture was stirred for 24 h,\n2 2\nfollowing by centrifugation, rinsed with deionized water several times, and freeze-dried. The obtained\nsolid was denoted as PM-BS12-Rh , PM-BS21-Rh , and PM-BS30Rh , where x referred to the content\nx x x\nof Rh element. The Rh element content of PM was characterized by ICP test”.\nPage 14, line 23\n“A 100 W LED lamp was used as the light, the light was turned on for 60 min after dark treatment for\n5 min” was revised as “A 100 W LED lamp was used as the light source, and the light was turned on\nfor 60 min after dark treatment for 5 min”.\nPage 14, line 32\n“NAD+ (10 mmol L-1)” was revised as “NAD+ (2, 5, and 10 mmol L-1)”.\nPage 15, line 3\n“A dual-channel reactor was well-established for EG synthesis. The reactor consisted of five parts: upper\nfixed plate, protective gasket, quartz stone window, photo-enzyme catalytic diversion channel, multi-\nenzyme cascade diversion channel, and temperature control water channel cover plate. A 100 ml solution\ncontaining methanol, MgSO , ThDP, TEOA/PBS, and NAD+ was continuously pumped into the reactor\n4\nvia a peristaltic pump. The enzymes (AOX/CAT/GALS) were put in the multi-enzyme cascade diversion\nchannel and PEM filtrated to form a PEM-PAN membrane was put in the photo-enzyme catalytic\ndiversion channel. The reactor was carried out for EG synthesis for ten light and dark cycles. The\nillumination time of each cycle was one hour, and the dark processing time was three hours.\nThe initial synthesis rate of EG is calculated by n /(m *t), where n refers to the mole mass of\nEG PEM EG\ngenerated EG under first one hour light illumination, m refers to mass of PEM, and t refers to light\nPEM\nillumination (one hour). The total turnover number (TTN) is calculated by n /n , where n\nproduct catalyst product\nrefers to the molar mass of the production of EG and n refers to the molar mass of ADH. The\ncatalyst\nphotonic quantum efficiency in the system of EG production from methanol is calculated by the equation\nof (2*m*N *h*c)/(S*P*t*λ)*100%, where m represents the molar amount of the synthesis EG, N\nA A\nrepresents the Avogadro constant, h is Planck constant, c is the light speed, S stands for irradiation area,\nP represents the intensity of the irradiation light, t is irradiation time, and λ stands for wavelength of the\nlight35” was added.\nIn the supporting information:\nPage 3, line 6\n“The data of inductively coupled plasma optical emission spectroscopy (ICP-OES) was performed on\niCAP7400 of Thermo, China” was added.\nPage 11\nSupplementary Fig. 7 was changed.\nPage 15\nSupplementary Fig. 11 was changed.\nPage 15, line 2\n“The pronton adsorption…” was revised as “The proton adsorption…”.\nPage 24\nSupplementary Fig. 20 was changed.\nPage 33, line 2\n“Second derivative of the FITR spectra of …” was revised as “Second derivative of the FTIR spectra\nof …”.\nPage 40\nSupplementary Fig. 36 was added.\nPage 46\nSupplementary Table 2 was added.\nPage 47\nSupplementary Table 3 was added.\nPage 48\nSupplementary Table 4 was changed.\nResponse to comments\nReviewer #2: The reviewer is willing to recommend the revised manuscript to be\npublished in Nat. Commun.\nResponse: Thank the reviewer for the highly positive remarks and support of\npublication of this work."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The reviewer is willing to recommend the revised manuscript to be published in Nat. Commun."}, "author_response": "Response: Thank the reviewer for the highly positive remarks and support of\npublication of this work."}]

Decision letter