Method for regenerating coenzyme NADH (Nicotinamide Adenine Dinucleotide Hydrocarbon) through in-vitro light driving of thylakoid

Abstract

The invention provides a method for regenerating NADH (Nicotinamide Adenine Dinucleotide) by using an in-vitro light-driven thylakoid membrane, which comprises the following steps: carrying out in-vitro coupling on a spinach thylakoid membrane and Ferredoxin: NAD + reductase, and realizing NADH regeneration through light energy driving. Compared with a currently reported coenzyme NADH regeneration method, the method has the advantages of being simple, high in regeneration rate, high in controllability, capable of avoiding accumulation of by-products, low in cost, environmentally friendly and the like, and has wide application prospects in the aspects of medicine production, industrial manufacturing, high-value chemical synthesis and the like.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for regenerating coenzyme NADH by light-driven thylakoid membrane in vitro. Background Art

[0002] NADH (nicotinamide adenine dinucleotide, reduced form), as one of the important cofactors of biological oxidoreductases, plays a key role in various metabolic reactions in organisms. Oxidoreductases are the largest of the six major enzymes classified in enzymology. Because of their good specificity and selectivity in the catalytic process, they are widely used in the fields of pharmaceutical production, industrial manufacturing, and high-value chemical synthesis. At present, the known oxidoreductases with NADH as a cofactor account for about 80% of all oxidoreductases (Chem. Rev. 2011, 111, 4111-4140.), and play a vital role in the reduction of carbonyls, carboxylic acids, C=C double bonds, nitro groups, and CN multiple bonds (Chem, 2017, 2, 621-654.). For example, alcohol dehydrogenase ADH in oxidoreductase can catalyze the reduction of various carbonyl compounds (including aromatic ketones, aliphatic ketones, ketoesters and haloketones, etc.) into optically active alcohols (Org. Biomol. Chem. 2010, 8, 1540-1550.). In pharmaceutical production, NADH-dependent alcohol dehydrogenase is used to efficiently reduce 3,5-bis(trifluoromethyl)acetophenone (BTAP) to synthesize an important chiral intermediate of the antiemetic drug aprepitant (J. Mol. Catal. B: Enzym. 2004, 28, 25-32.). Izumori et al. in Japan used arabinitol dehydrogenase (NADH-dependent) to catalyze arabinitol to produce L-xylulose (J. Ferment. Bioeng. 1991, 72, 488-490.). However, NADH is expensive, and an effective regeneration method is essential. Cofactor regeneration can not only greatly reduce the reaction cost, but also improve the reaction efficiency, while avoiding the complex preparation, separation and extraction process of the cofactor. Therefore, NADH regeneration has also become a key technology in many industrial and research applications. Technological innovations around NADH regeneration have emerged around the world. At present, NADH regeneration methods can be divided into four categories, namely chemical reduction, electrochemical method, bioenzyme method and photocatalytic regeneration method. Chemical reduction is one of the main methods used for NADH regeneration in the early days. Chemical reduction usually uses hydrogen as a reducing agent to convert NAD +The method has the problems of harsh reaction conditions, high toxicity of reducing agents and difficulty in controlling by-products, which limits its widespread promotion in large-scale industrial applications. The bioenzymatic method usually uses formate dehydrogenase and glucose dehydrogenase to catalyze the NADH regeneration reaction: Formate dehydrogenase (FDH): Formate dehydrogenase catalyzes the oxidation of formate to produce carbon dioxide and converts NAD + Reduced to NADH (Moscow Univ. Chem. Bull. 2023, 78, 151-169.); Glucose dehydrogenase (GDH): Glucose dehydrogenase catalyzes the oxidation of glucose to produce gluconolactone and simultaneously converts NAD + Reduction to NADH (Chembiochem. 2020, 21(18), 2680-2688.). However, this method usually requires sacrificial substrates such as formic acid and glucose, and has poor atom economy. Electrochemical regeneration of NADH includes indirect electrochemistry and direct electrochemistry (Chem, 2017, 2(5): 621-654.). Direct electrochemical regeneration of NADH is to apply a high overpotential to the working electrode to directly convert NAD + However, high overvoltage will not only convert NAD + It is converted into 1,4-NADH with biological enzyme catalytic activity, and it is also converted into byproducts without biological enzyme catalytic activity: 1,2-NADH, 1,6-NADH and dimer (NAD2), which is difficult to promote and apply. Indirect electrochemical regeneration of NADH is to introduce an electron mediator into the direct electrochemical system to complete the NAD under a lower applied voltage. + The photocatalytic method uses light energy to drive the catalytic reaction and regenerate NADH. The photocatalyst absorbs photons to generate electron-hole pairs, which participate in the NAD + The reduction reaction is used to reduce it to NADH. Commonly used photocatalysts include metal oxides (such as TiO2), metal sulfides (such as CdS) and organic photocatalysts (such as phthalocyanine compounds) (Progress in Chemistry, 2022, 34 (11), 2351-2360.). However, these methods have problems such as high energy consumption, low product purity / quality, high equipment requirements, high investment and production costs, and environmental pollution. Therefore, it is urgent to develop a NADH biological regeneration method with simple regeneration method, strong controllability, high safety, and green environmental protection. Summary of the invention

[0003] In order to improve the above technical problems, the present invention provides a method for regenerating NADH by light-driven thylakoid membrane in vitro, comprising:

[0004] (1) Isolation and extraction of thylakoid membranes;

[0005] (2) Ferredoxin (Fdx, Ferredoxin) and Ferredoxin:NAD + Expression and purification of reductase (MaFNR);

[0006] (3) The thylakoid membrane is coupled to MaFNR and mixed with Fdx in a reaction system to perform light-driven thylakoid membrane regeneration of NADH.

[0007] In one embodiment, the aforementioned thylakoid membrane is a thylakoid membrane derived from spinach;

[0008] Preferably, the Fdx is Fdx derived from spinach, and the MaFNR is Ferredoxin:NAD derived from Marinomonas mediterranea. + Reductase.

[0009] In one embodiment, HEPES buffer, magnesium salt, potassium salt, hydrogen phosphate, ADP, betaine, thylakoid membrane, sodium ascorbate and cofactors are also added to the reaction system of the aforementioned step (3).

[0010] In one embodiment, the pH of the HEPES buffer is 7.6;

[0011] Preferably, the concentration of the HEPES buffer is 50 mM.

[0012] In one embodiment, the magnesium salt is magnesium chloride or magnesium sulfate;

[0013] Preferably, the magnesium salt is magnesium chloride;

[0014] More preferably, the concentration of the magnesium salt is 5 mM.

[0015] In one embodiment, the potassium salt is potassium chloride or potassium sulfate,

[0016] Preferably, the potassium salt is potassium chloride;

[0017] More preferably, the concentration of the potassium salt is 10 mM.

[0018] In one embodiment, the hydrogen phosphate salt is dipotassium hydrogen phosphate or disodium hydrogen phosphate, etc.

[0019] Preferably, the hydrogen phosphate salt is dipotassium hydrogen phosphate;

[0020] More preferably, the concentration of the hydrogen phosphate is 10 mM.

[0021] In one embodiment, the cofactor is NAD + ;

[0022] Preferably, the NAD + The concentration of the cofactor is 1-10 mM.

[0023] In one embodiment, the temperature of the light-driven thylakoid membrane regeneration NADH reaction is 30°C.

[0024] In one embodiment, the light intensity of the light-driven thylakoid membrane regeneration NADH reaction is 50-100 μmol photons m -2 s -1 .

[0025] In one embodiment, the time of the light-driven thylakoid membrane regeneration NADH reaction is 0, 10, 20, 30, 40, or 50 minutes.

[0026] In one embodiment, the concentration of ADP in the light-driven thylakoid membrane regeneration NADH reaction is 3-30 mM.

[0027] In one embodiment, the amount of sodium ascorbate used in the light-driven thylakoid membrane regeneration NADH reaction is 1-10 mM.

[0028] In one embodiment, the amount of betaine used in the light-driven thylakoid membrane regeneration NADH reaction is 0.1-0.5M.

[0029] In one embodiment, the amount of Fdx used in the light-driven thylakoid membrane regeneration NADH reaction is 5-10 μM.

[0030] In one embodiment, the amount of thylakoid membrane used in the light-driven thylakoid membrane regeneration NADH reaction is 10-20 μg chlorophyll equivalent / mL.

[0031] In one embodiment, the amount of MaFNR used in the light-driven thylakoid membrane regeneration of NADH is 0-4 μM.

[0032] Beneficial Effects

[0033] The present invention relates to a method for regenerating NADH by using extracorporeal light-driven thylakoid membranes. The method comprises: +The reductase is coupled in vitro and NADH is regenerated by light energy. Compared with the currently reported coenzyme NADH regeneration method, this method has the advantages of simple regeneration method, fast regeneration rate, strong controllability, avoidance of by-product accumulation, low cost, and green environmental protection. It has broad application prospects in pharmaceutical production, industrial manufacturing, and high-value chemical synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The electrophoresis pattern of MaFNR protein is shown.

[0035] Figure 2 Shown is the effect of different MaFNR concentrations on the NADH synthesis rate.

[0036] Figure 3 Shown are the changes in light response of the regenerated NADH in spinach thylakoid membranes after the addition of MaFNR.

[0037] Figure 4 Figure 2 shows the regeneration of NADH in spinach thylakoid membranes after adding MaFNR to the membranes. 1 H-NMR verification diagram.

[0038] Figure 5 Different concentrations of MaNFR, Fdx, and NADP are shown + NADH and NADPH regeneration rates under different conditions, a: NADH and NADPH generation rates under different MaNFR concentrations; b: NADH and NADPH generation rates under different Fdx concentrations; c: NADP + The production rates of NADH and NADPH under different conditions.

[0039] Figure 6 It shows that after adding MaFNR to the illuminated spinach thylakoid membrane, TsFDH is driven to catalyze CO2 to synthesize formic acid. a: Schematic diagram of NADH-regenerated thylakoid membrane driving TsFDH to catalyze CO2 to synthesize formic acid; b: HPLC analysis of NADH-regenerated thylakoid membrane driving TsFDH to catalyze CO2 to synthesize formic acid; c: Time curve of NADH-regenerated thylakoid membrane driving TsFDH to catalyze CO2 to synthesize formic acid. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0041] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0042] pET28a vector was from Novagen, Madison, WI;

[0043] Escherichia coli expression strain BL21 (DE3), Invitrogen, Carlsbad, CA;

[0044] β-Nicotinamide adenine dinucleotide (NAD + ) and β-nicotinamide adenine dinucleoside phosphate (NADP + ) from, Aladdin, Shanghai, China;

[0045] Ferredoxin (Fdx, Ferredoxin) and Ferredoxin: NAD + The reductase (MaFNR) was obtained by prokaryotic expression according to the genetic engineering method.

[0046] The present invention has discovered a Ferredoxin:NAD that can couple with the spinach thylakoid membrane to regenerate NADH + The reductase is derived from Marinomonas mediterranea, and its amino acid sequence number is WP_013663187 (NCBI database), and the amino acid sequence is as follows (SEQ ID NO: 1):

[0047] MKQRCVIVGGGHAGAQVSTSLRKLGWEGKITVFTNEYSFPYHRPPLSKSFLLGNTTEHELLIRPEIAYAKDDIDVLINQEVRDIYPERHSLVMQDGSNIHYDKL VLCMGSKMRRLDVLGSDLRGIFYIKTIRDIRMLSEYLEGGQKNIVMVGGGYVSLETAAVLRKMGHLVTILVRSNRILNGSTSAPVSDFLLEQHTRNGVNIVTQK SVSHFVEDSDSAEHKMRQVICTDGSRYGADLVVIGVGSDADSELARKAGLDVTDGIVVDQYGRTSQEDIFAAGDCTSQLQSNGIAMQIASVPNALEQAKSVAST ICGKPKSSNAVQWFWSDQYENKLQMAGLNHGHDQTVLREKSHDQFSLWYLRDGRVIASECINSPADFMLSKRMIAAKTCVPSHVLADVSCSLNEYAKPVKRMSEA

[0048] After obtaining the amino acid sequence, it was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene synthesis and cloned into the pET28a expression vector. Then, the plasmid was transformed into Escherichia coli BL21 (DE3) (Invitrogen, Carlsbad, CA) for protein expression and purification. The results of protein purification are shown in Figure 1 shown.

[0049] The present invention also requires the addition of ferredoxin (Fdx, Ferredoxin) from Spinacia oleracea, and the optimized DNA sequence is as follows (SEQ ID NO: 2):

[0050] ATGGCAGCATATAAAGTGACGCTGGTGACTCCGACCGGTAACGTAGAATTTCAGTGTCCTGATGACGTCTATATCCTTGATGCTGCTGAAGAGGAAGGCATCGATTTACCATACTCCTGTCGCGCCGGTTCGTGCTCAAGCTGCGCCG GTAAACTGAAGACGGGATCATTGAACCAAGACGATCAGAGCTTCCTGGACGACGACCAGATTGATGAAGGGTGGGTTCTGACATGCGCGGCGTACCCGGTGAGCGATGTTACCATTGAGACCCATAAGGAAGAAGAACTCACCGCGTAA

[0051] Example 1: Obtaining spinach thylakoid membranes

[0052] The specific steps for separation and extraction of spinach thylakoid membrane are as follows:

[0053] (1) In a dark environment, add 1 ml of pre-cooled buffer A (330 mM sorbitol, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl2, 0.1% (w / v) bovine serum albumin) to 1 g of fresh spinach leaves and blend them in a blender.

[0054] (2) The spinach leaf homogenate obtained in the above step was filtered through eight layers of gauze, and the filtrate was centrifuged at 3000×g at 4°C for 10 min.

[0055] (3) Resuspend the precipitate obtained in the above step with buffer B (300 mM sorbitol, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl2 and 10 mM sodium L-ascorbate).

[0056] (4) The resuspended precipitate was overlaid on an 80%:40% Percoll gradient (80%:80% v / v Percoll, 10 mM sodium L-ascorbate, 300 mM sucrose, 66 mM MOPS-KOH pH 7.6 and 40%:40% v / v Percoll, 10 mM sodium L-ascorbate, 300 mM sucrose, 25 mM MOPS-KOH pH 7.6), and centrifuged at 8000×g for 10 min at 4°C.

[0057] (5) Aspirate the upper thylakoid band, add 10 times the volume of buffer B, centrifuge at 3000 × g at 4°C for 10 min, and discard the supernatant.

[0058] (6) Resuspend the precipitate in buffer C (10 mM HEPES-KOH, 10 mM MgCl2, 10 mM sodium L-ascorbate, 10% DMSO), divide into EP tubes, and store at -80°C.

[0059] (7) Before using thylakoid membranes in subsequent experiments, they need to be washed 2 to 3 times with buffer D (330 mM sorbitol, 10 mM HEPES-KOH pH 7.6, 10 mM MgCl2, and 10 mM sodium ascorbate).

[0060] Example 2: Obtaining Ferredoxin (Fdx) and Ferredoxin:NAD + Reductase (MaFNR)

[0061] Ferredoxin (Fdx, Ferredoxin) and Ferredoxin:NAD + The expression and purification of reductase (MaFNR) are as follows:

[0062] (1) The Fdx and MaNFR gene sequences were synthesized by GENEWIZ and cloned into the pET28a vector, and then transformed into Escherichia coli BL21 (DE3). Single colonies on LB solid medium plates were picked and placed in 5 mL of 50 μg mL -1 Kana resistance LB liquid medium, cultured overnight in a 37°C shaker. The next day, the bacterial solution was added to 500 mL of 50 μg mL -1 Kana resistance LB liquid medium was cultured in a shaker at 37°C until OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mM for induction, and change the shaking temperature to 16°C for 18-20 hours.

[0063] (2) After collecting the cells by centrifugation at 5000 rpm for 20 minutes, resuspend the precipitate with buffer A (50 mM HEPES, 50 mM NaCl, pH 7.5). The cell suspension was lysed in a high-pressure homogenizer and centrifuged at 8000 rpm for 20 minutes to collect the supernatant. Purify the protein using a Ni-NTA column: rinse the Ni-NTA column with buffer A for 2 column volumes (CV), and then inject the protein supernatant into the column. Wash the impurities with buffer B (30 mM imidazole added to buffer A) for 3 to 5 CVs, and then wash the target protein with buffer C (500 mM imidazole added to buffer A). The eluted protein was concentrated using a 10 kD ultrafiltration tube, replaced with buffer A three times, detected by SDS-PAGE using a 12% protein gel, and the protein concentration was detected by the Bradford method.

[0064] Example 3: Effect of different MaFNR concentrations on NADH synthesis rate

[0065] The reaction system was 0.6 ml, including 50 mM HEPES-KOH (pH 7.6), 3 mM ADP, 8 μM Fdx, 5 mM K2HPO4, 1 mM NAD + , 0-0.5mM NADP + , different concentrations of MaNFR (0-4 μM), 10 mM sodium L-ascorbate, 0.5 M betaine, 10 mM KCl, 5 mM MgCl2 and 5 μg TMs, and the reaction was initiated by light illumination with an intensity of 100 μmol photons m -2 s -1 , react at room temperature, detect the kinetic curve at 340nm wavelength, and the detection time is 10 minutes. NADH concentration is based on the NADH molar extinction coefficient of 6220M - 1 cm -1 The effect of different MaFNR concentrations on the NADH synthesis rate is as follows: Figure 2 As shown, within the MaFNR concentration range of 0-4 μM, the higher the MaFNR concentration, the faster the NADH synthesis rate.

[0066] Example 4: Changes in the light response of spinach thylakoid membranes regenerating NADH after mixing with Fdx and MaFNR

[0067] The reaction system was 0.6 ml, including 50 mM HEPES-KOH (pH 7.6), 3 mM ADP, 8 μM Fdx, 5 mM K2HPO4, 1 mM NAD +, 4 μM MaNFR, 10 mM sodium L-ascorbate, 0.5 M betaine, 10 mM KCl, 5 mM MgCl2, and 5 μg TMs. The cells were cycled in a 10-min dark cycle followed by a 10-min light cycle. The light intensity was 100 μmol photons m -2 s -1 , react at room temperature, detect the kinetic curve at 340nm wavelength, and the total detection time is 60 minutes. NADH concentration is based on the NADH molar extinction coefficient of 6220M -1 cm -1 The light response changes of the spinach thylakoid membrane after adding MaFNR to regenerate NADH are shown in Figure 3 shown.

[0068] Example 5: Spinach thylakoid membrane coupling Ferredoxin:NAD + Verification of NADH produced by reductase photoreaction

[0069] The reaction system was 0.6 ml, including 50 mM HEPES-KOH (pH 7.6), 3 mM ADP, 8 μM Fdx, 5 mM K2HPO4, 1 mM NAD + , 4 μM MaNFR, 10 mM sodium L-ascorbate, 0.5 M betaine, 10 mM KCl, 5 mM MgCl2, TMs concentration of 10-120 μg mL -1 , light intensity 100 μmol photons m -2 s -1 , react at room temperature, and after 1 hour of light reaction, centrifuge and take 300 μL of the supernatant, add 300 μL of heavy water, transfer to an NMR tube, and perform 400M 1 H-NMR analysis identified NADH, such as Figure 4 shown.

[0070] Example 6: Different concentrations of MaNFR, Fdx, and NADP + NADH and NADPH regeneration rates under

[0071] Different concentrations of MaNFR, Fdx, and NADP + NADH and NADPH regeneration rate under the following conditions: the reaction system was 0.6 ml, including 50 mM HEPES-KOH (pH 7.6), 3 mM ADP, 0-16 μM Fdx, 5 mM K2HPO4, 1 mM NAD + , 0-0.5mM NADP + , 0-4 μM MaNFR, 10 mM sodium L-ascorbate, 0.5 M betaine, 10 mM KCl, 5 mM MgCl2, 10 μg mL-1 TMs, light intensity 100 μmol photons m -2 s -1 , react at room temperature, the reaction sampling time is 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min, and the concentrations of NADH and NADPH produced by the light reaction are detected by high performance liquid chromatography.

[0072] Sample treatment: 100 μL of sample was added with 100 μL of solution A (acetonitrile: methanol: water = 40:40:20, containing 0.1 M formic acid), mixed and shaken for 10 seconds, ice-bathed for 3 minutes, and then 8.7 μL of solution B (15% ammonium bicarbonate, W / V) was added, mixed and ice-bathed for 20 minutes, centrifuged at 12000 rpm, 4°C for 15 minutes, and the supernatant was taken for HPLC analysis.

[0073] HPLC analysis conditions: Agilent 1260 high performance liquid chromatograph, SUPELCOSIL TM LC-18-T chromatographic column (5μm particle size, L×ID25cm×4.6mm) and UV detector. Mobile phase A is NaH2PO4 (50mM, pH 6.0), and mobile phase B is NaH2PO4 (50mM, pH 6.0) + 10% methanol. HPLC gradient program is: 100% A 9min; 0%-25% B 6min; 25-70% B 3min; 70%-100% B 2min; 100% B 6min; 100% A 5min. Detection wavelength 340nm, column oven temperature 25℃, flow rate 1mL / min.

[0074] Detected by high performance liquid chromatography, Figure 5 As shown, the NADPH / NADH ratio can be determined by MaNFR, Fdx, and NADP + The dosage can be regulated to achieve controllable photoregeneration of NADPH and NADH.

[0075] Example 7: Using NADH-regenerated spinach thylakoid membrane to drive formate dehydrogenase to catalyze carbon dioxide (CO2) to synthesize formic acid

[0076] 1mL reaction system: 50mM HEPES-KOH (pH 7.6), 50mM NaHCO3, 10mM Sodium ascorbate, 5mM MgCl2, 5mM KCl, 2mM ADP, 2mM NAD + , 8μM Fdx, 0.4μM MaNFR, 1mg TsFDH (from Thiobacillus sp.), 15μg TMs, 0.5M betaine, light intensity 100μmol photons m-2 s -1 , reaction temperature 30℃. 10μL 10% sulfuric acid was added to 100μL reaction sample to terminate the reaction, then centrifuged at 13000rpm for 10 minutes to remove protein, and the supernatant was taken for HPLC analysis. Shimadzu LC20A liquid chromatography was used to detect formic acid. The liquid chromatography column was Aminex HPX-87H (Bio-Rad) column, the mobile phase was 5mM sulfuric acid, the injection volume was 20μL, and the flow rate was 0.6mL min -1 , the temperature is 40°C, the detector is a differential detector, and the detection time is 20 minutes. Figure 6 As shown, after 6 hours, the production of formate could reach 2.9 mM.

[0077] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for regenerating NADH by light-driven thylakoid membranes in vitro, comprising: (1) Isolation and extraction of thylakoid membranes; (2) Ferredoxin (Fdx, Ferredoxin) and Ferredoxin:NAD + Expression and purification of reductase (MaFNR); (3) The thylakoid membrane is coupled to MaFNR and then mixed with Fdx in a reaction system to perform light-driven thylakoid membrane regeneration NADH reaction.

2. The method according to claim 1, wherein the thylakoid membrane is a thylakoid membrane derived from spinach; Preferably, the Fdx is Fdx derived from spinach, and the MaFNR is Ferredoxin:NAD derived from Marinomonas mediterranea. + Reductase.

3. The method according to claim 1 or 2, wherein the reaction system of step (3) further comprises HEPES buffer, magnesium salt, potassium salt, hydrogen phosphate, ADP, betaine, thylakoid membrane, sodium ascorbate and cofactors.

4. The method according to claim 3, wherein the cofactor is NAD + ; Preferably, the NAD + The concentration of the cofactor is 1-10 mM.

5. The method according to any one of claims 1 to 4, wherein the temperature of the light-driven thylakoid membrane regeneration NADH reaction is 30°C.

6. The method according to any one of claims 1 to 5, wherein the light intensity of the light-driven thylakoid membrane regeneration NADH reaction is 50 to 100 μmol photons m -2 s -1 .

7. The method according to any one of claims 1 to 6, wherein the amount of betaine used in the light-driven thylakoid membrane regeneration NADH reaction is 0.1-0.5 M.

8. The method according to any one of claims 1 to 7, wherein the amount of Fdx used in the light-driven thylakoid membrane regeneration NADH reaction is 5 to 10 μM.

9. The method according to any one of claims 1 to 8, wherein the amount of thylakoid membrane used in the light-driven thylakoid membrane regeneration NADH reaction is 10 to 20 μg chlorophyll equivalent / mL.

10. The method according to any one of claims 1 to 9, wherein the amount of MaFNR used in the light-driven thylakoid membrane regeneration of NADH is 0 to 4 μM.

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