Method for synthesizing p-hydroxybenzaldehyde from p-hydroxybenzoic acid through light-driven enzyme catalysis

Abstract

The invention provides a method for synthesizing p-hydroxybenzaldehyde from p-hydroxybenzoic acid through light-driven enzyme catalysis. A used reaction system comprises carboxylic acid reductase (CAR), a photosynthesis unit, ferredoxin (Fdx) and phosphotransferase (PAP). According to the method, light energy is utilized to drive spinach capsule membranes to co-regenerate NADPH and ATP, and the problem that carboxylic acid reductase needs coenzyme supply of NADPH and ATP at the same time is solved. According to the method, a light energy driven enzyme catalysis method is adopted, a biological photosynthetic reaction is used for replacing a traditional high-energy-consumption and high-pollution chemical process, and green and efficient synthesis of p-hydroxybenzaldehyde is achieved through cooperation of double-coenzyme light regeneration and enzyme specific catalysis.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, and in particular to a light-driven enzymatic method for the synthesis of p-hydroxybenzaldehyde from p-hydroxybenzoic acid. Background Technology

[0002] p-Hydroxybenzaldehyde ( p- Hydroxybenzaldehyde, p- P-hydroxybenzaldehyde (p-hydroxybenzaldehyde) possesses exceptional reactivity due to its hydroxyl and carboxyl group structure, making it a crucial intermediate in organic synthesis. Currently, the global demand for p-hydroxybenzaldehyde is approximately 22,000 tons per year, with a market value of around 1 billion yuan. As a key aromatic aldehyde compound, it holds an irreplaceable position in the pharmaceutical, pesticide, fragrance, and polymer material fields. For example, in the pharmaceutical industry, p-hydroxybenzaldehyde is frequently used to synthesize antibiotics such as amoxicillin and cefotaxime, and is also a core intermediate in cardiovascular drugs (such as p-hydroxyphenylacetamide). In agriculture, p-hydroxybenzaldehyde is used to synthesize the herbicides benzonitrile and dichlorvos, as well as the microbial inhibitor dichloro-p-hydroxybenzaldehyde. In the fragrance and food industry, p-hydroxybenzaldehyde is commonly used in the synthesis of valuable fragrances such as vanillin and anisaldehyde, making it a vital fragrance raw material. Furthermore, it has wide applications in the textile and electroplating industries; for instance, p-hydroxybenzaldehyde can be used to improve the dyeability of polyethylene glycol fibers and can also be used as a gloss agent and leveling agent in the electroplating industry.

[0003] However, the industrial production of p-hydroxybenzaldehyde has long relied on chemical synthesis routes, primarily including the Gattermann process (JPS62153240A), trichloroacetaldehyde process (US4584410A), and formaldehyde process using phenol as a raw material; esterification, Mn2O3 oxidation, and oxygen-catalyzed oxidation processes using p-cresol as a raw material; and synthesis methods using p-nitrotoluene (JPS62155236A) and p-aminobenzaldehyde as raw materials. All of these chemical synthesis methods require high temperature and pressure, precious metal catalysis, or highly corrosive reagents, and suffer from problems such as numerous byproducts and severe waste generation. To overcome the limitations of chemical methods, bio-enzyme catalysis has emerged as an alternative due to its high selectivity and mild conditions. Carboxylic acid reductase (CAR) can catalyze the direct reduction of p-hydroxybenzoic acid to p-hydroxybenzaldehyde, but it still faces key bottlenecks, such as the coenzyme dependence of carboxylic acid reductase and the lack of an economical and efficient NADPH / ATP co-regeneration strategy. This dual-coenzyme regeneration bottleneck has become a key constraint on industrialization.

[0004] There are currently three main methods for coenzyme regeneration: one is by constructing NAD(P)H or ATP regeneration modules, such as lactate dehydrogenase (… Biotechnol. Bioeng. , 2011, 108 (2): 465-469), alcohol dehydrogenase ( Biotechnol. Bioeng. , 2006, 93 (6): 1216-1220), formate dehydrogenase ( Biotechnol. Lett. , 2014, 36 (4):835-841) and glucose dehydrogenase ( J. Ind. Microbiol. Biotechnol. , 2007, 34 (1): 83-90) and other NAD(P)H regeneration modules, as well as ATP regeneration modules such as glucose and pyruvate ( , 2007, 34 (1): 83-90) Biotechnol. Bioeng. , 2001, 74(4): 309-316; BMC Biotechnol. (2009, 9: 58), this method usually requires the addition of a sacrificial substrate, resulting in the accumulation of byproducts and compromising atom economy; the second method uses photosensitizers and electrodes to couple oxidoreductases, and uses photogenerated electrons or electroreduction to convert the oxidoreductase electron acceptor NADP. + / NAD + Reducing to NADPH / NADH ( ACS Catal. , 2019, 9(12): 11492-11501; Angew. Chem. Int. Ed. , 2018, 57(27): 7958-7985; Curr. Opin. Biotechnol (., 2022, 73: 14-21), this method has poor biocompatibility, insufficient green sustainability, and can only regenerate NAD(P)H; thirdly, it utilizes ATP synthase and proton pumps (such as rhodopsin) to construct artificial vesicles or liposomes, driving ATP synthase to synthesize ATP through proton gradient potential. Nat. Biotechnol. , 2018, 36(6): 530-535), but this method has poor ATP synthase orientation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a light-driven enzymatic synthesis method for p-hydroxybenzaldehyde from p-hydroxybenzoic acid. This method solves the bottleneck problem of high cost and low efficiency in the dual-coenzyme regeneration of NADPH and ATP in existing carboxylic acid reductase processes.

[0006] In one aspect, the present invention provides a composition for catalytic reduction of carboxylic acids, comprising: Carboxylic acid reductase (CAR) is used to catalyze the formation of aldehydes from carboxylic acids; A photosynthetic unit is used to provide the NADPH and ATP coenzymes required for catalytic reactions using light energy; the photosynthetic unit is preferably a thylakoid membrane (TMs). Fritoxin (Fdx) mediates and enhances the conversion of photosystem I (PSI, on the thylakoid membrane) to Fdx-NADP. + Electron transport by reductases (FNR, on the thylakoid membrane); Phosphotransferase (PAP) is used to catalyze the conversion of AMP to ADP.

[0007] In one embodiment of the present invention, the carboxylic acid reductase is derived from... Segniliparus rugosus In one embodiment of the present invention, the amino acid sequence number of the carboxylic acid reductase is WP_007468889.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with that sequence.

[0008] In one embodiment of the present invention, the phosphotransferase is derived from... Acinetobacter johnsonii In one embodiment of the present invention, the amino acid sequence number of the phosphotransferase is WP_178927831.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with that sequence.

[0009] In one embodiment of the present invention, the ferrolin is derived from... Spinacia oleracea The amino acid sequence number of the ferroredoxin is NP_001413440.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with that sequence.

[0010] In one embodiment of the present invention, the amino acid sequence of the ferricopentatoxin is as follows (SEQ ID NO:2): MAAYKVTLVTPTGNVEFQCPDDVYILDAAEEEGIDLPYSCRAGSCSSCAGKLKTGSLNQDDQSFLDDDQIDEGWVLTCAAYPVSDVTIETHKEEELTA*.

[0011] In one embodiment of the present invention, the thylakoid membrane is derived from spinach.

[0012] In one embodiment of the present invention, the concentration of the carboxylic acid reductase is 0-1 mg / mL. -1 Preferably 0.5-1 mg / mL -1 The concentration of the thylakoid membrane is 100-200 μg chlorophyll equivalent mL. -1 The concentration of the ferroredoxin is 5-10 μM, and the concentration of the phosphotransferase is 0-1 mg / mL. -1 Preferably 0.5-1 mg / mL -1 .

[0013] In one embodiment of the present invention, the carboxylic acid reductase (CAR), ferroredoxin (Fdx), and / or phosphotransferase (PAP) are expressed in nucleic acid form, vector form, or host form. In a specific embodiment of the present invention, the nucleic acid may include genes, mRNA, oligonucleotides, etc.; the vector may be plasmids, viral vectors, etc.; and the host may be Escherichia coli, yeast cells, insect cells, or mammalian cells.

[0014] In a second aspect, the present invention provides a reaction system for the reduction of carboxylic acids, comprising the above-described composition.

[0015] In one embodiment of the present invention, the reaction system further includes: phosphate buffer, magnesium salt, potassium salt, cofactor, PolyP, and betaine.

[0016] In one embodiment of the present invention, the concentration of the buffer solution (e.g., phosphate-buffered saline, PBS) is 50-150 mM, and the pH is 7.0-7.8. Preferably, the pH of the buffer solution is 7.4. Preferably, the concentration of the buffer solution in the reaction system is 100 mM.

[0017] In one embodiment of the present invention, various magnesium salts can be used, such as magnesium chloride and magnesium sulfate. Preferably, the magnesium salt is magnesium chloride. Preferably, the concentration of the magnesium salt is 5-15 mM, used to provide an ionic environment for the reduction of carboxylic acid. Preferably, the concentration of the magnesium salt in the reaction system is 10 mM.

[0018] Those skilled in the art will understand that various potassium salts can be used in this invention, such as potassium chloride and potassium sulfate. Preferably, the potassium salt is potassium chloride. Preferably, the concentration of the potassium salt is 5-15 mM, used to provide an ionic environment for the reduction of carboxylic acids. Preferably, the concentration of the potassium salt in the reaction system is 10 mM.

[0019] In one embodiment of the present invention, the cofactors are ADP and NADP. + As a coenzyme for electron transfer and energy conversion, it promotes the reduction of carboxylic acid substrates. Preferably, the concentration of cofactor ADP in the reaction system is 0.5-1 mM, and NADP... + The concentration is 0.5~1 mM.

[0020] In one embodiment of the present invention, the concentration of polyphosphoric acid (PolyP) is 0~10 mM, and it is used as a phosphoric acid donor to provide phosphorus for AMP to generate ADP.

[0021] In one embodiment of the present invention, the concentration of betaine is 0.5~2 M, which is used to enhance the stability of the thylakoid membrane.

[0022] In one embodiment of the present invention, the reaction system further includes a substrate, which is a carboxylic acid, preferably an aromatic acid compound. Further, the substrate is p-hydroxybenzoic acid, and the concentration of p-hydroxybenzoic acid is 10-50 mM.

[0023] In one embodiment of the present invention, the reaction system drives NADP through photosynthetic units. + The benzoic acid is reduced to NADPH, thereby achieving the biocatalytic conversion of benzoic acid into the corresponding carboxylic acid reduction product.

[0024] In a third aspect, the present invention provides a method for producing aromatic aldehydes, comprising reacting the above-described composition or reaction system under light conditions to obtain aromatic aldehydes.

[0025] In one embodiment of the present invention, the aromatic aldehyde is p-hydroxybenzaldehyde.

[0026] In one embodiment of the present invention, the reaction temperature is 15°C to 45°C, preferably 30°C; the reaction pH is 6.0 to 8.0; and the reaction time is 0 hours to 10 hours. For example, it can be 0.1, 2, 4, 6, 8, or 10 hours.

[0027] In one embodiment of the present invention, the reaction light intensity is 50~100 µmol photons m -2 s -1 .

[0028] In one embodiment of the present invention, a purification step is further included. In a specific embodiment of the present invention, purification is performed using high-performance liquid chromatography (HPLC).

[0029] In one embodiment of the present invention, the conditions for high-performance liquid chromatography (HPLC) are as follows: mobile phase A is 0.1% acetic acid in water, and mobile phase B is methanol. The HPLC gradient program is: 0-4 min 20% B, 4-24 min 20%-40% B, 24-27 min 40%-100% B, 27-29 min 100% B, 29-30 min 100%-20% B, and 30-35 min 20% B. The injection volume is 10 μL, and the flow rate is 1 mL / min. -1 Column temperature 30℃.

[0030] In one embodiment of the present invention, the molar conversion rate of p-hydroxybenzaldehyde relative to p-hydroxybenzoic acid is at least 40%.

[0031] In a fourth aspect, the present invention provides the use of the above-described composition and reaction system in the preparation of aromatic aldehyde compounds.

[0032] In one embodiment of the present invention, the aromatic aldehyde compound is p-hydroxybenzaldehyde.

[0033] Compared with the prior art, the present invention has the following beneficial effects: This method utilizes light energy to drive the co-regeneration of NADPH and ATP in the spinach thylakoid membrane, solving the problem of coenzyme supply for carboxylic acid reductase, which requires both NADPH and ATP. This invention replaces the traditional high-energy-consuming and high-polluting chemical process with a light-driven enzyme catalysis method, achieving the green and efficient synthesis of p-hydroxybenzaldehyde through the synergistic effect of dual-coenzyme photoregeneration and enzyme-specific catalysis. This technology not only overcomes the industry bottleneck of multiple coenzyme requirements for carboxylic acid reductase but also provides an innovative paradigm for the sustainable manufacturing of aromatic aldehydes. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the mechanism for the synthesis of p-hydroxybenzaldehyde from p-hydroxybenzoic acid catalyzed by a light-driven thylakoid membrane-CAR coupling system.

[0035] Figure 2 For SDS-PAGE detection of SrCAR and PAP, M stands for Marker.

[0036] Figure 3 The results are the HPLC detection results of p-hydroxybenzaldehyde in Example 1.

[0037] Figure 4 This is the mass spectrometry chromatogram of p-hydroxybenzaldehyde in Example 1.

[0038] Figure 5 NMR of p-hydroxybenzaldehyde in Example 1 1 H spectrum. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0040] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0041] Experimental materials: pET28a vector, Novagen, Madison, WI; Percoll used for extracting thylakoid membranes, Solarbio, China; Escherichia coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA; β-nicotinamide adenine dinucleotide phosphate (NADP) + Aladdin, Shanghai, China; In this invention, ferroredoxin (Fdx), phosphotransferase (PAP), and carboxylic acid reductase (CAR) were all obtained through prokaryotic expression using genetic engineering methods.

[0042] This invention utilizes light energy to drive a thylakoid membrane-CAR coupling system to catalyze the synthesis of p-hydroxybenzaldehyde from p-hydroxybenzoic acid. The catalytic reaction mechanism is as follows: Figure 1 As shown, carboxyl reductase (CAR) and phosphotransferase (PAP) are derived from... Segniliparus rugosus and Acinetobacter johnsonii The amino acid sequences were obtained as WP_007468889.1 and WP_178927831.1 (NCBI database). After obtaining the amino acid sequences, they were sent to Suzhou Genewise Biotechnology Co., Ltd. for gene synthesis and cloned into the pET28a expression vector. Then, the plasmid was transformed into E. coli BL21(DE3) for protein expression and purification.

[0043] This invention also requires additional additions Spinacia oleracea The optimized DNA sequence of the source Ferredoxin (Fdx) is as follows (SEQ ID NO:1): ATGGCAGCATATAAAGTGACGCTGGTGACTCCGACCGGTAACGTAGAATTTCAGTGTCCTGATGACGTCTATATCCTTGATGCTGCTGAAGAGGAAGGCATCGATTTACCATACTCCTGTCGCGCCGGTTCGTGCTCAAGCTGCGCCG GTAAACTGAAGACGGGATCATTGAACCAAGACGATCAGAGCTTCCTGGACGACGACCAGATTGATGAAGGGTGGGTTCTGACATGCGCGGCGTACCCGGTGAGCGATGTTACCATTGAGACCCATAAGGAAGAAGAACTCACCGCGTAA The spinach thylakoid membrane is a membrane structure within the chloroplasts of spinach (Spinacia oleracea), primarily composed of thylakoids, and is the core site of the light-dependent reactions (light-dependent phase) of photosynthesis. The thylakoid membrane is rich in photosynthetic pigments (such as chlorophyll), protein complexes (such as photosystems I and II, and the cytochrome b6f complex), and components of the electron transport chain. The spinach thylakoid membrane is responsible for capturing light energy, conducting photocatalytic water splitting, and generating ATP and NADPH for subsequent dark reactions (carbon fixation). The spinach thylakoid membrane described in this invention can be prepared by the following method: 1) Mix large-leaf spinach with pre-cooled buffer A under dark conditions, and break it up with a mixer to obtain a homogenate; after filtering the homogenate, centrifuge the filtrate under low temperature conditions and collect the precipitate; 2) Resuspend the precipitate obtained in step 1) with buffer B, and cover the resuspended solution on an 80%:40% Percoll gradient solution, centrifuge under low temperature conditions, and separate the upper thylakoid band; 3) Mix and dilute the upper thylakoid band obtained in step 2) with buffer B, centrifuge under low temperature conditions, discard the supernatant; resuspend the obtained precipitate with buffer C and store it at low temperature. Buffer A contains 330 mM sorbitol, 50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2, and 0.1% (w / v) bovine serum albumin; Buffer B contains 300 mM sorbitol, 50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2, and 10 mM L-ascorbate sodium; the 80% Percoll gradient solution contains 80% (v / v) Percoll, 10 mM L-ascorbate sodium, 300 mM sucrose, and 66 mM MOPS-KOH (pH 7.6); the 40% Percoll gradient solution contains 40% (v / v) Percoll, 10 mM L-ascorbate sodium, 300 mM sucrose, and 25 mM MOPS-KOH (pH 7.6); Buffer C contains 10 mM HEPES-KOH, 10 mM MgCl2, and 10 mM L-ascorbate sodium; the 80% Percoll gradient solution contains 80% (v / v) Percoll, 10 mM L-ascorbate sodium, 300 mM sucrose, and 25 mM MOPS-KOH (pH 7.6); and the 40% Percoll gradient solution ... L-ascorbic acid sodium and 10% DMSO.

[0044] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. A particularly preferred set of parameters (and, unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shifted vacancy penalty of 5. Alternatively, a PAM120 weighted remainder table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used, employing the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), to determine the percentage of identity between two amino acid sequences or nucleotide sequences.

[0045] Example 1: Isolation and extraction of spinach thylakoid membranes 1. Take 500 g of large-leaf spinach and, under dark conditions, add 200 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). Blend the homogenate with a mixer. Filter the homogenate through eight layers of gauze. Centrifuge the filtrate at 3000×g for 10 min at 4°C and collect the precipitate.

[0046] 2. Resuspend the precipitate from step 1 in buffer B (300 mM sorbitol, 50 mM HEPES-KOH pH 7.6, 5 mM MgCl2, and 10 mM L-ascorbic acid sodium). Spread evenly on an 80%:40% Percoll gradient solution. Centrifuge at 8000×g for 10 min at 4 °C using the 80%:40% Percoll gradient solution (80%: 80% v / v percoll, 10 mM L-ascorbic acid sodium, 300 mM sucrose, 66 mM MOPS-KOH pH 7.6 and 40%: 40% v / v percoll, 10 mM L-ascorbic acid sodium, 300 mM sucrose, 25 mM MOPS-KOH pH 7.6).

[0047] 3. Aspirate the upper thylakoid band, add 10 times the volume of buffer B, centrifuge at 3000×g for 10 min at 4 °C, and discard the supernatant. Resuspend the precipitate in buffer C (10 mM HEPES-KOH, 10 mM MgCl2, 10 mM L-ascorbic acid sodium, 10% DMSO), and aliquot into EP tubes. Store at -80 °C. Before using the thylakoid membrane in subsequent experiments, wash 2-3 times with buffer D (330 mM sorbitol, 10 mM HEPES-KOH pH 7.6, 10 mM MgCl2, and 10 mM ascorbic acid sodium).

[0048] Example 2 Expression and purification of ferroredoxin (Fdx), phosphotransferase (PAP), and carboxylic acid reductase (CAR) 1. Gene cloning, transformation, and expression: The synthesized Fdx, PAP, and CAR genes were cloned into the pET28a vector and transformed into *E. coli* BL21(DE3). A single transformed colony was picked and inoculated into 5 mL of LB broth containing kanamycin (50 μg mL⁻¹), and cultured overnight at 37°C with shaking. The next day, the overnight culture was transferred 1:100 to 500 mL of LB broth containing the same concentration of kanamycin, and cultured at 37°C with shaking until OD (dose expiratory volume). 600 The concentration was increased to 0.6-0.8. Then, IPTG was added to a final concentration of 0.1 mM to induce protein expression, and the culture temperature was lowered to 16℃ for 18-20 hours.

[0049] 2. Cell harvesting, lysis, and purification: Bacterial cells were collected by centrifugation at 5000 rpm for 20 min, and the precipitate was resuspended in buffer A (50 mM HEPES, 50 mM NaCl, pH 7.5). After cell lysis by high-pressure homogenization, the supernatant was collected by centrifugation again (8000 rpm, 20 min). Purification was performed using a Ni-NTA affinity chromatography column: the column was first equilibrated with buffer A (2 column volumes, CV), followed by loading the protein supernatant. Impurities were eluted with buffer B containing 30 mM imidazole (buffer A as the base solution) (3–5 CV), and the target protein was eluted with buffer C containing 500 mM imidazole (buffer A as the base solution). The collected eluent was concentrated using a 10 kDa ultrafiltration tube and replaced three times with buffer A. Phosphotransferase (PAP) and carboxyl reductase (CAR) were analyzed for protein purity by 12% SDS-PAGE, and protein concentration was determined using the Bradford assay. The extinction coefficient of ferritin (Fdx) at 420 nm was 9.68 mM cm⁻¹. -1 Concentration determination was performed. The results of protein purification are as follows: Figure 2 As shown Example 3: Photocatalytic synthesis of p-hydroxybenzaldehyde from p-hydroxybenzoic acid using a thylakoid membrane-CAR coupling system. 1 mL reaction system: 100 mM PBS buffer (pH 7.4), 10 mM MgCl2, 10 mM KCl, 1 mM NADP + 1 mM ADP, 50 mM p-hydroxybenzoic acid, 10 mM PolyP, 0.5 M betaine, 200 μg / mL -1 TMs, 1 mg mL -1 SrCAR, 1 mg mL -1 PAP, 10 μM Fdx, light intensity 100 µmol photons m -2 s -1 The reaction was carried out at 30℃. 100 μL of acetonitrile was added to 100 μL of the reaction sample to terminate the reaction. The mixture was then centrifuged at 13000 rpm for 10 minutes to remove protein, and the supernatant was collected for HPLC analysis. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected using liquid chromatography (HPLC).

[0050] HPLC analysis conditions: A Shimadzu LC-2050 high-performance liquid chromatograph, an Agilent ZORBAX SB-C18 column (4.6 × 250 mm 5 μm), and a UV detector were used. Mobile phase A was 0.1% acetic acid in water, and mobile phase B was methanol. The gradient analysis program was as follows: 0–4 min 20% B, 4–24 min 20%–40% B, 24–27 min 40%–100% B, 27–29 min 100% B, 29–30 min 100%–20% B, and 30–35 min 20% B. The injection volume was 10 μL, and the flow rate was 1 mL / min. -1 The column temperature was 30℃, the detection wavelength was 254 nm, and the detection time was 35 min.

[0051] Detected by high performance liquid chromatography, such as Figure 3 As shown, the concentration of p-hydroxybenzaldehyde was 20 mM at 6 hours. Therefore, the molar conversion of p-hydroxybenzaldehyde relative to p-hydroxybenzoic acid was 40% (molar conversion is the actual number of moles of product / theoretical number of moles of substrate converted to the same carbon product × 100%).

[0052] Under the above liquid chromatography conditions, 100 μl of sample was collected after 15.6 min and analyzed by mass spectrometry using a TripleTOF 6600 mass spectrometer in positive ion mode. Figure 4 As shown, after spectral library search and comparison analysis, it was confirmed to be p-hydroxybenzaldehyde.

[0053] The p-hydroxybenzaldehyde sample was separated and purified using the above liquid chromatography conditions, and then concentrated by removing water using a vacuum concentrator at 45°C. Finally, it was analyzed using 400M NMR. 1 The structure was identified by 1H spectroscopy (using deuterated DMSO as the solvent), and the results are as follows. Figure 5 As shown, 1 HNMR (400 MHz, DMSO) δ=10.61 (s, 1H), 9.80 (s, 1H), 7.78 (d, J =8.6, 2H), 6.95(d, J =8.5, 2H). After comparison with the 1H spectrum of p-hydroxybenzaldehyde, it was confirmed to be p-hydroxybenzaldehyde.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A composition for catalyzing the reduction of carboxylic acids, characterized in that, include: Carboxylic acid reductase (CAR) is used to catalyze the formation of aldehydes from carboxylic acids; A photosynthetic unit is used to provide the NADPH and ATP coenzymes required for catalytic reactions using light energy; the photosynthetic unit is preferably a thylakoid membrane (TMs). Fritoxin (Fdx) mediates and enhances the conversion of photosystem I (PSI) to Fdx-NADP. + Electron transfer in reductase (FNR); Phosphotransferase (PAP) catalyzes the conversion of AMP to ADP.

2. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The carboxylic acid reductase comes from Segniliparus rugosus ; Preferably, the amino acid sequence number of the carboxylic acid reductase is WP_007468889.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with that sequence.

3. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The phosphotransferase is derived from Acinetobacter johnsonii ; Preferably, the amino acid sequence number of the phosphotransferase is WP_178927831.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with that sequence.

4. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The ferrolin is derived from Spinacia oleracea ; Preferably, the amino acid sequence number of the ferroredoxin is NP_001413440.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with this sequence. Preferably, the amino acid sequence of the ferricopentatoxin is as follows: MAAYKVTLVTPTGNVEFQCPDDVYILDAAEEEGIDLPYSCRAGSCSSCAGKLKTGSLNQDDQSFLDDDQIDEGWVLTCAAYPVSDVTIETHKEEELTA*.

5. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The thylakoid membrane is derived from spinach.

6. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The concentration of the carboxylic acid reductase is 0-1 mg / mL. -1 Preferably 0.5-1 mg / mL -1 The concentration of the thylakoid membrane is 100-200 μg chlorophyll equivalent mL. -1 The concentration of the ferroredoxin is 5-10 μM, and the concentration of the phosphotransferase is 0-1 mg / mL. -1 Preferably 0.5-1 mg / mL -1 .

7. The composition for catalytic reduction of carboxylic acids as described in claim 1, characterized in that, The carboxylic acid reductase (CAR), ferroredoxin (Fdx), and / or phosphotransferase (PAP) are expressed in nucleic acid form, vector form, or host form.

8. A reaction system for the reduction of carboxylic acids, comprising the composition according to any one of claims 1-7; Preferably, the reaction system further includes: Buffer solution, magnesium salt, potassium salt, cofactor, PolyP, betaine; Preferably, the concentration of the buffer solution is 50-150 mM, and the pH is 7.0-7.8; Preferably, the concentration of the magnesium salt is 5-15 mM; Preferably, the concentration of the potassium salt is 5-15 mM; Preferably, the cofactors are ADP and NADP. + The concentration of ADP was 0.5-1 mM, and the concentration of NADP was... + The concentration is 0.5~1 mM; Preferably, the concentration of the polyphosphoric acid (PolyP) is 0~10 mM; Preferably, the concentration of betaine is 0.5~2 M; Preferably, the reaction system further includes a substrate, which is a carboxylic acid, preferably an aromatic acid compound; further, the substrate is p-hydroxybenzoic acid, and the concentration of p-hydroxybenzoic acid is 10-50 mM.

9. A method for producing aromatic aldehydes, comprising reacting the above-described composition or reaction system under light conditions to obtain aromatic aldehydes; Preferably, the aromatic aldehyde is p-hydroxybenzaldehyde; Preferably, the reaction temperature is 15°C to 45°C; and the reaction pH is 6.0 to 8.

0. Preferably, the reaction light intensity is 50~100 µmol photons m -2 s -1 ; Preferably, the method further includes a purification step; preferably, purification is performed using high performance liquid chromatography. Preferably, the high-performance liquid chromatography (HPLC) conditions are as follows: mobile phase A is 0.1% acetic acid in water, and mobile phase B is methanol. The HPLC gradient program is: 0-4 min 20% B, 4-24 min 20%-40% B, 24-27 min 40%-100% B, 27-29 min 100% B, 29-30 min 100%-20% B, and 30-35 min 20% B. The injection volume is 10 μL, and the flow rate is 1 mL / min. -1 Column temperature 30℃; Preferably, the molar conversion rate of p-hydroxybenzaldehyde relative to p-hydroxybenzoic acid is at least 40%.

10. The use of the composition according to any one of claims 1-7 and the reaction system according to claim 8 in the preparation of aromatic aldehyde compounds; Preferably, the aromatic aldehyde compound is p-hydroxybenzaldehyde.

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