Method for synthesizing gastrodin by catalyzing phenol through multi-enzyme cascade system

Abstract

The invention discloses a method for synthesizing gastrodin by catalyzing phenol through a multi-enzyme cascade system. The multi-enzyme cascade system comprises carboxylase, carboxylic acid reductase, alcohol dehydrogenase, glucose dehydrogenase, phosphokinase and glycosyl transferase. Phenol is used as a starting raw material and is converted into a key intermediate p-hydroxybenzoic acid through catalysis of carboxylase, the p-hydroxybenzoic acid is reduced into p-hydroxybenzaldehyde through carboxylic acid reductase, then the p-hydroxybenzaldehyde is reduced into p-hydroxybenzyl alcohol through alcohol dehydrogenase, and finally gastrodin is synthesized through catalysis of glycosyl transferase. According to the method, one-pot reaction is adopted, in-situ regeneration of coenzyme NAD (P) H and ATP is achieved through glucose dehydrogenase and phosphokinase, reaction conditions are mild, selectivity is high, environment friendliness is achieved, and a new way is provided for sustainable production of phenolic glycoside compounds.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for synthesizing gastrodin from phenol using a multi-enzyme cascade system. Background Technology

[0002] Gastrodin (p-hydroxybenzyl alcohol-β-D-glucoside) is a phenolic glycoside compound with important biological activity and commercial value. It is the core active ingredient of the traditional Chinese medicine Gastrodia elata, exhibiting significant sedative, analgesic, and neuroprotective effects, and its demand continues to grow in the treatment of nervous system diseases and in functional health products.

[0003] Currently, the commercial production of gastrodin mainly relies on plant extraction. This method is constrained by the long plant growth cycle, low content of the target component, complex extraction and separation processes, and potential issues related to resource sustainability, resulting in high production costs, unstable supply, and difficulty in meeting large-scale market demand. Although chemical synthesis routes have been explored, they are often cumbersome, require the use of precious metal catalysts or organic solvents, are difficult to control in terms of regio and stereoselectivity, and are accompanied by environmental pressures, which do not conform to the principles of green chemistry and sustainable development.

[0004] As the most abundant renewable aromatic polymer in nature, lignin, along with cellulose and hemicellulose, constitutes the main components of plant cell walls. Although its complex three-dimensional network structure poses a significant challenge to its high-value utilization, depolymerizing it into simple aromatic platform compounds such as phenol, and then directionally synthesizing high-value-added fine chemicals, is of vital strategic significance for realizing the full utilization of biomass resources, promoting a circular economy, and reducing dependence on fossil resources.

[0005] Therefore, this invention provides a green biomanufacturing method for the synthesis of gastrodin from phenol via a multi-enzyme cascade catalysis to solve the aforementioned problems. This method uses lignin-derived phenol as a raw material and constructs an in vitro multi-enzyme catalytic system including carboxylase, reductase, and glycosyltransferase to achieve efficient and precise conversion of phenol to the target phenolic glycoside under mild conditions, providing an innovative solution for the sustainable production of this important natural product. Summary of the Invention

[0006] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for synthesizing gastrodin from phenol using a multi-enzyme cascade system. This invention constructs a multi-enzyme cascade catalytic system using renewable lignin-derived phenol as a substrate, enabling the green synthesis of gastrodin, an important phenolic glycoside active molecule.

[0007] To address the aforementioned technical problems, this invention discloses a method for synthesizing gastrodin from phenol using a multi-enzyme cascade system. The specific technical solution is as follows: This invention provides a method for synthesizing gastrodin from phenol using a multi-enzyme cascade system. The multi-enzyme cascade system includes phenol, carboxylase, carboxyl reductase, alcohol dehydrogenase, glycosyltransferase, phosphokinase, and glucose dehydrogenase. Using phenol as a substrate, gastrodin is synthesized through a reaction catalyzed by carboxylase, carboxyl reductase, alcohol dehydrogenase, glycosyltransferase, phosphokinase, and glucose dehydrogenase.

[0008] The carboxylic acid reductase includes any one of MmCAR, MAB2962, MAB3367, MAB4714, or MSM5739; wherein, The GenBank accession number for the amino acid sequence of the MmCAR is WP_012393886. The amino acid sequence of MAB2962 has a GenBank accession number of CAM63040. The amino acid sequence of MAB3367 has a GenBank accession number of CAM63443. The amino acid sequence of MAB4714 has a GenBank accession number of CAM64782. The amino acid sequence of the MSM5739 has a GenBank accession number of ABK71854.

[0009] In some embodiments of the present invention, the carboxylic acid reductase is MAB2962.

[0010] The alcohol dehydrogenase is either HLADH or ADH6; wherein, The GenBank accession number for the amino acid sequence of HLADH is AAB26666.1; The GenBank accession number for the amino acid sequence of ADH6 is CAA90836.1.

[0011] In some embodiments of the present invention, the alcohol dehydrogenase is ADH6.

[0012] The carboxylase (Ec_pHBD) mentioned above is derived from Enterobacter cloacae P240, whose amino acid sequence has the GenBank accession number BAE97712.1; And / or, the glycosyltransferase (UGT73B6) is derived from... Rhodiola sachalinensis Its amino acid sequence has a GenBank accession number of AY547304.1; And / or, the phosphokinase (PPK2) is derived from Rhizobium meliloti (strain 1021)(Ensifer meliloti) (Sinorhizobium meliloti), whose amino acid sequence is available in GenBank under accession number CAC41944.1; And / or, the glucose dehydrogenase (GDH) is derived from Saccharolobus solfataricus (Sulfolobus solfataricus), whose amino acid sequence has the GenBank accession number CAA09918.1.

[0013] In the aforementioned multi-enzyme cascade system, the initial concentrations of phenol, carboxylase, carboxylase, carboxylreductase, alcohol dehydrogenase, glycosyltransferase, phosphokinase, and glucose dehydrogenase are 5-20 mM, 1-10 U / mL, 1-10 U / mL, 1-5 U / mL, and 1-5 U / mL, respectively. In some embodiments of the invention, the initial concentrations of carboxylase, carboxylreductase, alcohol dehydrogenase, glucose dehydrogenase, phosphokinase, glycosyltransferase, and phenol are 10 mM. The activity unit (U) of a carboxylase is defined as the amount of enzyme required to catalyze the formation of p-hydroxybenzoic acid from 1 μmol of phenol per minute; the activity unit (U) of a carboxyl reductase is defined as the amount of enzyme required to catalyze the formation of p-hydroxybenzaldehyde from 1 μmol of p-hydroxybenzoic acid per minute; the activity unit (U) of an alcohol dehydrogenase is defined as the amount of enzyme required to catalyze the formation of p-hydroxybenzyl alcohol from 1 μmol of p-hydroxybenzaldehyde per minute; the activity unit (U) of a glycosyltransferase is defined as the amount of enzyme required to catalyze the formation of glycosides from 1 μmol of phenol analog substrate per minute; and the activity unit (U) of a glucose dehydrogenase is defined as the amount of enzyme required to catalyze the formation of NADPH from 1 μmol of NADP+ per minute. The light absorption coefficient of NADPH at 340 nm is 6.3 mmol. -1 L cm -1 One unit of phosphokinase activity (U) is defined as the amount of enzyme required to catalyze the production of ATP from 1 μmol of AMP per minute.

[0014] In some embodiments of the present invention, the carboxylase, carboxyl reductase, alcohol dehydrogenase, glucose dehydrogenase, phosphokinase, and glycosyltransferase are obtained by constructing the genes of the relevant enzymes into a plasmid vector, transforming it into *E. coli* BL21 competent cells, inducing expression, and then purifying the enzyme. Notably, the carboxyl reductase requires co-expression with phosphoadenosyltransferase (Sfp) to be active. The Sfp gene is derived from... Bacillus subtilis (strain 168), whose amino acid sequence is accessed in GenBank under the number CAA44858.1. Specifically, Sfp and carboxylate reductase were co-expressed on the same plasmid vector.

[0015] Specifically, the plasmid vector includes, but is not limited to, the pET 28a(+) vector, and the Escherichia coli includes, but is not limited to, Escherichia coli BL21(DE3).

[0016] The specific method for inducing expression is as follows: The engineered bacteria are inoculated into a culture medium and cultured at 30–37°C until OD... 600 When the concentration of IPTG reaches 0.6–0.8, a final concentration of 0.1–1 mmol / L is added, and expression is induced at 16–25°C for 12–24 h. The wet bacterial cells are then collected by centrifugation for purification. Preferably, the engineered bacteria are inoculated into a culture medium and cultured at 37°C until the OD reaches 0.6–0.8. 600 When the bacterial concentration was 0.6–0.8, IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 25°C for 12 h. The wet bacterial cells were then collected by centrifugation for purification. More preferably, single colonies of the engineered bacteria were picked and inoculated into 20 mL of LB liquid medium containing 50 μg / mL antibiotic. The culture was incubated at 37°C with shaking at 200 rpm for 12 h to obtain a seed culture. This seed culture was then inoculated into 400 mL of LB liquid medium containing 50 μg / mL antibiotic at a 5% v / v inoculation rate and incubated at 37°C with shaking at 200 rpm until the OD reached 0.5%. 600 =0.6-0.8, add IPTG to a final concentration of 0.1 mmol / L and induce at 25 °C for 12 h, then collect the wet bacterial cells by centrifugation. More preferably, the purification involves washing and lysing the wet bacterial cells, followed by purification using a protein purification column and gradient elution.

[0017] The multi-enzyme cascade system includes bicarbonate, metal ion salts, coenzymes, ATP, sodium hexametaphosphate, glucose, and uridine diphosphate glucose (UDPG). The multi-enzyme cascade system also includes a solvent, including KPi buffer. In some embodiments of the present invention, the pathway for synthesizing gastrodin is as follows: phenol is converted to p-hydroxybenzoic acid in the presence of carboxylase and bicarbonate; p-hydroxybenzoic acid is converted to p-hydroxybenzaldehyde by carboxyl reductase, metal ions, NAD(P)H coenzyme, and ATP; p-hydroxybenzaldehyde is reduced to p-hydroxybenzyl alcohol by alcohol dehydrogenase and NAD(P)H; and p-hydroxybenzyl alcohol is converted to gastrodin using UDP-glucose as a glycosyl donor under the catalysis of glycosyltransferase. In the multi-enzyme cascade system of the present invention, sodium hexametaphosphate and phosphokinase are used to regenerate ATP, and glucose dehydrogenase and glucose are used to regenerate NAD(P)H. In some embodiments of the present invention, the solvent is a KPi buffer solution with a pH of 5.8-10.0, preferably a 50 mM potassium phosphate (KPi) buffer solution with a pH of 7.0.

[0018] The bicarbonate salt includes potassium bicarbonate and / or sodium bicarbonate; the metal ion salt includes Zn. 2+ Mg 2+ Mn 2+ Fe 2+ or Co 2+ Any one of the following salts; the coenzyme is NADPH and / or NADH. In some embodiments of the present invention, the bicarbonate is potassium bicarbonate, and the metal ion salt is Mg. 2+ Salt, preferably MgCl 2。

[0019] In the aforementioned multi-enzyme cascade system, the initial concentrations of bicarbonate, metal ion salt, and coenzyme are 2.5-3 M, 0.5-1.5 mM, 0.5-5 mM, 1-15 mM, sodium hexametaphosphate, glucose, and uridine diphosphate glucose. In some embodiments of the invention, the initial concentrations of coenzyme, metal ion salt, bicarbonate, glucose, sodium hexametaphosphate, UDPG, and ATP are 10 mM.

[0020] The reaction is carried out at a temperature of 25-37°C for 6-36 hours. In some embodiments of the invention, the reaction is conducted at a rotation speed of 50-300 rpm; in other embodiments, the reaction is carried out at 30°C and 200 rpm for 24 hours.

[0021] The multi-enzyme cascade system synthesizes gastrodin through a synchronous catalytic reaction.

[0022] Beneficial effects: This invention achieves a precise and efficient conversion from phenol to gastrodin, a high-value phenolic glycoside, by constructing a multi-enzyme cascade system. The pathway first converts phenol into the key intermediate p-hydroxybenzoic acid under the catalysis of a carboxylase, and then sequentially synthesizes gastrodin through the catalysis of carboxyl reductase, alcohol dehydrogenase, and glycosyltransferase. This multi-enzyme cascade strategy significantly improves the overall reaction efficiency and atom economy, while effectively reducing the formation of byproducts due to the high selectivity of enzyme catalysis.

[0023] This invention not only achieves flexible synthesis of important phenolic glycoside active molecules from a single raw material, but also, by using lignin-based phenol as the starting material and a coenzyme regeneration system constructed with glucose dehydrogenase and phosphokinase, the entire process combines raw material renewability with process economy. Gastrodin has wide applications in high-end cosmetics, pharmaceuticals, and health products, with enormous market potential. Therefore, this invention provides a technologically advanced solution with significant competitive advantages for the green and sustainable production of phenolic glycoside natural products. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 This is a schematic diagram of the reaction pathway of the present invention. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0027] The specific activities of the carboxylase, carboxyl reductase, glucose dehydrogenase, phosphokinase, alcohol dehydrogenase, and glycosyltransferase described in the following examples are 22 U / mg, 20 U / mg, 21 U / mg, 14 U / mg, 12 U / mg, and 15 U / mg, respectively. Here, one unit (U) of carboxylase activity is defined as the amount of enzyme required to catalyze the formation of p-hydroxybenzoic acid from 1 μmol of phenol per minute; one unit (U) of carboxyl reductase activity is defined as the amount of enzyme required to catalyze the formation of p-hydroxybenzaldehyde from 1 μmol of p-hydroxybenzoic acid per minute; and one unit (U) of glucose dehydrogenase activity is defined as the amount of enzyme required to catalyze the formation of NADPH from 1 μmol of NADP+ per minute. The light absorption coefficient of NADPH at 340 nm is 6.3 mmol. -1 L cm -1 The activity unit (U) of a phosphokinase is defined as the amount of enzyme required to catalyze the production of ATP from 1 μmol of AMP per minute; the activity unit (U) of an alcohol dehydrogenase is defined as the amount of enzyme required to catalyze the production of p-hydroxybenzaldehyde from p-hydroxybenzyl alcohol from 1 μmol of p-hydroxybenzaldehyde per minute; the activity unit (U) of a glycosyltransferase is defined as the amount of enzyme required to catalyze the production of glycosides from 1 μmol of phenol analog substrates per minute.

[0028] In the following examples, the substrate conversion rate % = [(initial substrate concentration - remaining substrate concentration) / initial substrate concentration] * 100%.

[0029] This invention utilizes high-performance liquid chromatography (HPLC) for the quantification and analysis of substrates and products. An Agilent EclipsePlus C18 (250 mm × 4.6 mm × 5 μm) column was used. Phase A consisted of 0.1% v / v trifluoroacetic acid aqueous solution, and Phase B consisted of acetonitrile. The column temperature was 40 °C, the flow rate was 0.4 mL / min, and detection was performed using a UV detector at a wavelength of 230 nm. The gradient elution program is shown in Table 1.

[0030] Table 1 Gradient elution program for high performance liquid chromatography

[0031] Example 1: Construction and Induced Expression of Related Genetically Engineered Bacteria in a Cascade System With carboxylase Ec For example, pHBD was synthesized by General Biotechnology (Anhui) Co., Ltd. The enzyme is derived from Enterobacter cloacae P240, and its amino acid sequence has the GenBank accession number BAE97712.1.

[0032] The synthesized carboxylase EcThe pHBD gene was cloned into the NdeI-XhoI restriction site of the pET28a(+) vector to obtain a recombinant plasmid. This plasmid was then transformed into *E. coli* BL21(DE3) competent cells and cultured at 37 °C with shaking for 1 h. The cells were then plated onto LB agar plates containing 50 μg / mL Kana resistance. After culturing at 37 °C for 16 h, single colonies were picked from the plates, confirming the successful construction of the carboxylase-engineered bacteria. Single colonies of the carboxylase-engineered bacteria were inoculated into 20 mL of LB agar containing 50 μg / mL Kana resistance and cultured at 37 °C for 12 h as a seed culture. This seed culture was then inoculated into 400 mL of LB agar containing 50 μg / mL Kana resistance at a 5% v / v inoculation rate and cultured at 37 °C and 200 rpm until OD (digestion). 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mmol / L and induce culture at 25 ℃ for 12 h to obtain bacterial suspension. Centrifuge at 8000 rpm for 5 min at 4 ℃ to collect bacterial cells. Wash the bacterial cells with 50 mM, pH 7 KPi buffer (2.829 g / L potassium dihydrogen phosphate and 5.087 g / L dipotassium hydrogen phosphate) and store at 4 ℃. Resuspend the washed bacterial sludge in 20 mL of 50 mM, pH 7.4 KPi buffer and sonicate the resuspended bacterial cells on ice (3 s on, 5 s off, 300 W power) for 15 min. Then centrifuge (9000 rpm, 4 ℃, 40 min), collect the supernatant, and filter the supernatant through a 0.22 μm aqueous filter membrane to obtain crude carboxylase enzyme solution.

[0033] 2 mL of Ni-NTA agarose purification resin packing material was loaded into a gravity-type protein purification column (purchased from Yisheng Biotechnology). The packing material was equilibrated three times with KPi buffer. Then, an appropriate amount of crude enzyme solution was slowly added, and the column was gently shaken for about 5 minutes to promote the full adsorption of the enzyme to the packing material. Gradient elution was performed using 20–500 mM imidazole buffer (17.01 g of imidazole was dissolved in 500 mL of ultrapure water to prepare 500 mM imidazole buffer, which was then diluted to prepare imidazole buffers of other concentrations). The specific process of gradient elution is as follows: the recombinant protein was recovered using a gradient method, and the proportion of elution buffer was gradually increased until the recombinant protein was eluted. Specifically, 20 mM imidazole buffer was added to wash twice, followed by 50 mM imidazole buffer to wash once to remove impurities; then 200 mM imidazole buffer was used to elute the target protein, and the eluent was collected in a centrifuge tube; finally, 500 mM imidazole buffer was used to remove the remaining protein in the purification column packing material. The collected eluent was concentrated and desalted using a Millipore ultrafiltration tube to obtain a pure enzyme solution. The solution was then aliquoted into 200 μL tubes and stored at -80°C. The purity and size were initially verified by polyacrylamide gel electrophoresis (SDS-PAGE).

[0034] The construction, induction expression, and extraction and purification methods of the genetically engineered bacteria involved in the following reaction system for carboxylic acid reductase, alcohol dehydrogenase, and glycosyltransferase are the same as above. Additionally, when constructing the genetically engineered bacteria, the carboxylic acid reductase requires the additional expression of phosphoadenosyltransferase Sfp (its amino acid sequence GenBank accession number is CAA44858.1), because carboxylic acid reductase requires co-expression with phosphoadenosyltransferase Sfp to be active. All construction processes are the same as above, except that when cloning the carboxylic acid reductase gene into the pET28a(+) vector, the phosphoadenosyltransferase Sfp gene is also cloned into the pET28a(+) vector, resulting in a recombinant plasmid containing both the carboxylic acid reductase gene and the Sfp gene. This plasmid is then introduced into competent cells for subsequent induction expression, extraction, and purification steps. The construction method can be found in Chinese Patent CN112795586A.

[0035] Example 2: Carboxylase catalyzes the production of p-hydroxybenzoic acid from phenol. 1 mL of 50 mM KPi buffer at pH 7.0 contains 10 mM phenol and 5 U / mL carboxylase. Ec pH BD and 2.7M potassium bicarbonate were used as the enzyme reaction system.

[0036] The enzyme reaction system was subjected to a reaction at 30 °C and 200 rpm in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by high-performance liquid chromatography. After 24 h of reaction, the yield of p-hydroxybenzoic acid reached 18.51%.

[0037] Example 3: Carboxylic acid reductase catalyzes the formation of p-hydroxybenzaldehyde from p-hydroxybenzoic acid. 1 mL of 50 mM pH 7.0 KPi buffer contains 10 mM p-hydroxybenzoic acid, 5 U / mL carboxylic acid reductase CAR (selected from MmCAR, MAB2962, MAB3367, MAB4714 or MSM5739), 1 mM MgCl2, 1 mM NADPH, 2 U / mL glucose dehydrogenase GDH, 100 mM glucose, 2 U / mL phosphokinase PPK2, 10 mM sodium hexametaphosphate and 10 mM ATP as the enzyme reaction system. The GenBank accession number for the amino acid sequence of MmCAR is WP_012393886; the GenBank accession number for the amino acid sequence of MAB2962 is CAM63040; the GenBank accession number for the amino acid sequence of MAB3367 is CAM63443; the GenBank accession number for the amino acid sequence of MAB4714 is CAM64782; and the GenBank accession number for the amino acid sequence of MSM5739 is ABK71854. The GenBank accession number for the amino acid sequence of the glucose dehydrogenase GDH described in this invention is CAA09918.1, and the GenBank accession number for the amino acid sequence of the phosphokinase PPK2 is CAC41944.1.

[0038] The enzyme reaction system was placed at 30℃ and 200 rpm for reaction in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by liquid chromatography. The yield of p-hydroxybenzaldehyde from p-hydroxybenzoic acid in the reaction is shown in Table 2. Table 2 shows that the MAB2962 catalytic reaction had the highest efficiency after 24 h of reaction, with a p-hydroxybenzaldehyde yield of 91.07%.

[0039] Table 2. Conversion rates of p-hydroxybenzoic acid and yields of p-hydroxybenzaldehyde catalyzed by different carboxyl reductases.

[0040] Example 4: Alcohol dehydrogenase catalyzes the production of p-hydroxybenzaldehyde from p-hydroxybenzaldehyde. One mL of 50 mM KPi buffer (pH 7.0) contained 10 mM p-hydroxybenzaldehyde, 2 U / mL alcohol dehydrogenase ADH (selected from HLADH or ADH6), 1 mM NADPH, 2 U / mL glucose dehydrogenase GDH, and 100 mM glucose as the enzyme reaction system. The GenBank accession number for the amino acid sequence of HLADH is AAB26666.1; the GenBank accession number for the amino acid sequence of ADH6 is CAA90836.1.

[0041] The enzyme reaction system was placed at 30 °C and 200 rpm for reaction in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by liquid chromatography. The yield of p-hydroxybenzaldehyde to p-hydroxybenzyl alcohol in the reaction is shown in Table 3. Table 3 shows that the ADH6 catalytic reaction had the highest efficiency after 24 h of reaction, with a p-hydroxybenzyl alcohol yield of 99.54%.

[0042] Table 3. Conversion rate of p-hydroxybenzaldehyde and yield of p-hydroxybenzyl alcohol catalyzed by different alcohol dehydrogenases.

[0043] Example 5: Glycosyltransferase catalyzes the production of gastrodin from p-hydroxybenzyl alcohol. A 1 mL 50 mM pH 7.0 KPi buffer containing 10 mM p-hydroxybenzyl alcohol, 5 U / mL glycosyltransferase UGT73B6, 1 mM MgCl2, and 10 mM uridine diphosphate glucose (UDPG) was used as the enzyme reaction system. The amino acid sequence of the glycosyltransferase UGT73B6 described in this invention has the GenBank accession number AY547304.1.

[0044] The enzyme reaction system was placed at 30 °C and 200 rpm for reaction in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10,000 rpm for 2 min, filtered (filter membrane diameter of 0.22 μm), and analyzed by liquid chromatography. After 24 h of reaction, the yield of gastrodin was 24.90%.

[0045] Example 6: Multi-enzyme cascade catalysis of phenol to p-hydroxybenzaldehyde 1 mL of 50 mM KPi buffer at pH 7.0 contains 10 mM phenol and 5 U / mL carboxylase. Ec The enzyme reaction system consisted of pHBD, 5 U / mL carboxylic acid reductase MAB2962, 2 U / mL glucose dehydrogenase GDH, 2 U / mL phosphokinase PPK2, 1 mM MgCl2, 1 mM NADPH, 100 mM glucose, 2.7 M potassium bicarbonate, 10 mM sodium hexametaphosphate, and 10 mM ATP.

[0046] The enzyme reaction system was placed at 30 °C and 200 rpm for reaction in sealed 2 mL centrifuge tubes. 100 μL of the reaction solution from 2 to 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by liquid chromatography. The concentrations of each component in the reaction are shown in Table 4. After 24 h of reaction, the yield of p-hydroxybenzaldehyde was 3.16 mM, with a yield of 31.60%.

[0047] Table 4. Concentrations of various substances in the reaction system for the formation of p-hydroxybenzaldehyde from phenol at different time points.

[0048] Example 7 Multi-enzyme cascade catalysis of phenol to p-hydroxybenzyl alcohol 1 mL of 50 mM KPi buffer at pH 7.0 contains 10 mM phenol and 5 U / mL carboxylase. Ec The enzyme reaction system consisted of pHBD, 5 U / mL carboxylic acid reductase MAB2962, 2 U / mL glucose dehydrogenase GDH, 2 U / mL phosphokinase PPK2, 2 U / mL alcohol dehydrogenase ADH6, 2.7 M potassium bicarbonate, 1 mM MgCl2, 1 mM NADPH, 100 mM glucose, 10 mM sodium hexametaphosphate, and 10 mM ATP.

[0049] The enzyme reaction system was placed at 30 °C and 200 rpm for reaction in sealed 2 mL centrifuge tubes. 100 μL of the reaction solution from 2 to 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by liquid chromatography. The concentrations of each component in the reaction are shown in Table 5. After 24 h of reaction, the yield of p-hydroxybenzyl alcohol was 2.34 mM, with a yield of 23.4%.

[0050] Table 5. Concentrations of various substances in the reaction system for the formation of p-hydroxybenzyl alcohol from phenol at different time points.

[0051] Example 8: Synthesis of Gastrodin from Phenol via Multi-Enzyme Cascade Catalysis 1 mL of 50 mM KPi buffer at pH 7.0 contains 10 mM phenol and 5 U / mL carboxylase. Ec A multi-enzyme cascade system was constructed using pHBD, 5 U / mL carboxylic acid reductase MAB2962, 2 U / mL glucose dehydrogenase GDH, 2 U / mL phosphokinase PPK2, 2 U / mL alcohol dehydrogenase ADH6, 5 U / mL glycosyltransferase UGT73B6, 2.7 M potassium bicarbonate, 1 mM MgCl2, 1 mM NADPH, 100 mM glucose, 10 mM sodium hexametaphosphate, 10 mM ATP, and 10 mM UDPG. The reaction pathway of this multi-enzyme cascade system catalyzing the synthesis of gastrodin from phenol is as follows: Figure 1 As shown.

[0052] The multi-enzyme cascade system was reacted at 30 °C and 200 rpm in sealed 2 mL centrifuge tubes. 100 μL of the reaction solution from 2 to 24 h was taken, quenched with 900 μL of methanol, sonicated, centrifuged at 10,000 rpm for 2 min, filtered (filter membrane diameter 0.22 μm), and analyzed by liquid chromatography. The concentrations of each component in the reaction are shown in Table 6. After 24 h of reaction, the gastrodin yield was 1.64 mM, with a yield of 16.40%.

[0053] Table 6. Concentrations of various substances in the reaction system for the formation of gastrodin from phenol at different time points.

[0054] This invention provides a method for synthesizing gastrodin from phenol using a multi-enzyme cascade system. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for synthesizing gastrodin from phenol using a multi-enzyme cascade system, characterized in that, The multi-enzyme cascade system includes phenol, carboxylase, carboxyl reductase, alcohol dehydrogenase, glycosyltransferase, phosphokinase, and glucose dehydrogenase; using phenol as a substrate, gastrodin is synthesized through a reaction catalyzed by carboxylase, carboxyl reductase, alcohol dehydrogenase, glycosyltransferase, phosphokinase, and glucose dehydrogenase.

2. The method according to claim 1, characterized in that, The carboxylic acid reductase includes any one of MmCAR, MAB2962, MAB3367, MAB4714, or MSM5739; wherein, The GenBank accession number for the amino acid sequence of the MmCAR is WP_012393886. The amino acid sequence of MAB2962 has a GenBank accession number of CAM63040. The amino acid sequence of MAB3367 has a GenBank accession number of CAM63443. The amino acid sequence of MAB4714 has a GenBank accession number of CAM64782. The amino acid sequence of the MSM5739 has a GenBank accession number of ABK71854.

3. The method according to claim 1, characterized in that, The alcohol dehydrogenase is either HLADH or ADH6; wherein, The GenBank accession number for the amino acid sequence of HLADH is AAB26666.1; The GenBank accession number for the amino acid sequence of ADH6 is CAA90836.

1.

4. The method according to claim 1, characterized in that, The amino acid sequence of the carboxylase has a GenBank accession number of BAE97712.1; And / or, the GenBank accession number for the amino acid sequence of the glycosyltransferase is AY547304.1; And / or, the GenBank accession number for the amino acid sequence of the phosphokinase is CAC41944.1; And / or, the GenBank accession number for the amino acid sequence of the glucose dehydrogenase is CAA09918.

1.

5. The method according to claim 1, characterized in that, In the aforementioned multi-enzyme cascade system, the initial concentration of phenol is 5-20 mM, the initial concentration of carboxylase is 1-10 U / mL, the initial concentration of carboxyl reductase is 1-10 U / mL, the initial concentration of alcohol dehydrogenase is 1-5 U / mL, the initial concentration of glycosyltransferase is 1-10 U / mL, the initial concentration of phosphokinase is 1-5 U / mL, and the initial concentration of glucose dehydrogenase is 1-5 U / mL.

6. The method according to claim 1, characterized in that, The multi-enzyme cascade system comprises bicarbonate, metal ion salt, coenzyme, ATP, sodium hexametaphosphate, glucose, and uridine diphosphate glucose; wherein the bicarbonate comprises potassium bicarbonate and / or sodium bicarbonate; and the metal ion salt comprises Zn. 2+ Mg 2+ Mn 2+ Fe 2+ or Co 2+ Any of the salts; the coenzyme is NADPH and / or NADH.

7. The method according to claim 6, characterized in that, In the aforementioned multi-enzyme cascade system, the initial concentration of bicarbonate is 2.5-3 M, the initial concentration of metal ion salt is 0.5-1.5 mM, the initial concentration of coenzyme is 0.5-5 mM, the initial concentration of ATP is 1-15 mM, the initial concentration of sodium hexametaphosphate is 1-15 mM, the initial concentration of glucose is 10-100 mM, and the initial concentration of uridine diphosphate glucose is 1-15 mM.

8. The method according to claim 1, characterized in that, The multi-enzyme cascade system includes a solvent, and the solvent includes KPi buffer.

9. The method according to claim 1, characterized in that, The reaction is carried out at a temperature of 25-37℃ for a time of 6-36 hours.

10. The method according to any one of claims 1 to 9, characterized in that, The aforementioned multi-enzyme cascade system synthesizes gastrodin through a simultaneous catalytic reaction.

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