A method for synthesizing danshensu using a chemoenzymatic cascade system

Abstract

The present invention discloses a method for synthesizing danshensu using a chemoenzymatic cascade system. The first enzymatic cascade system or the second enzymatic cascade system is subjected to a catalytic reaction to generate (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl) propionic acid, and then a demethylation reaction is carried out using hydrobromic acid to finally generate D-danshensu. The technical solution provided by the present invention synthesizes danshensu using vanillin as a raw material. Vanillin has a low cost and stable chemical properties; the reaction conversion rate of the chemoenzymatic cascade system for synthesizing danshensu is high, the conversion rate of vanillin can reach 100%, and it has excellent chemoselectivity. The yield of danshensu reaches 90%, all of which are D-danshensu, and no other by-products are generated, showing good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and particularly relates to a method for synthesizing danshensu using a chemoenzymatic cascade system. Background Art

[0002] Danshensu is a natural compound extracted from the plant Salvia miltiorrhiza Bunge of the Labiatae family, and has various pharmacological effects and biological activities. Due to its unique pharmacological properties, danshensu has received extensive attention in the medical field, and plays an important role especially in the treatment of cardiovascular diseases. With the in-depth study of the pharmacological effects of danshensu, its application prospects in the pharmaceutical field are becoming increasingly broad. However, the extraction cost of natural danshensu is relatively high, and the extraction amount is limited. Therefore, the research on the synthesis of danshensu has attracted much attention.

[0003] At present, various methods for synthesizing danshensu have been reported, including chemical synthesis, biosynthesis, etc. Chemical synthesis methods mainly construct the molecular structure of danshensu through organic synthesis means, but there are problems such as cumbersome reaction steps and low product purity. Biosynthesis methods utilize technologies such as biocatalytic enzymes to achieve the efficient synthesis of danshensu, and have advantages such as good selectivity, greenness, and environmental friendliness.

[0004] In the field of biocatalytic synthesis of danshensu, most methods use L-DOPA as a substrate, and obtain D-danshensu through two-step enzyme catalysis of deamination and reduction. However, the structure of L-DOPA itself is unstable and easily oxidized, and L-DOPA is expensive, with too high a cost, which brings great obstacles to industrial synthesis. Zhao Guangrong et al. constructed engineered Escherichia coli to ferment and produce danshensu using glucose as a substrate; by means of modular optimization strategies and strategies for knocking out genes regulating the production pathway to improve the yield of danshensu, finally, the engineered Escherichia coli for metabolic engineering fermented for 60 h to obtain a danshensu yield of 7.1 g / L and a productivity of 0.47 mol·mol -1 glucose. Wang Jian et al. used low-cost phenylpyruvic acid (PPA) as a substrate to construct an artificial thermophilic cascade reaction composed of D-mandelate dehydrogenase (ManDH), phenylalanine 4-hydroxylase (PAH), and hydroxyphenylacetic acid 3-hydroxylase (HpaH) for cell-free production of danshensu (SAA). However, in order to recycle cofactors NADH and 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH4), this system introduced three enzymes, formate dehydrogenase, dihydropteridine reductase (DHPR), and pterin-carbinolamine dehydrogenase (PCD), which increased the cost of the cascade system and the catalytic efficiency was not very high.

[0005] Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most "tasteful" and popular aromatic chemicals in the world. It is widely used as a flavor in foods and beverages and as a fragrance ingredient in perfumes and cosmetics. It is even regarded as an attractive chemical platform for synthetic materials, fine chemicals, and pharmaceuticals. Lignin is the most abundant source of aromatic hydrocarbons in natural flavors and is thus a promising renewable raw material for the production of bio-based vanillin. In fact, vanillin has been produced from sulfate lignin through various oxidative depolymerization processes for many years. Therefore, using vanillin as a raw material to synthesize danshensu has the characteristics of low-cost and easy availability of the substrate, which can greatly reduce the production cost. Summary of the Invention

[0006] Object of the Invention: In view of the deficiencies of the prior art, the present invention provides a method for synthesizing danshensu using a chemoenzymatic cascade system.

[0007] To solve the above technical problems, the present invention discloses a method for synthesizing danshensu from any one of vanillin, glycine, and threonine using a chemoenzymatic cascade system. Vanillin is catalyzed by an aldehyde lyase or transaldolase dependent on pyridoxal phosphate (PLP) to obtain 3-methoxy-4-hydroxybenzserine, which is then catalyzed by a threonine dehydratase or benzserine dehydratase dependent on pyridoxal phosphate to generate 3-methoxy-4-hydroxyphenylpyruvic acid. Then, it is catalyzed by NADH-dependent D-lactate dehydrogenase to generate (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionic acid. Finally, through the demethylation reaction of HBr, D-danshensu is obtained. The specific technical solution is as follows:

[0008] A method for synthesizing danshensu using a chemoenzymatic cascade system, characterized in that the first enzyme cascade system or the second enzyme cascade system is subjected to a catalytic reaction to generate (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionic acid, and then a demethylation reaction is carried out using hydrobromic acid to finally generate D-danshensu;

[0009] The first enzyme cascade system described above includes: any one of vanillin, glycine, aldehyde lyase, threonine dehydratase, and benzserine dehydratase, D-lactate dehydrogenase, formate dehydrogenase, sodium formate, cofactor, and solvent;

[0010] The second enzyme cascade system described above includes: any one of vanillin, threonine, transaldolase, threonine dehydratase, and benzserine dehydratase, D-lactate dehydrogenase, formate dehydrogenase, sodium formate, cofactor, and solvent;

[0011] Among them, the enzyme cascade system is preferably the second enzyme cascade system.

[0012] Among them, the aldolase is a pyridoxal phosphate-dependent threonine aldolase or phenylserine aldolase, preferably any one of the following a1 to a5, and further preferably a1; the transaldolase is a pyridoxal phosphate-dependent threonine transaldolase, preferably a6 or a7 below, and further preferably a6.

[0013] a1, L-threonine aldolase PpLTA from Pseudomonas putida, Uniprot serial number is 1V72;

[0014] a2, L-threonine aldolase EcTA from Escherichia coli, GenBank serial number is WP_000566376.1;

[0015] a3, threonine aldolase PfLTA from Pseudomonas fluorescens, GenBank serial number is EXF96374.1;

[0016] a4, threonine aldolase RsLTA from Ralstonia solanacearum, GenBank serial number is KFZ94941.1;

[0017] a5, phenylserine aldolase PsLTA from Pseudomonas sp., GenBank serial number is AFY19474.1;

[0018] a6, threonine transaldolase PsLTTA from Pseudomonas sp., GenBank serial number is WP_065949345.1.

[0019] a7, threonine transaldolase ChLTTA from Chitiniphilus shinanonensis, GenBank serial number is WP_018749561.1.

[0020] Among them, the threonine dehydratase is a pyridoxal phosphate-dependent threonine dehydratase, and the phenylserine dehydratase is a pyridoxal phosphate-dependent phenylserine dehydratase, preferably any one of the following b1 to b5, further preferably b1 or b3, and most preferably b3.

[0021] b1, threonine dehydratase PxLTD from Paraburkholderia xenovorans, GenBank serial number is WP_011488286.1;

[0022] b2, threonine dehydratase CgLTD derived from Corynebacterium glutamicum, GenBank accession number: SJM46940.1;

[0023] b3, phenylserine dehydratase RpLTD derived from Ralstonia pickettii, GenBank accession number: BAC53614.1;

[0024] b4, phenylserine dehydratase BbLTD derived from Bordetella bronchiseptica, GenBank accession number: VTQ90664.1;

[0025] b5, phenylserine dehydratase PtLTD derived from Pandoraea terrae, GenBank accession number: VVE58836.1;

[0026] Among them, the D-lactate dehydrogenase is NADH-dependent D-lactate dehydrogenase, preferably any one of the following c1-c5, more preferably c1, c2 or c3, and most preferably c1.

[0027] c1, D-specific 2-hydroxyacid dehydrogenase LfD2-HDH derived from Lactobacillus frumenti, GenBank accession number: ON209401.1;

[0028] c2, D-lactate dehydrogenase Lp-D-LDH derived from Lactobacillus pentosus, GenBank accession number: CCC17764.1;

[0029] c3, D-lactate dehydrogenase D-LDH82319 derived from Limosilactobacillus reuteri, GenBank accession number: UFK68252.1;

[0030] c4, D-lactate dehydrogenase Lf-LDH derived from Lactobacillus fermentum, GenBank accession number: WP_338432499.1;

[0031] c5, D-lactate dehydrogenase PaLDH derived from Pediococcus acidilactici, GenBank accession number: ARW28447.1.

[0032] Among them, the solvent is potassium phosphate buffer with a pH of 7 to 8, preferably pH 7.5 and a concentration of 50 mM.

[0033] Among them, in the enzyme cascade system, the initial concentration of vanillin is 10 to 50 mM, preferably 10 mM; the initial concentration of glycine is 10 eq of vanillin; the initial concentration of threonine is 1 to 5 eq of vanillin, preferably 3 eq. The initial concentration of aldolase or transaldolase is 1 to 5 U / mL, preferably 3 U / mL; the initial concentration of threonine dehydratase or phenylserine dehydratase is 1 to 10 U / mL, preferably 2 U / mL; the initial concentration of D-lactate dehydrogenase is 1 to 10 U / mL, preferably 1 U / mL; the initial concentration of formate dehydrogenase is 1 to 10 U / mL; the initial concentration of sodium formate is 5 eq of vanillin.

[0034] Among them, the cofactor is pyridoxal phosphate and NAD + in the composition; in the enzyme cascade system, the initial concentration of pyridoxal phosphate is 0.1 to 0.5 mM, preferably 0.01 eq of vanillin; the initial concentration of NAD + is 0.1 to 0.5 mM, preferably 0.01 eq of vanillin.

[0035] Among them, for the catalytic reaction, the reaction pH is 6 to 8, the reaction temperature is 20 to 40 °C, and the reaction time is 1 to 12 h. Preferably, the pH is 7 to 8, the reaction temperature is 30 to 35 °C, and the reaction time is 4 to 12 h. More preferably, the pH is 7.5, the reaction temperature is 30 °C, and the reaction time is 4 h.

[0036] Among them, the hydrobromic acid is a 40 wt% aqueous hydrobromic acid solution; the volume of the aqueous hydrobromic acid solution used is 0.5 to 2.5 mL / mg based on the mass of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid, preferably 2 mL / mg.

[0037] Among them, the demethylation reaction is carried out under microwave conditions, the microwave power is 100 to 250 W, and the reaction time is 5 to 30 min. Preferably, the microwave power is 100 W and the reaction time is 10 min.

[0038] Among them, for the demethylation reaction, a catalyst, hexadecyltributylphosphonium bromide (TBHDPB), is added, and the addition amount is 100% or less of the mass of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid, preferably 50%.

[0039] Among them, the chemoenzymatic cascade system synthesizes danshensu through a one-pot two-step catalytic reaction.

[0040] Among them, the catalytic reaction is carried out in an air atmosphere.

[0041] Beneficial effects:

[0042] (1) The technical solution provided by the present invention synthesizes danshensu using vanillin as a raw material. Compared with levodopa and 3,4-dihydroxyphenylpyruvic acid, vanillin has a lower price and lower cost; and vanillin has more stable chemical properties; in this chemoenzymatic cascade reaction, the conversion rate of the substrate can reach 100%, and no by-products are generated, with excellent chemoselectivity.

[0043] (2) The method of the present invention optimizes the sources and dosages of aldolase, transaldolase, threonine dehydratase, phenylserine dehydratase, and D-lactate dehydrogenase in the chemoenzymatic cascade system, the dosages of cofactors, reaction temperature, reaction time, reaction solvent, etc. Finally, the conversion rate of 10 mM vanillin is 100%, and the yield of danshensu reaches 90%.

[0044] (3) The method for synthesizing danshensu of the present invention has a high reaction conversion rate and good chemoselectivity, and all are D-danshensu, with good application prospects. Description of the drawings

[0045] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0046] Figure 1 It is the liquid chromatogram of the reaction solution obtained after the chemoenzymatic cascade reaction under different reaction time conditions in Example 4. Green represents a reaction time of 1 h, yellow represents a reaction time of 2 h, blue represents a reaction time of 4 h, purple represents a reaction time of 8 h, and orange represents a reaction time of 12 h;

[0047] Figure 2 It is the total reaction flow chart for synthesizing D-danshensu by reacting vanillin and threonine as substrates, where LTTA represents transaldolase, LTD represents threonine dehydratase or phenylserine dehydratase, and D-LDH represents D-lactate dehydrogenase;

[0048] Figure 3 It is the liquid chromatogram of the reaction solution obtained after the chemoenzymatic cascade reaction (vanillin concentration is 10 mM) in Example 6 and the reaction solution after the demethylation reaction with HBr in Example 7;

[0049] Figure 4 It is the mass spectrum of D-danshensu (m / z: 198) synthesized in Example 7. Specific embodiments

[0050] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified. The recombinant Escherichia coli strains used were all synthesized by Anhui General Biology Co., Ltd., and the enzymes used were all pure enzymes synthesized and purified under conventional conditions.

[0051] The specific activities of aldolase, transaldolase, threonine dehydratase, phenylserine dehydratase, D-lactate dehydrogenase, and formate dehydrogenase in the following examples were 20 U / mg, 20 U / mg, 30 U / mg, 30 U / mg, 32 U / mg, and 0.4 U / mg, respectively.

[0052] One unit (U) of aldolase or transaldolase activity is defined as the amount of enzyme required to catalyze the formation of 4-hydroxy-3-methoxyphenylserine from 1 μmol of vanillin per minute.

[0053] One unit (U) of threonine dehydratase or phenylserine dehydratase activity is defined as the amount of enzyme required to catalyze the formation of 4-hydroxy-3-methoxyphenylpyruvate from 1 μmol of 4-hydroxy-3-methoxyphenylserine per minute.

[0054] One unit (U) of D-lactate dehydrogenase activity is defined as the amount of enzyme required to catalyze the formation of NAD from 1 μmol of NADH per minute. + The amount of enzyme required. The molar absorption coefficient of NADH at 340 nm is 6.01 mmol -1 L cm -1 .

[0055] One unit (U) of formate dehydrogenase activity is defined as the amount of enzyme required to catalyze the formation of NADH from 1 μmol of NAD per minute. + The amount of enzyme required. The molar absorption coefficient of NADH at 340 nm is 6.01 mmol -1 L cm -1 .

[0056] In the following examples, the aldolase is a pyridoxal phosphate-dependent threonine aldolase or phenylserine aldolase, and the transaldolase is a pyridoxal phosphate-dependent threonine transaldolase. Among them, PpLTA is an L-threonine aldolase derived from Pseudomonas putida, with the Uniprot serial number 1V72; EcTA is an L-threonine aldolase derived from Escherichia coli, with the GenBank serial number WP_000566376.1; PfLTA is a threonine aldolase derived from Pseudomonas fluorescens, with the GenBank serial number EXF96374.1; RsLTA is a threonine aldolase derived from Ralstonia solanacearum, with the GenBank serial number KFZ94941.1; PsLTA is a phenylserine aldolase derived from Pseudomonas sp., with the GenBank serial number AFY19474.1; PsLTTA is a threonine transaldolase derived from Pseudomonas sp., with the GenBank serial number WP_065949345.1; ChLTTA is a threonine transaldolase derived from Chitiniphilus shinanonensis, with the GenBank serial number WP_018749561.1.

[0057] In the following examples, the threonine dehydratase is a pyridoxal phosphate-dependent threonine dehydratase, and the phenylserine dehydratase is a pyridoxal phosphate-dependent phenylserine dehydratase. Among them, PxLTD is a threonine dehydratase derived from Paraburkholderia xenovorans, with the GenBank serial number WP_011488286.1; CgLTD is a threonine dehydratase derived from Corynebacterium glutamicum, with the GenBank serial number SJM46940.1; RpLTD is a phenylserine dehydratase RpLTD derived from Ralstonia pickettii, with the GenBank serial number BAC53614.1; BbLTD is a phenylserine dehydratase derived from Bordetella bronchiseptica, with the GenBank serial number VTQ90664.1; PtLTD is a phenylserine dehydratase derived from Pandoraea terrae, with the GenBank serial number VVE58836.1.

[0058] In the following examples, the D-lactate dehydrogenase is NADH-dependent D-lactate dehydrogenase, where LfD2-HDH is D-specific 2-hydroxyacid dehydrogenase derived from Lactobacillus frumenti, with GenBank accession number ON209401.1; Lp-D-LDH is D-lactate dehydrogenase derived from Lactobacillus pentosus, with GenBank accession number CCC17764.1; D-LDH82319 is D-lactate dehydrogenase derived from Limosilactobacillus reuteri, with GenBank accession number UFK68252.1; Lf-LDH is D-lactate dehydrogenase derived from Lactobacillus fermentum, with GenBank accession number WP_338432499.1; PaLDH is D-lactate dehydrogenase derived from Pediococcus acidilactici, with GenBank accession number ARW28447.1.

[0059] In the following examples, the liquid-phase detection conditions are as follows: Agilent 1260 DAD high-performance liquid chromatography, the chromatographic column is Eclipse Plus C18, 5 μm, 4.6×250 mm, mobile phase A (methanol): mobile phase B (water) = 30:70, flow rate 0.5 mL / min, detection wavelength 280 nm.

[0060] The product salvianic acid obtained in the present invention is D-salvianic acid (D-DSS), and its specific rotation in aqueous solution is 0.1258. The detection conditions are as follows: 589 nm sodium lamp, temperature 20 °C, specific rotation: 34.358.

[0061] In the following examples, the calculation formula for the substrate conversion rate is: substrate conversion rate = (substrate initial concentration - substrate concentration in the reaction solution) / substrate initial concentration × 100%; the calculation formula for the product yield is: product yield = (actual product concentration generated / theoretical product concentration generated) × 100%.

[0062] Example 1 Optimization of Aldolase and Transaldolase

[0063] The enzyme reaction system consists of 1 mL of 50 mM KPi buffer at pH 7 containing 10 mM vanillin, 3 U of aldolase or transaldolase (selected from one of PpLTA, EcTA, PfLTA, RsLTA, PsLTA, PsLTTA or ChLTTA), 0.1 mM pyridoxal phosphate, and 100 mM glycine or 20 mM threonine (when the enzyme added to the reaction system is aldolase, glycine is used as the substrate, and when the enzyme added to the reaction system is transaldolase, threonine is used as the substrate). The enzyme reaction system is placed in a shaker at 25 °C and 200 rpm for reaction. The reaction is carried out in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 12 h of reaction is taken, 400 μL of methanol is added to quench for 1 min, centrifuged at 10000 rpm for 2 min, and the supernatant is filtered (the pore size of the filter membrane is 0.22 μm) and then subjected to liquid phase detection. The substrate conversion rates in the reaction are shown in Table 1. It can be seen from Table 1 that when the reaction is carried out for 12 h, the catalytic efficiency of PsLTTA is the highest, and the conversion rate of vanillin is 70%.

[0064] Table 1 Conversion rates of vanillin catalyzed by different aldolases or transaldolases

[0065]

[0066]

[0067] Example 2 Optimization of threonine dehydratase and phenylserine dehydratase

[0068] The enzyme cascade reaction system consists of 1 mL of 50 mM KPi buffer at pH 7 containing 10 mM vanillin, 3 U of transaldolase PsLTTA, 3 U of threonine dehydratase or phenylserine dehydratase (selected from one of PxLTD, CgLTD, RpLTD, BbLTD, PtLTD), 0.1 mM pyridoxal phosphate, and 20 mM threonine. The multi-enzyme cascade reaction system is placed in a shaker at 25 °C and 200 rpm for reaction. The reaction is carried out in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 12 h of reaction is taken, 400 μL of methanol is added to quench for 1 min, centrifuged at 10000 rpm for 2 min, and the supernatant is filtered (the pore size of the filter membrane is 0.22 μm) and then subjected to liquid phase detection. The substrate conversion rates in the reaction are shown in Table 2. It can be seen from Table 2 that when the reaction is carried out for 12 h, the catalytic efficiency of the cascade of PsLTTA and RpLTD is the highest, and the conversion rate of vanillin is 100%.

[0069] Table 2 Yields of 4-hydroxy-3-methoxyphenylpyruvic acid produced by the cascade of different dehydratases and PsLTTA

[0070]

[0071] Optimization of Example 3D - Lactate Dehydrogenase

[0072] 1 mL of 50 mM KPi buffer with a pH of 7 contains 10 mM 4 - hydroxy - 3 - methoxyphenylpyruvic acid, 3 U of D - lactate dehydrogenase (selected from one of LfD2 - HDH, Lp - D - LDH, D - LDH82319, Lf - LDH, PaLDH), 1 U of formate dehydrogenase (FDH, from Candida boidinii., purchased from sigma - adlrich), 50 mM sodium formate, and 0.1 mM NAD + , as the enzyme reaction system. The said enzyme reaction system was placed in a shaker at 25 °C and 200 rpm for reaction. The reaction was carried out in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 4 h of reaction was taken, 400 μL of methanol was added to quench for 1 min, centrifuged at 10000 rpm for 2 min, and the supernatant was filtered (membrane pore size 0.22 μm) and then subjected to liquid - phase detection. The substrate conversion rates in the reaction are shown in Table 4. It can be seen from Table 3 that when the reaction time is 4 h, the catalytic reaction efficiency of LfD2 - HDH is the highest, and the conversion rate of 4 - hydroxy - 3 - methoxyphenylpyruvic acid is 100%.

[0073] Table 3 Conversion rates of different lactate dehydrogenases catalyzing the reaction of 4 - hydroxy - 3 - methoxyphenylpyruvic acid

[0074]

[0075] Example 4 Optimization of Reaction Time

[0076] 1 mL of 50 mM KPi buffer with a pH of 7 contains 10 mM vanillin, 20 mM threonine, 0.1 mM pyridoxal phosphate, 3 U of PsLTTA, 2 U of RpLTD, 1 U of LfD2 - HDH, 1 U of FDH, 0.1 mM NAD + and 50 mM sodium formate, as the multi - enzyme cascade reaction system. The said multi - enzyme cascade reaction system was placed in a shaker at 25 °C and 200 rpm for reaction. The reaction was carried out in a sealed 2 mL centrifuge tube. 100 μL of the reaction solution after 1 h, 2 h, 4 h, 8 h, and 12 h of reaction were taken respectively, 400 μL of methanol was added to quench for 1 min, centrifuged at 10000 rpm for 2 min, and the supernatant was filtered (membrane pore size 0.22 μm) and then subjected to liquid - phase detection. The liquid - phase chromatograms of the reaction solutions obtained at different reaction times are as Figure 1 shown, from Figure 1It can be seen that the rate-limiting step of the reaction lies in the first reaction catalyzed by transaldolase. There is no accumulation of intermediate products during the whole process, and only the chromatographic peaks of the substrate vanillin and the product (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl) propionic acid (mM) appear. The concentration changes of each component during the reaction are shown in Table 4. It can be seen from Table 4 that the reaction proceeds very fast. The reaction ends after 4 h, and the conversion rate of vanillin is as high as 96.5%, and the yield of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl) propionic acid is as high as 96.5%.

[0077] Table 4 List of the concentration change results of each component during the multi-enzyme cascade reaction

[0078]

[0079]

[0080] Example 5 Optimization of the ratio of vanillin to threonine

[0081] 1 mL of 50 mM KPi buffer with a pH of 7 contains 10 mM vanillin, 10 - 50 mM threonine, 0.1 mM pyridoxal phosphate, 3 U PsLTTA, 2 U RpLTD, 1 U LfD2-HDH, 1 U formate dehydrogenase, 0.1 mM NAD + and 50 mM sodium formate, serving as the multi-enzyme cascade reaction system. The multi-enzyme cascade reaction system is placed in a shaker at 25 °C and 200 rpm for reaction. The reaction is carried out in a sealed 2 mL centrifuge tube. Take 100 μL of the reaction solution after 4 h of reaction, add 400 μL of methanol to quench for 1 min, centrifuge at 10000 rpm for 2 min, and take the supernatant for filtration (the pore size of the filter membrane is 0.22 μm) and then perform liquid phase detection. The concentration changes of each component in the reaction are shown in Table 5. It can be seen from Table 5 that when the concentration ratio of vanillin to threonine is 1:3, the conversion rate of vanillin is the highest, reaching 100%. When the addition amount of threonine is less than 3 eq of vanillin, it is not conducive to the conversion of vanillin.

[0082] Table 5 Influence of the addition amount of threonine on the conversion of vanillin

[0083]

[0084] Example 6 Optimization of the concentration of the substrate vanillin

[0085] 1 mL of 50 mM KPi buffer at pH 7 contains 10 - 50 mM vanillin, 30 - 150 mM threonine, 0.1 - 0.5 mM pyridoxal phosphate (the molar ratio of vanillin:threonine: pyridoxal phosphate is 10:30:0.1), 3 UPsLTTA, 2 URpLTD, 1 U LfD2-HDH, 1 U formate dehydrogenase, 0.1 - 0.5 mM NAD + (the molar ratio of vanillin:NAD + is 100:1), 50 - 250 mM sodium formate (the concentration of sodium formate is maintained at 5 eq of vanillin), as a multi-enzyme cascade reaction system. The multi-enzyme cascade reaction system is placed at 25 °C and 200 rpm for reaction. The reaction is carried out in a sealed 2 mL centrifuge tube. Take 100 μL of the reaction solution after 4 h of reaction, add 400 μL of methanol to quench, ultrasonically mix, centrifuge at 10000 rpm for 2 min, filter (the filter membrane diameter is 0.22 μm) for liquid phase detection. The concentration changes of each component in the reaction are shown in Table 6. It can be seen from Table 6 that when the concentration of vanillin in the reaction system is greater than 20 mM, the conversion rate of vanillin gradually decreases. At a substrate concentration of 20 mM, the conversion rate of vanillin is 87.16%, and at a substrate concentration of 50 mM, the conversion rate of vanillin is 57.23%.

[0086] Table 6 Vanillin conversion rate and product yield at different vanillin substrate concentrations during the multi-enzyme cascade reaction

[0087]

[0088] Example 7 Demethylation experiment with hydrobromic acid

[0089] Add 1 mL of the completely reacted vanillin enzyme cascade reaction solution in Example 6 (obtained by reacting with 10 mM vanillin as the substrate, containing 2 mg of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid) to a microwave reaction flask, and then add 1 - 5 mL of 40 wt% aqueous hydrobromic acid solution. The microwave power is 100 - 250 W, and the reaction time is 5 - 30 min. Take samples and centrifuge at 10000 rpm for 2 min, filter (the filter membrane diameter is 0.22 μm) for liquid phase detection. The total reaction process is as Figure 2 shown. The liquid chromatograms of the reaction solution before and after the hydrobromic acid demethylation reaction are as Figure 3 shown.

[0090] As shown in Table 7, the optimal reaction conditions are 4 mL of HBr dosage, 10 min of reaction time, 100 W of microwave power, and 1 mg of catalyst TBHDPB dosage. Under these conditions, the conversion rate of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid is 100%, and the yield of D-salvianolic acid also reaches 90%. The mass spectrum of the obtained D-salvianolic acid is asFigure 4 as shown

[0091] Table 7 Changes in Substrate Conversion Rate and Danshensu Yield under Different Reaction Conditions in the Demethylation Experiment of Hydrobromic Acid

[0092]

[0093]

[0094] The present invention provides an idea and method for synthesizing danshensu using a chemoenzymatic cascade system. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented using existing technologies.

Claims

1. A method for synthesizing danshensu using a chemoenzymatic cascade system, characterized in that, The first enzyme cascade system or the second enzyme cascade system is subjected to a catalytic reaction to generate (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl) propionic acid, and then a demethylation reaction is carried out using hydrobromic acid, and finally D-salvianolic acid A is generated; The first enzyme cascade system described above includes: any one of vanillin, glycine, aldolase, threonine dehydratase, and phenylserine dehydratase, D-lactate dehydrogenase, formate dehydrogenase, sodium formate, cofactor, and solvent; The second enzyme cascade system described above includes: vanillin, threonine, transaldolase, any one of threonine dehydratase and phenylserine dehydratase, D-lactate dehydrogenase, formate dehydrogenase, sodium formate, cofactor, and solvent; The aldolase is any one of the following a1 to a5; the transaldolase is the following a6 or a7: a1, L-threonine aldolase PpLTA from Pseudomonas putida, Uniprot serial number 1V72; a2, L-threonine aldolase EcTA from Escherichia coli, GenBank serial number WP_000566376.1; a3, threonine aldolase PfLTA from Pseudomonas fluorescens, GenBank serial number EXF96374.1; a4, threonine aldolase RsLTA from Ralstonia solanacearum, GenBank serial number KFZ94941.1; a5, phenylserine aldolase PsLTA from Pseudomonas sp., GenBank serial number AFY19474.1; a6, threonine transaldolase PsLTTA from Pseudomonas sp., GenBank serial number WP_065949345.1; a7, threonine transaldolase ChLTTA from Chitiniphilus shinanonensis, GenBank serial number WP_018749561.1; The threonine dehydratase is the following b1; the phenylserine dehydratase is the following b3: b1, threonine dehydratase PxLTD from Paraburkholderia xenovorans, GenBank serial number WP_011488286.1; b3, phenylserine dehydratase RpLTD from Ralstonia pickettii, GenBank serial number BAC53614.1; The D-lactate dehydrogenase is any one of the following c1 to c5: c1, D-specific 2-hydroxyacid dehydrogenase LfD2-HDH from Lactobacillus frumenti, GenBank serial number ON209401.1; c2, D-lactate dehydrogenase Lp-D-LDH derived from Lactobacillus pentosus, GenBank accession number CCC17764.1; c3, D-lactate dehydrogenase D-LDH82319 derived from Limosilactobacillus reuteri, GenBank accession number UFK68252.1; c4, D-lactate dehydrogenase Lf-LDH derived from Lactobacillus fermentum, GenBank accession number WP_338432499.1; c5, D-lactate dehydrogenase PaLDH derived from Pediococcus acidilactici, GenBank accession number ARW28447.

1.

2. The method according to claim 1, wherein The solvent is potassium phosphate buffer with a pH of 7 - 8.

3. The method according to claim 1 or 2, characterized in that, In the described enzyme cascade system, the initial concentration of vanillin is 10 - 50 mM; the initial concentration of glycine is 10 eq of vanillin; the initial concentration of threonine is 1 - 5 eq of vanillin; the initial concentration of aldolase or transaldolase is 1 - 5 U / mL; the initial concentration of threonine dehydratase or phenylserine dehydratase is 1 - 10 U / mL; the initial concentration of D-lactate dehydrogenase is 1 - 10 U / mL; the initial concentration of formate dehydrogenase is 1 - 10 U / mL; the initial concentration of sodium formate is 5 eq of vanillin.

4. The method according to claim 1, characterized in that, The cofactors described above are pyridoxal phosphate and NAD + in the composition; in the enzyme cascade system, the initial concentration of the pyridoxal phosphate is 0.1 to 0.5 mM, and the initial concentration of the NAD + is 0.1 to 0.5 mM.

5. The method according to claim 1, wherein For the described catalytic reaction, the reaction pH is 6 - 8, the reaction temperature is 20 - 40 °C, and the reaction time is 1 - 12 h.

6. The method according to claim 1, characterized in that, The hydrobromic acid is a 40 wt% aqueous solution of hydrobromic acid; the volume of the hydrobromic acid aqueous solution used is 0.5 - 2.5 mL / mg based on the mass of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid.

7. The method according to claim 1, characterized in that The demethylation reaction is carried out under microwave conditions with a microwave power of 100 - 250 W and a reaction time of 5 - 30 min.

8. The method according to claim 7, wherein For the demethylation reaction, a catalyst cetyltributylphosphonium bromide is added, and the addition amount is less than 100% of the mass of (R)-2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid.

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