CN111662936A

CN111662936A - Method for producing tanshinol by enzyme method

Info

Publication number: CN111662936A
Application number: CN202010719293.4A
Authority: CN
Inventors: 李超; 张振东; 王戬
Original assignee: Shanghai First Maternity and Infant Hospital
Current assignee: Shanghai First Maternity and Infant Hospital
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2020-09-15

Abstract

The invention relates to a method for producing tanshinol by an enzyme method, which takes phenylpyruvic acid as a substrate, artificially designs a synthetic route, clones related enzyme coding genes in the designed metabolic route from thermophilic bacteria, ferments and produces a series of enzymes and purifies the enzymes, then adds related starting materials, and oscillates and converts at 50-300 r/min under the conditions of 30-65 ℃ and pH6.0-8.0 to obtain the tanshinol. The method of the invention has the following characteristics: (1) the enzyme catalysis is adopted, so that the cytotoxic effect of a substrate product caused by using cells and the generation of other byproducts are avoided; (2) the operation is simple, and the subsequent product is simple to separate and purify; (3) the conversion rate of the substrate phenylpyruvic acid to generate the danshensu is high and can reach over 84.9 percent, and the product danshensu can be accumulated to a higher concentration. The invention lays a foundation for realizing the high-efficiency production of the tanshinol.

Description

Method for producing tanshinol by enzyme method

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for producing tanshinol by an enzyme method, which is a method for producing tanshinol at high temperature by designing an artificial synthesis way, constructing thermophilic enzymes from different bacterial sources to carry out in-vitro cell-free enzymatic reaction.

Background

Plant Phenolic Acids (PAs) are the main source of natural antioxidants, originally extracted from various parts of the plant, including seeds, nuts, roots, bark, leaves, etc. Various high-added-value, low-molecular-weight PAs, such as vanillic acid, 4-hydroxybenzoic acid, salicylic acid, gallic acid, cinnamic acid, 4-coumaric acid, ferulic acid, phenylacetic acid, 3, 4-dihydroxybenzoic acid, and protocatechuic acid, have been obtained by extraction from various types of plants, wherein the content of each PA depends on the type of the specific plant and the site of the material. In recent years, there have been many studies attempting to obtain such compounds through chemical synthesis, enzymes, and metabolic engineering. However, unlike the enumerated examples above, which illustrate the complete biosynthetic pathway, the natural synthetic pathway of tanshinol in plants remains unclear.

The tanshinol is derived from the herb Salvia miltiorrhiza (Salviaminetiorrhiza), has great pharmacological activity including antitumor activity, antioxidant activity and anti-inflammatory property, and can improve cardiovascular diseases. It is also a precursor for the synthesis of drugs such as Danshensu Bingpinan Zhi and Danshensu ethyl ester (isoproyl 3- (3,4-dihydroxyphenyl) -2-hydroxyproanoate). So far, the synthesis of danshensu has the following modes: 1) directly extracting from Saviae Miltiorrhizae radix; 2) chemical synthesis; 3) constructing engineering bacteria by using escherichia coli, and synthesizing the engineering bacteria by using 4-hydroxyphenylpyruvic acid (4HPP) as a raw material; 4) takes catechol/pyruvic acid/ammonia or 3, 4-dihydroxyphenyl-l-alanine (l-DOPA) as raw material, and utilizes the catalysis of escherichia coli whole cells for synthesis. Although the research on the synthesis of danshensu has made a great progress, it cannot be used for industrial large-scale and efficient production. This may be due to the restriction of intracellular metabolic flux, inevitable toxicity to microbial cells, excessive production of by-products, insufficient supply of cofactors in whole-cell biocatalysis, etc., resulting in lower danshensu conversion efficiency and yield. Therefore, it is necessary to search for a new synthetic method instead of the existing method and to solve the above-mentioned problems.

One possible solution is to use a cell-free catalytic system, which does not require intact cells, but only purified enzymes. This method can eliminate the phase existing in the synthesis process of cellConcerns such as substrate or product toxicity to the cell, formation of by-products or other metabolic pathways induced by the substrate. The invention designs and constructs an artificial thermophilic enzymatic reaction consisting of d-mandelate dehydrogenase (ManDH), phenylalanine 4-hydroxylase (PAH) and 4-hydroxyphenylacetic acid 3-hydroxylase (HpaH) for cell-free synthesis of tanshinol. The enzyme reaction system can utilize phenylpyruvic acid (PPA) at low cost as a substrate by introducing a novel thermostable enzyme ManDH identified from thermophilic Thermococcus barophilus and PAH identified from Thermomonospora curvata. Meanwhile, in order to solve the problem of continuous consumption of cofactor during the catalytic cycle, there have been introduced the compounds including Nicotinamide Adenine Dinucleotide (NADH) and 6, 7-dimethyl-5, 6,7, 8-tetrahydropterin (DMPH)₄) A dual cofactor recycling system. There are no reports on the present invention.

Disclosure of Invention

The invention aims to provide a method for synthesizing tanshinol by using in-vitro cell-free enzymatic reaction aiming at the defects of the prior art. Two enzymes with good thermostability are disclosed, which are D-mandelate dehydrogenase from Thermococcus barophilus and phenylalanine 4-hydroxylase of Thermomonospora curvata, respectively. By utilizing a synthesis way designed by combining the two enzymes with 4-hydroxyphenylacetic acid 3-hydroxylase and a cofactor recycling method, the finally optimized enzyme system can efficiently synthesize the tanshinol under the condition of high temperature (not less than 50 ℃) by taking cheap phenylpyruvic acid as a substrate.

Specifically, the present invention provides the following:

on one hand, the invention provides two kinds of proteins with good thermal stability and D-mandelate dehydrogenase and phenylalanine 4-hydroxylase activities, wherein the proteins are respectively derived from Thermococcus barophilus and Thermomonosporacurvata and are respectively proteins synthesized by gene sequences shown in SEQ ID NO.1 and SEQ ID NO. 2. The gene sequence with the sequence number of SEQ ID NO.1 consists of 302 amino acid residues, and the gene sequence with the sequence number of SEQ ID NO.2 consists of 297 amino acid residues. The protein can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The nucleic acid molecule encoding the above protein may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.

In another aspect, the invention also provides a method for producing tanshinol by using the protein to construct an in vitro cell-free enzyme reaction system. The enzyme system is shown in the attached figure 1, and is described in detail as follows:

phenylpyruvic acid is firstly reduced into phenyllactic acid by NADH dependent ManDH; then, the phenyllactic acid is oxidized to tanshinol by pterin-dependent aromatic amino acid hydroxylating enzyme (AAAH) and HpaH with oxygen via 4-hydroxyphenylpyruvate (4 HPLA). Because phenyllactic acid and 4-hydroxyphenyllactic acid lack specific hydroxylation enzymes in the nature, based on structural similarity, PAH is preferably used for carrying out hydroxylation reaction on C-4 position of phenyllactic acid to synthesize 4-hydroxyphenyllactic acid, and HpaH is selected for carrying out hydroxylation reaction on C-3 position of 4-hydroxyphenyllactic acid to synthesize danshensu. Finally, 1mol of phenylpyruvic acid plus 1mol of oxygen can produce 1mol of danshensu and 2mol of water.

Preferably, the hydrogen peroxide (H) is generated by the catalytic reaction of PAH and HpaH in the enzyme reaction system as described above₂O₂) The hydrogen peroxide can be decomposed into water and oxygen by adding catalase so as to realize the recycling of the oxygen in the reaction system.

More preferably, in the enzymatic reaction system as described above, recycling of NADH in the reaction system is achieved by adding formate and Formate Dehydrogenase (FDH).

More preferably, in the enzymatic reaction system as described above, the DMPH in the reaction system is achieved by adding pterin-4 a-methanolamine dehydratase (PCD) and dihydrobiopterin reductase (DHPR)₄Recycling;

further, in the method for producing danshensu using phenylpyruvic acid (PPA) as a substrate as described above:

the use concentration of the substrate phenylpyruvic acid in the final reaction system is preferably 1-50 mM;

the use concentration of the liquid enzymes ManDH, PAH and HpaH in the final reaction system is preferably 5-10U, 0.3-1U and 20-40U;

the use concentration of the catalase in the final reaction system is preferably 500-1500U;

the cofactors NADH, DMPH in the final reaction system₄FAD and FAD are preferably used in concentrations of 0.5-3 mM, 0.05-0.4 mM and 0.2-1 mM;

the using concentration of the Formate Dehydrogenase (FDH) in the final reaction system is preferably 3-9U;

the use concentration of the pterin-4 a-methanolamine dehydratase (PCD) and the dihydrobiopterin reductase (DHPR) in the final reaction system is preferably 0.1-2 mg;

still further, the reaction conditions of the reaction system as described above: the temperature is controlled to be 30-65 ℃, and preferably 50 ℃; controlling the pH value to be 6.0-8.0, preferably 7.0; the rotation speed is controlled to be 50-300 rpm, preferably 200-250 rpm; the culture time is 8-20 h.

The invention has the advantages that:

the invention provides corresponding enzyme method process conditions, so that the danshensu can be produced by cheap raw materials at high temperature, and the conversion rate of a substrate product can reach 84.9 percent at most. Therefore, the method for producing the tanshinol can save cost, simplify operation flow, have short production period and have certain industrial application potential.

Drawings

FIG. 1 is a way of synthesizing danshensu from phenylpyruvic acid.

FIG. 2 is a protein purification process for ManDH, PAH, HpaB and HpaC. M, marker; coli BL21(pET28a) supernatant; coli BL21 supernatant containing target protein expression; 3, the purified target protein.

FIG. 3 is a protein purification process for FDH, PCD and DHPR. M, marker; coli BL21(pET28a) supernatant; coli BL21 supernatant containing target protein expression; 3, the purified target protein.

FIG. 4 is a time-course diagram of production of danshensu from phenylpyruvic acid. Tangle-solidup, phenylpyruvic acid; a xxx, danshensu.

FIG. 5 is a chromatogram and a mass spectrum of tanshinol produced by the present invention, which are verified by using a liquid chromatograph and a LC-MS.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

The strains and reagents used in the examples of the invention were as follows:

coli DH5 α and BL21(DE3), host bacteria for gene cloning and protein expression, respectively, were cultured in LB liquid medium at 37 ℃ in a shaker at 200rpm or LB solid medium in an incubator at 37 ℃. If necessary, 50. mu.g/mL kanamycin was added to LB medium.

The LB culture medium formula is as follows: 10g of peptone, 5g of yeast extract, 10g of NaCl, pH 7.0, constant volume of distilled water to 1L, and sterilizing at 121 ℃ for 20 minutes; 15g/L agar powder is added into the LB solid culture medium;

the plasmid used by the invention is pET28a (+), and is used for transferring the protein coding gene in the related pathway into BL21(DE3) for expression.

Example 1 construction of plasmid pET-ManDH carrying D-mandelic acid dehydrogenase Gene and protein acquisition

(1) Extraction of pET28a plasmid

Escherichia coli DH5 α (pET28a) carrying pET28a plasmid was inoculated at 1% inoculum size into 5mL LB liquid medium containing kanamycin (50. mu.g/mL) and cultured at 37 ℃ for 12 hours on a shaker at 200 rpm. The cultured microbial cells were used to extract pET28a plasmid.

(2) Amplification of D-mandelic acid dehydrogenase Gene ManDH

The codon of E.coli K12 was used to optimize the nucleotide sequence of ManDH (GenBank: WP-05693439) in Thermococcus barophilus as shown in SEQ ID NO. 1. The ManDH after the whole gene synthesis was inserted into pMD18-T, and PCR amplification was performed using the forward primer (5'-GCGCGAGCTCATGAAAATCTGCATCCTGGG-3') (SEQ ID NO.8) and the reverse primer (5'-CGCCAAGCTTTTATTTAGACTGGGTCAGCA-3') (SEQ ID NO.9), respectively.

(3) Construction of pET-ManDH plasmid

The recovered ManDH fragment and pET28a plasmid were digested with SacI and HindIII, and ligated with T4DNA ligase to obtain recombinant plasmid pET-ManDH.

(4) Prokaryotic expression of ManDH

Transforming the pET-ManDH obtained in the step 3 into E.coli BL21(DE3), shaking the bacteria at 37 ℃ until the OD 600nm is 0.6-0.8, adding IPTG with the final concentration of 1mM for induction, shaking the bacteria at 16 ℃ for 16h, collecting the bacteria, suspending the bacteria in 50mM phosphate buffer solution (PBS; pH 7.4), ultrasonically breaking the cells, collecting the supernatant, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis, wherein the SDS-PAGE is shown in figure 2 a.

(5) Purification of ManDH

And (4) carrying out affinity chromatography purification on the prokaryotic expression product detected by SDS-PAGE obtained in the step (4). And (3) passing the supernatant through a chromatographic column filled with Ni-NTA gel, washing the non-specifically bound hybrid protein by using a washing buffer solution (25mM Tris hydrochloric acid buffer solution, 500mM NaCl and 50mM imidazole, wherein the concentrations are the final concentrations in the solution and the pH value is 8.0), and finally eluting the target protein by using an elution buffer solution (25mM Tris hydrochloric acid buffer solution, 500mM NaCl, 20-500 mM imidazole, wherein the concentrations are the final concentrations in the solution and the pH value is 8.0) to obtain the purified target protein. The buffer was then changed to 50mM phosphate buffer (pH 7.0) using a molecular sieve gel column to obtain the purified protein of interest. As shown in FIG. 2a by SDS-PAGE, the desired band was about 35kDa in size.

Example 2 construction of plasmid pET-PAH carrying phenylalanine-4-hydroxylase Gene and protein acquisition

(1) Extraction of the pET28a plasmid: the same as in example 1.

(2) Amplification of phenylalanine-4-hydroxylase Gene PAH

PAH (GenBank: WP _012854034) in Thermomonospora curvata was optimized according to the codon of E.coli K12, and its nucleotide sequence was shown in SEQ ID NO. 2. The PAH after the whole gene synthesis was inserted into pMD18-T, and PCR amplification was performed using the forward primer (5'-ACGCGGATCCATGATGGAAGAAGCTCAGTA-3') (SEQ ID NO.10) and the reverse primer (5'-GCCCGAGCTCTTAAGAACCAGCAGAAGCAG-3') (SEQ ID NO.11), respectively.

(3) Construction of pET-PAH plasmid

The recovered PAH fragment and pET28a plasmid were digested with BamHI and SacI, and ligated with T4DNA ligase to obtain the recombinant plasmid pET-PAH.

(4) Prokaryotic expression of PAH

The same as in example 1. The result of SDS-PAGE analysis is shown in FIG. 2 b.

(5) Purification of ManDH

The same as in example 1. The SDS-PAGE result showed that the size of the desired band was about 35kDa, as shown in FIG. 2 b.

Example 3 construction of plasmids pET-hpaB and pET-hpaC carrying 4-hydroxyphenylacetate 3-hydroxylase Gene and protein acquisition

(1) Extraction of the pET28a plasmid: the same as in example 1.

(2) Amplification of 4-Hydroxyphenylacetic acid 3-hydroxylase genes hpaB and hpaC

hpaB (GenBank, TPY _2462) and hpaC (GenBank, TPY _2460) in Sulfobacillus acidophilus were optimized according to the codon of E.coli K12, and the nucleotide sequences thereof are shown in SEQ ID nos. 3 and 4. The hpaB and hpaBC after the whole gene synthesis were inserted into pMD18-T, and PCR amplification was performed using the forward primer 1 (5'-TACGGAGCTCATGGGTATCCGTACCGGTGA-3') (SEQ ID NO.12) and the reverse primer 1 (5'-TCCGCATATGTTAAGAACGAGCTTCTTTGG-3') (SEQ ID NO.13), and the forward primer 2 (5'-GCGCGGATCCGTTATGATGAACGCACTGGA-3') (SEQ ID NO.14) and the reverse primer 2 (5'-CGCCGAGCTCTTAACGTTCCAGCAGCAGGA-3') (SEQ ID NO.15), respectively.

(3) Construction of pET-hpaB and pET-hpaC plasmids

The recovered hpaB, hpaC fragments, and pET28a plasmid were digested with SacI/NdeI and BamHI/SacI, respectively, and ligated with T4DNA ligase to obtain recombinant plasmids pET-hpaB and pET-hpaC.

(4) Prokaryotic expression of HpaBC

The same as in example 1. The results of SDS-PAGE electrophoretic analysis are shown in FIGS. 2c and 2 d.

(5) Purification of HpaBC

The same as in example 1. The SDS-PAGE results are shown in FIGS. 2c and 2d, and the target bands are about 56 kDa and 22kDa, respectively.

Example 4 construction of plasmid pET-fdh carrying formate dehydrogenase Gene and protein acquisition

(1) Extraction of the pET28a plasmid: the same as in example 1.

(2) Amplification of formate dehydrogenase Gene fdh

FDH (GenBank: EFW95288) in Ogataeaparalpha was optimized according to the codon of E.coli K12, and its nucleotide sequence is shown in SEQ ID NO. 5. The fdh after the whole gene synthesis was inserted into pMD18-T, and PCR amplification was performed using the forward primer (5'-GCGCGGATCCATGAAAGTTGTTCTGGTTCT-3') (SEQ ID NO.16) and the reverse primer (5'-CGGTAAGCTTTTATTTGTCAGCACCGTAAG-3') (SEQ ID NO.17), respectively.

(3) Construction of pET-fdh plasmid

The recovered fdh fragment and pET28a plasmid were digested with BamHI and HindIII, and ligated with T4DNA ligase to obtain a recombinant plasmid pET-fdh.

(4) Prokaryotic expression of FDH

The same as in example 1. The results of SDS-PAGE analysis are shown in FIG. 3 a.

(5) Purification of FDH

The same as in example 1. The SDS-PAGE result showed that the size of the target band was about 45kDa in FIG. 3 a.

Example 5 construction of plasmids pET-DHPR and pET-PCD carrying dihydrobiopterin reductase and pterin-4 a-methanolamine dehydratase genes and protein acquisition

(1) Extraction of the pET28a plasmid: the same as in example 1.

(2) Amplification of dihydrobiopterin reductase and pterin-4 a-methanolamine dehydratase genes DHPR and PCD

The DHPR (GenBank, WP _011172549) in Thermus thermophilus and the PCD (GenBank, NP _213022) in Aquifex aeolicus were optimized according to the codon of E.coli K12, and the nucleotide sequences thereof are shown as SEQ ID No.6 and No. 7. The DHPR and PCD after the whole gene synthesis were inserted into pMD18-T and PCR amplification was performed using the forward primer 1 (5'-CCGAGAATTCATGCGTACCGCTCTGGTTAC-3') (SEQ ID No.18) and the reverse primer 1 (5'-CGGTAAGCTTTTACAGGTTCCAACCACCAG-3') (SEQ ID No.19), and the forward primer 2 (5'-GCGCGGATCCATGGTTCGTAAACTGTCTGA-3') (SEQ ID No.20) and the reverse primer 2 (5'-CGGCAAGCTTTTAGTGTTTCAGGATTTCTT-3') (SEQ ID No.21), respectively.

(3) Construction of pET-DHPR and pET-PCD plasmids

The recovered DHPR, PCD fragment and pET28a plasmid were digested with EcoRI/HindIII and BamHI/EcoR, respectively, and ligated with T4DNA ligase to obtain recombinant plasmids pET-DHPR and pET-PCD.

(4) Prokaryotic expression of DHPR and PCD

The same as in example 1. The results of SDS-PAGE analysis are shown in FIGS. 3b and 3 c.

(5) Purification of DHPR and PCD

The same as in example 1. The SDS-PAGE results are shown in FIGS. 3b and 3c, and the target bands are approximately 28 and 14kDa in size, respectively.

EXAMPLE 6 identification of enzymatic Properties of the protein of interest obtained in examples 1 to 3

(1) Activity of ManDH on substrate phenylpyruvic acid

Reaction system: 50mM PBS (pH 7.0), 0.2mM NADH and various concentrations of phenylpyruvic acid; NADH oxidation was measured at a wavelength of 340nm and one unit of enzyme activity was defined as the amount of enzyme required to oxidize 1. mu. molLNADH per minute. The KM, kcat and catalytic efficiency (kcat/KM) of ManDH to phenylpyruvic acid are respectively detected to be 2.7mM and 96.5s^-1And 3.6 × 104s^-1M^-1。

(2) Activity of PAH on the substrate phenyllactic acid

Reaction system: 50mM PBS (pH 7.0), 100. mu.M FeSO4, 1000U/mL catalase, 500. mu.M DMPH4, 5mM DTT, and various concentrations of phenyllactic acid. One unit of enzyme activity is defined as the amount of enzyme required to consume 1. mu. mol of PLA per minute. The KM, kcat and catalytic efficiency (kcat/KM) of PAH to the phenyllactic acid are respectively 4.2mM and 90.0s^-1And 2.1 × 104s^-1M^-1。

(3) Activity of HpaBC on substrate 4-hydroxyphenyllactic acid

Reaction system: 50mM PBS (p)H7.0), 0.2mM NADH, 2. mu.M FAD and different concentrations of 4-hydroxyphenyllactic acid. One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1. mu. molLNADH per minute. The KM, kcat and catalytic efficiency (kcat/KM) of PAH to the phenyllactic acid are respectively 5.2mM and 68.8s^-1And 1.3 × 104s^-1M^-1。

(4) Determination of optimum reaction pH value of ManDH, PAH and HpaBC

A buffer system: 50mM citric acid-sodium citrate buffer, pH 3.0 to 7.0; pH 7.0 to 9.0, 50mM phosphate buffer. The experiment is repeated for three times, and the enzyme activity is expressed by relative activity (according to the maximum value of 100 percent, the enzyme activity value under other conditions is correspondingly converted to obtain the relative activity of the enzyme). The optimum reaction pH values of ManDH, PAH and HpaBC are 7.0, 6.5 and 7.5 respectively.

(5) Determination of optimal reaction temperature for ManDH, PAH and HpaBC

The enzyme activity from 30 ℃ to 100 ℃ is measured, the experiment is repeated for three times, and the enzyme activity is expressed by relative activity (the relative activity of the enzyme is obtained according to the maximum value of 100 percent and the corresponding conversion of the enzyme activity value under other conditions). The detection shows that the optimal reaction temperature of the ManDH, the PAH and the HpaBC is 85 ℃, 65 ℃ and 50 ℃.

(6) Thermal stability of ManDH, PAH and HpaBC

0.2mg/mL of ManDH, PAH and HpaBC was added to 500. mu.L of 50mM sodium phosphate buffer (pH 7.0), respectively, and incubated at 50 ℃ for 12 and 24 hours, followed by measurement of relative enzyme activity. The experiment is repeated for three times, and the enzyme activity is expressed by relative activity (according to the maximum value of 100 percent, the enzyme activity value under other conditions is correspondingly converted to obtain the relative activity of the enzyme). The enzyme activities of the remaining enzyme activities of the ManDH, the PAH and the HpaBC after 12/24 hours are respectively 105.1%/109.1%, 91.3%/81.0% and 86.6%/77.2% through detection.

Example 7 optimization of reaction conditions for Salvianic acid Synthesis System

Temperature, pH, phenylpyruvic acid concentration, PCD/DHPR concentration on the production of tanshinol. Respectively as follows:

(1) the influence of the temperature on the production of the tanshinol by the phenylpyruvic acid is detected, the best effect is achieved when the temperature is 50 ℃, and the yield of the tanshinol is 76.9%, 89.6%, 95.5% and 86.5% when the rest temperatures are 40 ℃, 45 ℃, 55 ℃ and 60 ℃ in sequence;

(2) the influence of the pH value on the production of the tanshinol from the phenylpyruvic acid is detected, the effect is best when the pH value is 7.0, and the yield of the tanshinol is 24.1%, 74.8%, 82.7% and 61.3% when the other pH values are 6.0, 6.5, 7.5 and 8.0 in sequence;

(3) the detection shows that the effect of the concentration of the phenylpyruvic acid on the production of the danshensu is the best when the initial adding amount of the phenylpyruvic acid is 50mM, and under the remaining adding amounts of 10, 20, 30, 40, 60 and 70mM, the yield of the danshensu is 25.1%, 53.9%, 78.2%, 96.1%, 83.9% and 74.6% when the adding amount is 50mM in sequence;

(4) the influence of the PCD/DHPR concentration on the production of the tanshinol by the phenyl pyruvic acid is detected, the PCD/DHPR has the best effect when the PCD/DHPR is used at the concentration of 1mg/mL, and the yield of the tanshinol is 0.6 percent, 4.0 percent, 13.2 percent, 38.1 percent and 100 percent when the other additive amount is 0.002, 0.005, 0.01, 0.1 and 2mg/mL in sequence when the additive amount is 1 mM;

EXAMPLE 8 production of Salvianic acid A under the optimal conditions in example 7

The optimal reaction conditions of the enzyme system are as follows: 50mM PBS (pH 7.0), 8.9U ManDH, 0.6U PAH, 27.4UHpaBC, 6U FDH, 1mg PCD, 1mg DHPR, 1000U catalase, 50 μ M FeSO4, 5.0mM DTT,50mM PPA, 0.5mM FAD, 2mM NADH, and 0.2mM DMPH₄(ii) a The reaction temperature was 50 ℃ and the rotation speed was 200 rpm. After the reaction is finished, taking reaction liquid, detecting and analyzing the concentrations of phenylpyruvic acid and danshensu by HPLC, and calculating the conversion rate of substrate products and the like.

As can be seen from FIG. 4, the enzyme method can consume about 46mM phenylpyruvic acid in about 12 hours, and simultaneously synthesize about 39mM tanshinol, and the conversion rate of the substrate product can reach 84.9%. The produced danshensu was verified by using a liquid chromatograph and a mass spectrometer, as shown in fig. 5.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> first women and infants health care institute in Shanghai City

<120> method for producing danshensu by enzyme method

<130>/

<160>21

<170>PatentIn version 3.3

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cgtccgctgg acatcgctgc tatgggtacc accggttacg acatcaccaa ataccaggac 780

gttctgttct gcgctgaatc tctggaccac ctggaagacg ttgttggtac cttctgggct 840

gactgcgacg acgactctat cgctgctctg gctgcttctg ctggttctta a 891

<210>3

<211>1476

<212>DNA

<213> Artificial sequence

<400>3

atgggtatcc gtaccggtga acagtacctg aacgctctgg acggtgctac ccgtgacttc 60

tggatccacg gtgaacgtgt taccggtaaa atcaccgaac acccggcttt ccgtaacatc 120

gctcagtctg ttgctcacct gtacgacatg cagcacgacc cggctatcca ggctgaaatg 180

acctacccgt ctccggctac cggtgacctg gttggcctga gcttcctgca gccgcatacc 240

aaagaagacc tggttcgtcg tcgtaccatg atgatgcact gggctcgtta cgctcacggt 300

atgatgggtc gtgctccgga ctacctgaac tctgaactga tggctctggc tgctgctgct 360

ccgttcttcg gtgaattcgg tgacaacatc cgtaaatact acgaatacgt tcgtgaacac 420

gacctgtgca ccacccacac cctgatcatg ccgcaggcta accgttctgc tccgccgtct 480

cagcaggctg acccgtacct ggctgctcgt gttgttgaaa aaacctctga aggtgttatc 540

atccgtggtg ctcgtatgct ggctaccctg ccgctggctg acgaaatcct ggttttcccg 600

tctaccgtta tccgtaacac cccggaagac aaaccgtacg ctttcgcttt cgctctgccg 660

ctgtctaccc cgggtctgcg tctgatctgc cgtgaaacct tcgactacgg taaatctcac 720

ttcgaccacc cgctgggctc gaggttcgaa gaaatggacg ctgttgttgt tttccacgac 780

gttctggttc cgtgggaacg tatcttcctg ctggaagacg ctgaccgttg caaccagctg 840

tacgctgcta ccgacgctgt tgttcacatg acccaccagg ttatcgttaa agacatcgct 900

aaagctgaat tcatcctggg tgttgctggt ctgatcgttg acaccatcgg tatcgaacag 960

ttccagcacg ttcaggaaaa agttgctgaa gttatcctgg ctctggaaac catgaaagct 1020

ttcatggttg ctggtgaagc taacgctaaa ctgaaccgtt acggtatcat gaccccggac 1080

tggaccccgt tcaacatggc tcgtaacacc ttcccgcgtc tgtacccgcg tctggttgaa 1140

atcatccagc agctggctgc tggtggtctg atggctatcc cgaccgaaaa agacttcgct 1200

cacccggaac tgcgtccgga cctcgacagg ttctaccagg ctcgtaacac cgacgcttgg 1260

gaacgtaccc gtctgttccg tctggcttgg gacgctgctg tttcttcttt cggttctcgt 1320

caggttctct acgaacgctt cttcttcgga gacccggttc gtatggctgg tgctctgtac 1380

cagtcttacg acaaagaacc ggctaaagct ctgatccgtg aattcctgaa ccagaccccg 1440

gacccgctgg ttcagaccaa agaagctcgt tcttaa 1476

<210>4

<211>528

<212>DNA

<213> Artificial sequence

<400>4

gttatgatga acgcactgga taccgaacgc gcatttcgtc aagcttgcgg tacctttgcg 60

accggtatta ccgttatcct gacccagcaa ggtgaagaaa ttcacggtat gaccgctaac 120

gcgtttatga gcgtttctct gaatccgcgt ctgattgcga ttagcgttaa ccgtaccagc 180

cgtatgcacg gctttctggc accggaaggg gttaccttca gcgttagcat tctgcagagc 240

acccaacgtc cggtttctga cgcatttagt cgtcgcggta ccgatattac cccgcagtgg 300

gttccgaccg cacatcaagt tccggttatt gcaggcgcat tagcttggtt tgtttgcgaa 360

aaagcacagg ctattgacgc aggcgatcat accattgtca ttggccgcgt tgtcgatttc 420

gcacagcata gcgaagatca gccgttaatc ttctatcgcg gccgctattt tgatcgcgtt 480

gaaaacggcg aacaggaagc actggaattc ctgctgctgg aacgttaa 528

<210>5

<211>1089

<212>DNA

<213> Artificial sequence

<400>5

atgaaagttg ttctggttct gtacgacgct ggtaaacacg ctcaggacga agaacgtctg 60

tacggttgca ccgaaaacgc tctgggtatc cgtgactggc tggaaaaaca gggtcacgaa 120

ctggttgtta cctctgacaa agaaggtgaa aactctgttc tggaaaaaaa catcccggac 180

gctgacgtta tcatctctac cccgttccac ccggcttaca tcaccaaaga acgtatcgac 240

aaagctaaaa aactgaaact gctggttgtt gctggtgttg gttctgacca catcgacctg 300

gactacatca accagtctgg tcgtgacatc tctgttctgg aagttaccgg ttctaacgtt 360

gtttctgttg ctgaacacgt tgttatgacc atgctggttc tggttcgtaa cttcgttccg 420

gctcacgaac agatcatctc tggtggttgg aacgttgctg aaatcgctaa agactctttc 480

gacatcgaag gtaaagttat cgctaccatc ggtgctggtc gtatcggtta ccgtgttctg 540

gaacgtctgg ttgctttcaa cccgaaagaa ctgctgtact acgactacca gtctctgtct 600

cgtgaagctg aagaaaaagt tggtgctcgt cgtgttcacg acatcaaaga actggttgct 660

caggctgaca tcgttaccat caactgcccg ctgcacgctg gttctaaagg tctggttaac 720

gctgaactgc tgaaacactt caaaaaaggt gcttggctgg ttaacaccgc tcgtggtgct 780

atctgcgttg ctgaagacgt tgctgctgct gttaaatctg gtcagctgcg tggttatggc 840

ggtgacgtgt ggtacccgca accggctccg aaagaccacc cgtggcgttc tatggctaac 900

aaatacggtg ctggtaacgc tatgaccccg cactactctg gttctgttat cgacgctcag 960

gttcgttacg ctcagggtac caaaaacatc ctggaatctt tcttcaccca gaaattcgac 1020

taccgtccgc aggacatcat cctgctgaac ggtaaataca aaaccaaatc ttacggtgct 1080

gacaaataa 1089

<210>6

<211>705

<212>DNA

<213> Artificial sequence

<400>6

atgcgtaccg ctctggttac cggttctgct aaaggtatcg gtcgtgctat cctgctggct 60

ctggctcgtg aaggttacgc tgttgctgtt cactaccgta cctctgaagc tctggctgaa 120

gctacccgtc aggaagctga agctctgggt gttaaagcta tcaaagttcg tgctgacctg 180

acccgtgaag aagaagttga ccgtctggtt gaagaagttc gttaccacct gggtggtgtt 240

ggtgttctgg ttaacaacgt tggtgactac ctgtacaaac cgatcgaaga agtttctctg 300

gaagaatggc gttggatcct ggacaccaac ctgaccgcta ccttcctgct gacccagcgt 360

gttctgccgc tgatggttgc tcagggtttc ggtcgtatcg ttaacctggg ttacgctggt 420

gctggtaacc tgctggctcg tacccacatc accccgtacg ttatcgctaa aaccggtgtt 480

atcctgtaca ccaaagctat cgctaaacgt ttcgctgctt ctggtatcac cgctaacgtt 540

gttgctccgg gtgttgctga aaactctgtt tctaaaccgc tgcacgaaat cccgatgggt 600

cgtctggctc tgctgcagga aatcgctcag gctgttctgt tcttcgttcg tgaaccgtac 660

ctgaccggtc aggttctgga agttgctggt ggttggaacc tgtaa 705

<210>7

<211>300

<212>DNA

<213> Artificial sequence

<400>7

atggttcgta aactgtctga agaagaagtt aaacgtgaac tggaaaacct ggaaggttgg 60

gaattctgca aagactacat ccagaaagaa ttctctacca aaaactggaa aaccaccatc 120

ttcgttgtta acgctatcgc ttctctggct gaagctcagt ggcaccaccc ggacctcgaa 180

gtgagcttca aaaaagttaa agttaaactg accacccacg aagctggtgg tatcaccgaa 240

cgtgacatca aactggctaa atctatcgac gaactggtta aagaaatcct gaaacactaa 300

<210>8

<211>30

<212>DNA

<213> Artificial sequence

<400>8

gcgcgagctc atgaaaatct gcatcctggg 30

<210>9

<211>30

<212>DNA

<213> Artificial sequence

<400>9

cgccaagctt ttatttagac tgggtcagca 30

<210>10

<211>30

<212>DNA

<213> Artificial sequence

<400>10

acgcggatcc atgatggaag aagctcagta 30

<210>11

<211>30

<212>DNA

<213> Artificial sequence

<400>11

gcccgagctc ttaagaacca gcagaagcag 30

<210>12

<211>30

<212>DNA

<213> Artificial sequence

<400>12

tacggagctc atgggtatcc gtaccggtga 30

<210>13

<211>30

<212>DNA

<213> Artificial sequence

<400>13

tccgcatatg ttaagaacga gcttctttgg 30

<210>14

<211>30

<212>DNA

<213> Artificial sequence

<400>14

gcgcggatcc gttatgatga acgcactgga 30

<210>15

<211>30

<212>DNA

<213> Artificial sequence

<400>15

cgccgagctc ttaacgttcc agcagcagga 30

<210>16

<211>30

<212>DNA

<213> Artificial sequence

<400>16

gcgcggatcc atgaaagttg ttctggttct 30

<210>17

<211>30

<212>DNA

<213> Artificial sequence

<400>17

cggtaagctt ttatttgtca gcaccgtaag 30

<210>18

<211>30

<212>DNA

<213> Artificial sequence

<400>18

ccgagaattc atgcgtaccg ctctggttac 30

<210>19

<211>30

<212>DNA

<213> Artificial sequence

<400>19

cggtaagctt ttacaggttc caaccaccag 30

<210>20

<211>30

<212>DNA

<213> Artificial sequence

<400>20

gcgcggatcc atggttcgta aactgtctga 30

<210>21

<211>30

<212>DNA

<213> Artificial sequence

<400>21

cggcaagctt ttagtgtttc aggatttctt 30

Claims

1. A method for producing tanshinol by an enzyme method is characterized by comprising the following steps:

the method comprises the following steps: designing and optimizing gene sequences of D-mandelate dehydrogenase (ManDH; GenBank: WP _05693439), phenylalanine-4-hydroxylase (PAH; GenBank: WP _012854034) and 4-hydroxyphenylacetic acid 3-hydroxylase (HpaH; GenBank: TPY _2462, TPY _2460) and synthesizing the target gene, wherein the gene sequence of the D-mandelate dehydrogenase is shown as a sequence table SEQ ID NO.1, the gene sequence of the phenylalanine-4-hydroxylase is shown as a sequence table SEQ ID NO.2, and the gene sequence of the 4-hydroxyphenylacetic acid 3-hydroxylase is shown as a sequence table SEQ ID NO. 3;

step two: amplifying full-length genes of D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetic acid 3-hydroxylase in the first PCR amplification step, respectively connecting the obtained gene segments to plasmids, transferring the plasmids into cells, screening the resistance of the cells, performing amplification culture step by step, inducing protein expression, respectively collecting wet cells containing the D-mandelate dehydrogenase, the phenylalanine-4-hydroxylase and the 4-hydroxyphenylacetic acid 3-hydroxylase, crushing the collected wet cells under high pressure, centrifuging, and gradually adding ammonium sulfate into supernate until protein solids are separated out; centrifuging, collecting protein, and purifying to obtain liquid enzymes of ManDH, PAH and HpaH respectively;

step three: will be described in detailMixing the prepared ManDH, PAH and HpaH liquid enzymes with a substrate phenylpyruvic acid with the concentration of 1-50mM, and adding catalase and formate with the concentration of 500-1500U, cofactor NADH with the concentration of 0.5-3 mM and DMPH with the concentration of 0.05-0.4 mM₄And FAD with the concentration of 0.2-1 mM is put into a constant-temperature shaking table for catalytic reaction, and the reaction conditions are as follows: the temperature is 30-65 ℃, the pH value is 6.0-8.0, and the time is 8-20 h, and the obtained reaction liquid is the solution containing the tanshinol;

wherein the concentrations of the liquid enzymes ManDH, PAH and HpaH in the third step are respectively 5-10U, 0.3-1U and 20-40U; in the third step, formate dehydrogenase (FDH; GenBank: EFW95288) with the concentration of 3-9U is used for recycling NADH; in the third step, pterin-4 a-methanolamine dehydratase (PCD; GenBank: NP-213022) with a concentration of 0.1-2 mg and dihydrobiopterin reductase (DHPR; GenBank: WP-011172549) with a concentration of 0.1-2 mg are selected for DMPH₄And (4) recycling.

2. The method of claim 1, wherein the genes encoding D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetic acid 3-hydroxylase further comprise:

(1) DNA molecules respectively shown in a sequence table SEQ ID NO.1, a sequence table SEQ ID NO.2 and a sequence table SEQ ID NO. 3; and/or;

(2) a DNA molecule which hybridizes to the DNA molecule of (1) under stringent conditions and encodes the D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetate 3-hydroxylase of claim 1; and/or;

(3) a DNA molecule having a homology of 80% or more with the DNA molecule of (1) or (2) and encoding D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetic acid 3-hydroxylase of claim 1.

3. The method of claim 1, wherein the genes encoding D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetic acid 3-hydroxylase are derived from: thermophilic bacteria Thermococcus barophilus, Thermomonospora curvata and Sulfobacillus acidophilus.

4. The method of claim 1, wherein the D-mandelate dehydrogenase, phenylalanine-4-hydroxylase and 4-hydroxyphenylacetic acid 3-hydroxylase gene sequences of E.coli strain DH5 α are selected in step one, primers are designed and cloned.

5. The method of claim 1, wherein the strain Escherichia coli BL21(DE3) is selected as the host strain for protein expression in step one.