CN108424937B

CN108424937B - Method for synthesizing tanshinol by enzyme method

Info

Publication number: CN108424937B
Application number: CN201810396471.7A
Authority: CN
Inventors: 张娟; 冯志彬; 段春利
Original assignee: Ludong University
Current assignee: Shandong Yangcheng Biotech Co ltd
Priority date: 2018-04-27
Filing date: 2018-04-27
Publication date: 2022-06-21
Anticipated expiration: 2038-04-27
Also published as: CN108424937A

Abstract

The invention discloses a novel synthetic method for synthesizing tanshinol, which is used for catalyzing levodopa to produce tanshinol by using tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase. Specifically, 0.5-1.5mol of levodopa (3, 4-dihydroxyphenylalanine), 10-30g/L of wet tyrosine ammonia lyase, 10-30g/L of wet phenylpyruvate reductase and 10-30g/L of wet glucose dehydrogenase are placed in a reactor to be uniformly mixed, the pH value is adjusted to be 7.0, the temperature is 30 ℃, and the catalytic reaction is carried out for 16-20h to obtain the tanshinol. The method uses tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase as substrates for the first time, adopts an enzymatic method to produce the danshensu, saves raw materials, and avoids the pollution of chemical synthesis.

Description

Method for synthesizing tanshinol by enzyme method

Technical Field

The invention belongs to the technical field of biosynthesis, and particularly relates to a method for synthesizing tanshinol by an enzymatic method.

Background

The danshensu is named as danshensu under the chemical name of beta- (3, 4-dihydroxyphenyl) lactic acid, and is an effective component of a traditional Chinese medicinal material, namely the red sage root, pharmacological researches show that the danshensu has the effects of reducing the range of myocardial infarction and reducing the course of disease, has protective effects on myocardial ischemia and reperfusion injury, can eliminate exogenous oxygen free radicals and protect the functions of mitochondria; in addition, the danshensu can obviously inhibit the aggregation of blood platelets, is effective to coronary heart disease, and is beneficial to the rehabilitation of body tissues and the correction of serious complications such as adult respiratory distress syndrome and the like; the tanshinol also has certain effects in resisting bacteria, diminishing inflammation and enhancing the immune function of the organism, and the survival rate can be improved by clinically applying the tanshinol to patients suffering from infectious shock; a large number of clinical researches also find that the danshensu can inhibit the synthesis of cell endogenous cholesterol and can be used for preventing and treating atherosclerosis; salvianic acid A plays a role in activating blood and dissolving stasis by inhibiting the aggregation of platelet releasers and prothrombin and reducing the coagulability of blood through the influence on the platelet releasers and the prothrombin. In addition, the tanshinol has certain therapeutic effects on hyperplastic scar, dilated coronary artery, liver injury, cerebral ischemia injury, cor pulmonale, tumor, altitude disease, and psoriasis. In conclusion, the danshensu has great market potential. At present, the production of danshensu mainly comprises two methods of plant extraction and chemical synthesis, wherein the plant extraction takes a danshen root medicinal material as a raw material, and is obtained by crushing, soaking in water, decocting, concentrating an extracting solution, precipitating by using ethanol, extracting by using diethyl ether and salting out a water phase. The main disadvantage of this method is that the excessive ethanol concentration during the extraction process lowers the active ingredient of danshensu, and although many studies have promoted the improvement of the method, the limitation of raw materials is the largest factor that limits the scale of production thereof. The chemical synthesis method originally originated from the report of Harington et al in 1931, and the danshensu is obtained by acetylating ketene compound, which can respectively use glycine or trans-cinnamic acid as raw materials, but the steps are complicated, the intermediate products are many, and the environmental pollution is serious. Therefore, the development of a new tanshinol synthesis method is vital, only one report is shown for the synthesis of tanshinol by utilizing a recombinant microbial technology at present, the report takes prephenate as a precursor, utilizes p-hydroxyphenylacetic acid metahydroxylase and D-lactate dehydrogenase to catalyze and synthesize tanshinol, and with the deep research, the recombinant microbial technology for producing tanshinol is concerned about due to high efficiency, low pollution and low cost, and the key enzyme of the reaction is selected and efficiently expressed, which is the key of success or failure.

Disclosure of Invention

The invention aims to provide a novel method for synthesizing tanshinol by an enzyme method, which is used for solving the problem of shortage of raw materials in the prior art and has the advantages of high efficiency, low pollution and low cost.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention takes levodopa as a substrate, and catalyzes and produces the tanshinol by Tyrosine Ammonia Lyase (TAL), phenylpyruvate reductase (PPR) and Glucose Dehydrogenase (GDH), wherein the glucose dehydrogenase is used for regenerating NADH, and the addition of NADH is avoided. The method comprises the following specific steps:

tyrosine ammonia lyase is used for catalyzing 3, 4-dihydroxyl phenyl alanine to generate 3, 4-dihydroxyl phenyl pyruvic acid, and then danshensu (3, 4-dihydroxyl phenyl lactic acid) is generated by reduction under the catalysis of phenylpyruvic acid reductase. In the process of reducing 3, 4-dihydroxyphenyl pyruvic acid to generate danshensu under the catalysis of phenylpyruvic acid reductase, NAD is generated simultaneously, coenzyme NADH is needed, and NADH is expensive. Therefore, in order to solve the problem, glucose dehydrogenase is added for regenerating NADH, because glucose exists in the whole reaction system, the glucose dehydrogenase is added to generate NADH by taking NAD as coenzyme during the process of generating gluconic acid, a circulating coenzyme regeneration system is formed, no additional NADH is required to be added, and the problem of high price of NADH is solved. The specific reaction process is as follows:

preferably, the tyrosine ammonia lyase is derived from Rhodobacter sphaeroides (Rhodobacter sphaeroides), but not limited thereto, and the tyrosine ammonia lyase contained in the Rhodobacter sphaeroides is capable of catalyzing deamination of phenylalanine and tyrosine. And tyrosine ammonia lyase of the bacterium is taken as a template, and the tyrosine ammonia lyase gene TAL-made expressed in escherichia coli can be synthesized after codon optimization.

The phenylpyruvic acid reductase is derived from Lactobacillus casei (Lactobacillus casei), but is not limited to the Lactobacillus casei, and the phenylpyruvic acid reductase contained in the Lactobacillus casei can catalyze the reduction of the ketonic acid into the lactic acid. And the phenylpyruvic acid reductase of the strain is used as a template, and the codon is optimized to synthesize the phenylpyruvic acid reductase gene PPR-made expressed in escherichia coli, wherein the enzyme is D-type phenylpyruvic acid reductase.

The glucose dehydrogenase of the present invention is derived from, but not limited to, Bacillus megaterium (Bacillus megaterium) wherein the glucose dehydrogenase contained therein performs regeneration of coenzyme NADH in catalyzing the production of gluconic acid from glucose. And glucose dehydrogenase gene GDH-made expressed in Escherichia coli can be synthesized by codon optimization using glucose dehydrogenase of the strain as a template.

In a preferred embodiment of the invention, the tyrosine ammonia lyase gene, the phenylpyruvate reductase gene and the glucose dehydrogenase gene which are synthesized after codon optimization are respectively cloned into an expression vector, introduced into escherichia coli to realize gene expression, then cells for expressing the tyrosine ammonia lyase, the cells for expressing the phenylpyruvate reductase and the cells for expressing the glucose dehydrogenase are collected, the thallus is placed at minus 80 ℃ for storage for 24 hours, and the thallus is directly added into a catalytic system for catalytic production of the tanshinol after being melted. Specifically, the method is as follows:

(1) heterogenous expression of tyrosine ammonia lyase TAL-made, phenylpyruvate reductase PPR-made and glucose dehydrogenase GDH-made: respectively constructing gene engineering bacteria of TAL-made, PPR-made and GDH-made, and respectively preparing wet bacteria for expressing TAL, PPR and GDH through fermentation;

(2) then 0.5-1.5mol of levodopa, 10-30g/L of wet tyrosine ammonia lyase, 10-30g/L of wet phenylpyruvate reductase and 10-30g/L of wet glucose dehydrogenase are placed in a reactor to be uniformly mixed, the pH value is adjusted to 7.0, the temperature is 30 ℃, and the catalytic reaction is carried out for 16-20h, thus obtaining the danshensu. Preferably, 15-20g/L of tyrosine ammonia lyase wet bacteria, 15-20g/L of phenylpyruvate reductase wet bacteria and 15-20g/L of glucose dehydrogenase wet bacteria are needed for catalyzing 0.5-1.5mol of substrate levodopa.

The specific method of the step (1) is as follows:

(a) constructing a TAL-made vector:

the TAL-made gene and the pET28a vector are respectively subjected to double enzyme digestion by utilizing BamHI and XhoI, and then the BamHI and the pET28a vector are connected to construct a plasmid pET28a-TAL-made for recombining and expressing the TAL-made;

(b) constructing a PPR-made vector:

carrying out double enzyme digestion on the PPR gene and the pET28a vector by using BamHI and XhoI respectively, and then connecting the two to construct a plasmid pET28a-PPR-made for recombinant expression of PPR-made;

(c) construction of GDH-made vector:

carrying out double enzyme digestion on a GDH-made gene and a pET28a vector by utilizing BamHI and XhoI respectively, and then connecting the two to construct a plasmid pET28a-GDH-made for recombinant expression of the GDH-made;

(d) constructing genetic engineering bacteria for respectively expressing TAL, PPR and GDH;

plasmids pET28a-TAL-made, pET28a-PPR-made and pET28a-GDH-made are respectively transferred into E.coli BL21(DE3), so as to obtain strains BL21-TAL, BL21-PPR and BL21-GDH which respectively express TAL, PPR and GDH in a recombinant mode;

(e) culturing a strain BL21-TAL in a fermentation culture medium, adding 0.1-0.5% of lactose, carrying out induction expression for 16h at 25-30 ℃, and centrifugally collecting thalli to obtain the TAL expression thalli;

culturing the strain BL21-PPR in a fermentation culture medium, adding 0.1-0.5% lactose, performing induction expression for 16h at 25-30 ℃, and centrifuging and collecting thalli to obtain the PPR expression thalli.

Culturing the strain BL21-GDH in a fermentation culture medium, adding 0.1-0.5% lactose, performing induced expression for 16h at 25-30 ℃, and centrifugally collecting thalli to obtain the GDH expression thalli.

Wherein the formula of the fermentation medium is as follows: 2% of glucose, 0.3% of ammonium sulfate, 0.5% of peptone, 2% of yeast extract powder and KH₂PO₄ 1.25％、MgSO₄0.1%, citric acid 0.15% and lactose 0.1-0.5%, and the fermentation conditions are as follows: the pH value is 7.0, the temperature is 37 ℃, the temperature is reduced to 25-30 ℃ after induction, and the thalli are stored for standby after the fermentation is finished and centrifuged at 6000rpm for 10min at minus 20 ℃.

Furthermore, the method for detecting the expression of the recombinant protein TAL comprises the following steps: culturing the obtained expression strain BL21-TAL in an LB liquid culture medium containing Kana at 37 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.1mM after the OD600 of the thalli reaches 0.4-0.6, carrying out induced expression for 16h at 25 ℃, collecting thalli before and after the IPTG induction is added respectively, and detecting the expression of the recombinant protein TAL by utilizing polyacrylamide gel electrophoresis.

The detection method of the expression of the recombinant protein PPR comprises the following steps: the obtained expression strain BL21-PPR is cultured in an LB liquid culture medium containing Kana at 37 ℃, after the bacterial body OD600 reaches 0.4-0.6, IPTG is added to the final concentration of 0.1mM, induction expression is carried out for 16h at 25 ℃, bacterial bodies before and after IPTG induction are added are respectively collected, and the expression of the recombinant protein PPR is detected by polyacrylamide gel electrophoresis.

The detection method of the recombinant protein GDH expression comprises the following steps: the obtained expression strain BL21-GDH is cultured in an LB liquid culture medium containing Kana at 37 ℃, after the OD600 of the thalli reaches 0.4-0.6, IPTG is added to the final concentration of 0.1mM, induction expression is carried out for 16h at 25 ℃, thalli before IPTG induction is added and after the induction is finished are respectively collected, and the expression of the recombinant protein GDH is detected by polyacrylamide gel electrophoresis.

The invention has the following advantages:

the invention belongs to the production of danshensu by adopting tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase as well as levodopa as a substrate for the first time, has originality and high molar conversion rate verified by experiments, is produced by adopting a biological enzyme method, has no pollution and no influence of byproducts, and is suitable for industrial production.

Because the reduction activity of the phenylpyruvate reductase selected by the invention needs coenzyme NADH, the invention does not directly use coenzyme NADH, but uses glucose dehydrogenase for the regeneration of NADH, thereby avoiding the addition of NADH and solving the problem of high price of NADH.

Drawings

FIG. 1 shows the results of the double restriction enzyme validation of plasmids pET28a-TAL-made, pET28a-PPR-made and pET28 a-GDH-made;

wherein lane M is DNA marker, lane 1 is BamHI/XhoI double digestion verification plasmid pET28 a-TAL-made; lane 2 shows BamHI/XhoI double restriction verification plasmid pET28 a-PPR-made; lane 3 shows the BamHI/XhoI double restriction verification plasmid pET28 a-GDH-made.

FIG. 2 shows SDS-PAGE detection of TAL recombinant proteins;

wherein, Lane M is a protein Marker, Lane 1 is a BL21-TAL pre-induction product, and Lane 2 is a BL21-TAL post-induction product;

FIG. 3 shows the result of SDS-PAGE detection of PPR recombinant protein;

in the figure, lane M is a protein Marker, lane 1 is the product before induction of BL21-PPR, and lane 2 is the product after induction of BL 21-PPR;

FIG. 4 shows the SDS-PAGE detection of the GDH recombinant protein;

in the figure, lane M is the protein Marker, lane 1 is the pre-induction product of BL21-GDH, and lane 2 is the post-induction product of BL 21-GDH.

FIG. 5 shows the retention time of compounds determined by high performance liquid chromatography.

Detailed Description

The present invention will be described in detail below with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The chromatograph used in the present invention is a model 1200 high performance liquid chromatograph manufactured by Agilent corporation, usa.

The materials used in the present invention are as follows: coli DH5 alpha, E.coli BL21(DE3), TransTaq DNA polymerase, T4DNA ligase, standard relative molecular weight (Mr) DNA (namely DNA marker, Mr 250-10,000), standard Mr protein and other reagents can be purchased from Beijing Quanji Biotechnology Limited company; dopa standard (Sigma-Aldrich, usa); kanamycin (Kana) was purchased from shanghai bio-technology limited; peptone and yeast extract (Oxoid, UK); other reagents were purchased from national reagents, ltd.

LB Medium/g.L^-1: peptone 10, yeast extract 5, sodium chloride 10, pH 7.0.

The following examples were designed to obtain the preferred procedure according to the method of orthogonal experiments.

Embodiment 1 a method for enzymatic synthesis of tanshinol, comprising the following steps:

heterologous expression of tyrosine ammonia lyase TAL-made, phenylpyruvate reductase PPR-made and glucose dehydrogenase GDH-made:

1. heterogenous expression and enzyme activity determination of tyrosine ammonia lyase TAL-made:

(1) obtaining TAL-made gene fragment

TAL-made gene (SEQ ID NO.2) is obtained by synthesis from a company after codon optimization suitable for escherichia coli expression according to Tyrosine Ammonia Lyase (TAL) gene sequence (accession number: CP015288, SEQ ID NO.1), the gene is completely identical with TAL gene amino acid sequence, and restriction enzyme cutting sites of BamHI and XhoI are introduced into the upstream and downstream of the gene sequence so as to facilitate subsequent vector construction.

(2) Construction of pET28a-TAL-made expression vector

And (3) connecting and transforming TAL-made gene synthesized by BamHI and XhoI through double enzyme digestion and pET28a vector, and constructing plasmid pET28a-TAL-made for recombining and expressing TAL. The constructed plasmid was verified by double digestion with BamHI and XhoI, and the products were electrophoretically detected to give 2 bands (5.9kb and 1.5kb) in agreement with the expectation (see FIG. 1, lane 1 for DNA molecular weight Marker, lane 2 for pET28a-TAL-made digestion electrophoresis). Transformation of pET28a-TAL-made into E.coli BL21(DE3) yielded TAL expressing strain BL 21-TAL.

The expression strain BL21-TAL was inoculated into LB medium (containing Kana 50ng/ml) and cultured at 37 ℃. When OD is reached₆₀₀When the value reaches 0.4-0.6, adding isopropyl-beta-D-thiogalactoside (IPTG) to a final concentration of 0.1mmol/L, inducing for 16h at 25 ℃, and detecting the expression of soluble protein by SDS-PAGE electrophoresis. The results show that TAL (Mr about 54.9KD) has obvious expression band after IPTG is added, and the size is consistent with the expected size. The results of SDS-PAGE detection of TAL expression before and after IPTG induction are shown in FIG. 2 (Lane M is protein molecular weight Marker, Lane 1 is BL21-TAL induction pre-product, and Lane 2 is BL21TAL induction post-product).

2. Heterologous expression and enzyme activity determination of phenylpyruvate reductase PPR-made:

(1) obtaining the PPR-made Gene fragment

A PPR-made gene (SEQ ID NO.4) which is completely identical with the amino acid sequence of the PPR gene and has BamHI and XhoI restriction sites introduced upstream and downstream of the gene sequence is synthesized by a company after codon optimization suitable for Escherichia coli expression is carried out according to a phenylpyruvate reductase (PPR) gene sequence (accession number: KP735960, SEQ ID NO.3), so that subsequent vector construction is facilitated.

(2) Construction of pET28a-PPR-made expression vector

The PPR-made gene synthesized by BamHI and XhoI through double enzyme digestion and pET28a vector are connected and transformed to construct plasmid pET28a-PPR-made for recombinant expression of PPR. The constructed plasmid was verified by double digestion with BamHI and XhoI, and the products were electrophoretically detected to give 2 bands (5.9kb and 0.9kb) in agreement with the expectation (see FIG. 1, lane 1 for DNA molecular weight Marker, lane 3 for pET28a-PPR-made digestion electrophoresis). pET28a-PPR-made was transformed into E.coli BL21(DE3) to give the PPR-expressing strain BL 21-PPR.

The expression strain BL21-PPR was inoculated into LB medium (containing Kana 50ng/ml) and cultured at 37 ℃. When OD is reached₆₀₀When the value reaches 0.4-0.6, adding isopropyl-beta-D-thiogalactoside (IPTG) to a final concentration of 0.1mmol/L, inducing for 16h at 25 ℃, and detecting the expression of soluble protein by SDS-PAGE electrophoresis. The results show that PPR (Mr about 33.7KD) has a clear expression band after IPTG addition, which is consistent with the expected size. The results of SDS-PAGE detection of PPR expression before and after IPTG induction are shown in FIG. 3 (lane M is protein molecular weight Marker, lane 1 is the product before BL21-PPR induction, and lane 2 is the product after BL21-PPR induction).

3. Heterologous expression and enzyme activity determination of glucose dehydrogenase GDH-made:

(1) obtaining GDH-made Gene fragment

GDH-made gene (SEQ ID NO.6) was obtained synthetically by a company after codon optimization for E.coli expression based on the Glucose Dehydrogenase (GDH) gene sequence (GenBank accession No.: J04805, SEQ ID NO.5), which is identical in amino acid sequence to the GDH gene, and restriction sites for BamHI and XhoI were introduced upstream and downstream of the gene sequence to facilitate subsequent vector construction.

(2) Construction of pET28a-GDH-made expression vector

The GDH-made gene synthesized by double digestion with BamHI and XhoI was ligated with pET28a vector, and after transformation, a plasmid pET28a-GDH-made was constructed to express recombinant GDH. The constructed plasmid was verified by double digestion with BamHI and XhoI, and the products of the digestion were electrophoretically detected to give 2 bands (5.9kb and 0.8kb) in agreement with the expectation (see FIG. 1, lane 1 is DNA molecular weight Marker, lane 4 is BamHI/XhoI double digestion verification plasmid pET28 a-GDH-male). pET28a-GDH-made was transformed into E.coli BL21(DE3) to obtain GDH-expressing strain BL 21-GDH.

The expression strain BL21-GDH was inoculated into LB medium (containing Kana 50ng/ml) and cultured at 37 ℃. When OD is reached₆₀₀When the value reaches 0.4-0.6, adding isopropyl-beta-D-thiogalactoside (IPTG) to a final concentration of 0.1mmol/L, inducing for 16h at 25 ℃, and detecting the expression of soluble protein by SDS-PAGE electrophoresis. The results show that GDH (Mr about 28.0KD) has a significant expression band after IPTG addition, consistent with the expected size. SDS-PAGE of GDH expression before and after IPTG induction is shown in FIG. 4 (Lane M is protein molecular weight Marker, Lane 1 is the product before induction of BL21-GDH, and Lane 2 is the product after induction of BL 21-GDH).

4. The strains BL21-TAL, BL21-PPR and BL21-GDH are fermented respectively

Culturing a strain BL21-TAL in a fermentation culture medium, adding 0.1% lactose, carrying out induction expression for 16h at 30 ℃, and centrifugally collecting thalli to obtain TAL expression thalli, wherein the expression thalli are stored at-20 ℃; culturing the strain BL21-PPR in a fermentation culture medium, adding 0.1% lactose, performing induction expression for 16h at 30 ℃, centrifuging and collecting thalli to obtain the PPR expression thalli, and storing the expression thalli at-20 ℃; culturing the strain BL21-GDH in a fermentation culture medium, adding 0.1% lactose, carrying out induction expression for 16h at 30 ℃, centrifuging and collecting thalli to obtain the GDH expression thalli, and storing the expression thalli at-20 ℃.

Secondly, preparing a catalytic reaction system to prepare a compound 1:

obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 0.5mol of levodopa, 10g/L of wet bacteria of amino acid deaminase, 10g/L of wet bacteria of phenylpyruvate reductase and 10g/L of wet bacteria of glucose dehydrogenase, adjusting the pH of a reaction system to be 8.0, and converting for 16h at the temperature of 30 ℃, wherein the content of the obtained danshensu is 48.9g/L, and the molar conversion rate is more than 49.0%. And identifying and quantifying the danshensu produced in the reaction mixture by using high performance liquid chromatography.

The reaction mixture was measured using HPLC method. Chromatographic conditions are as follows: chromatography column C₁₈Columns (4.6 mm. times.250 mm, 5 m); the mobile phase A is methanol, and the mobile phase B is 1% acetic acid; the flow rate is 0.7 ml/min; the column temperature is 30 ℃; the detection wavelength is 280 nm. The chromatographic conditions were as follows: 0-15min, 12% mobile phase A; then within 15min, mobile phase a increased linearly from 12% to 52%; under the above conditions, a peak (shown by an arrow in fig. 5A) corresponding to the retention time of the danshensu standard (shown by an arrow in fig. 5C) appeared in the reaction mixture, and fig. 5B is an HPLC spectrum of the substrate levodopa. Measuring the product yield by standard curve method, preparing 1, 2, 4, 8 and 10ng/ml series concentration standard solutions (tanshinol) respectively, and measuring by sample injection. Linear regression was performed using the solution concentration c as the abscissa and the peak area a as the ordinate to obtain 206.86c +60.63 (r)²＝0.9975)。

Example 2

The procedure was the same as in example 1 except for the following steps.

Obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 1.0mol of levodopa, 20g/L of wet bacteria of amino acid deaminase, 20g/L of wet bacteria of phenylpyruvate reductase and 20g/L of wet bacteria of glucose dehydrogenase, adjusting the pH of a reaction system to be 8.0, and converting for 16h at the temperature of 30 ℃, wherein the content of the obtained danshensu is 58.0g/L, and the molar conversion rate is more than 58.1%.

Example 3

The procedure was the same as in example 1 except for the following steps.

Obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 1.5mol of levodopa, 30g/L of wet bacteria of amino acid deaminase, 30g/L of wet bacteria of phenylpyruvate reductase and 30g/L of wet bacteria of glucose dehydrogenase, adjusting the pH of a reaction system to be 8.0, and converting for 16h at the temperature of 30 ℃, wherein the content of the obtained danshensu is 60.7g/L, and the molar conversion rate is more than 60.8%.

Example 4

The procedure was the same as in example 1 except for the following steps.

Obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 0.5mol of levodopa, 20g/L of wet bacteria of amino acid deaminase, 20g/L of wet bacteria of phenylpyruvate reductase and 20g/L of wet bacteria of glucose dehydrogenase, adjusting the pH of a reaction system to be 8.0, and converting for 20 hours at the temperature of 30 ℃, wherein the content of the obtained danshensu is 55.2g/L, and the molar conversion rate is more than 55.3%.

Example 5

The procedure was the same as in example 1 except for the following steps.

Obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 1.0mol of levodopa, 30g/L of wet bacteria of amino acid deaminase, 30g/L of wet bacteria of phenylpyruvate reductase and 30g/L of wet bacteria of glucose dehydrogenase, adjusting the pH of a reaction system to be 8.0, and converting for 20 hours at the temperature of 30 ℃, wherein the content of the obtained danshensu is 59.2g/L, and the molar conversion rate is more than 59.3%.

Example 6

The procedure was the same as in example 1 except for the following steps.

Obtaining expression thalli of TAL, PPR and GDH according to the method in the step one, and adding the following medicines into a 250ml triangular flask in sequence to prepare a catalytic system: 1.5mol of levodopa, 10g/L of wet bacteria of amino acid deaminase, 10g/L of wet bacteria of phenylpyruvate reductase and 10g/L of wet bacteria of glucose dehydrogenase, adjusting the pH value of a reaction system to be 8.0, and converting for 20 hours at 30 ℃, wherein the content of the obtained danshensu is 52.2g/L, and the molar conversion rate is more than 52.3%.

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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acgggacctg cctcgatcca ttcgatctcc acgaacgccg ccaatcagga tgtggtctcg 1320

cttgggacca tcgccgcgcg cctctgccgc gagaagatcg accgttgggc ggagatcctt 1380

gcgatcctcg ctctctgtct tgcacaagct gcggagctgc gctgcggcag cggcctagac 1440

ggggtgtctc ccgcggggaa gaagctggtg caggccctgc gcgagcagtt cccgccgctt 1500

gagacggacc ggcccctggg acaggaaatt gccgcgcttg ctacgcacct cttgcagcaa 1560

tctcccgtct ga 1572

<210> 2

<211> 1584

<212> DNA

<213> TAL-made

<400> 2

ggatccatgc tggccatgag tccgccgaaa ccggccgttg aactggatcg tcatattgat 60

ctggatcagg cacatgccgt ggcaagtggc ggtgcccgca ttgtgctggc accgcctgca 120

cgtgatcgct gtcgtgcaag cgaagcacgt ctgggcgccg ttattcgtga agcacgtcat 180

gtgtatggtc tgaccaccgg ttttggcccg ctggccaatc gcctgattag cggcgaaaat 240

gttcgtaccc tgcaggccaa tctggttcat catctggcaa gtggcgtggg tccggttctg 300

gattggacca ccgcacgtgc aatggttctg gcacgcctgg tgagtattgc ccagggtgca 360

agtggtgcca gtgaaggtac cattgcccgt ctgattgatc tgctgaatag cgaactggca 420

ccggcagtgc cgagtcgcgg cacagtgggt gcaagtggcg atctgacccc gctggcacat 480

atggttctgt gcctgcaggg ccgtggcgat tttctggatc gcgatggtac ccgcctggat 540

ggtgcagaag gcctgcgtcg cggtcgtctg cagccgttag atctgagtca tcgtgatgcc 600

ctggcactgg ttaatggtac cagcgccatg accggtattg ccctggtgaa tgcacatgca 660

tgccgtcatc tgggtaattg ggcagtggca ctgaccgcac tgctggccga atgtctgcgc 720

ggccgcaccg aagcatgggc cgcagcactg agtgatctgc gtccgcatcc gggtcagaaa 780

gatgccgcag cacgcctgcg tgcccgtgtg gatggtagcg cacgcgttgt tcgccatgtt 840

attgcagaac gtcgcctgga tgcaggtgac attggtaccg aaccggaagc cggccaggat 900

gcatatagtc tgcgttgcgc accgcaggtg ctgggcgctg gttttgatac cctggcatgg 960

catgatcgcg ttctgaccat tgaactgaat gcagttaccg ataatccggt ttttccgccg 1020

gatggcagtg tgccggccct gcatggcggt aattttatgg gtcagcatgt tgccctgacc 1080

agtgatgccc tggcgaccgc cgttaccgtt ctggccggtc tggcagaacg ccagattgcc 1140

cgcctgaccg atgaacgcct gaatcgtggt ctgccgccgt ttctgcatcg cggtccggca 1200

ggcctgaata gcggctttat gggtgcacag gtgaccgcca ccgcactgtt agccgaaatg 1260

cgcgccaccg gcccggccag cattcatagt attagcacca atgccgcaaa tcaggatgtg 1320

gttagcctgg gtaccattgc agcccgcctg tgccgtgaaa aaattgatcg ttgggccgaa 1380

attctggcaa ttctggccct gtgcctggcc caggcagccg aactgcgttg cggtagtggc 1440

ctggatggtg ttagtccggc cggtaaaaaa ctggtgcagg ccctgcgcga acagtttccg 1500

ccgctggaaa ccgatcgtcc gctgggccag gaaattgccg cactggccac ccatctgctg 1560

cagcagagcc cggtttaact cgag 1584

<210> 3

<211> 939

<212> DNA

<213> PPR

<400> 3

atgatgaaga ttctaaacag ctttagtctg aagccggagc agcgccaaac acttgaagca 60

gcgggacaca ccgtcatcga tgctgacaag cttgatgatg ccacggctca acaaattgac 120

gtggtttatg gttggaatgc tgcggctacc cgcgtgaact ttgaccgact tcagtttgtg 180

caggcgatgt ccgctggcgt tgattattta ccattggctg agttggctaa acaccatgtc 240

ttgctggcta atacaagcgg cattcacgcc gaacccattg cggagtatgt gcttggcgcc 300

ttgtttacga tcagtcgcgg tattttgcca gccattcgag cagatcgcga catgtggaca 360

ttacgccaag agcgaccgcc aatgacattg cttaagggca aaacagcagt catttttggc 420

accggtcata ttggatcaac gattgcgacc aaactccagg cattgggctt gcacacgatt 480

ggcgtgagtg cacatggccg tccggctgca ggatttgacc aagtgatgac ggatgtggcg 540

acccatgagg cagcagggcg agcagatgtc gtcatcaacg cgttaccatt aacgccagat 600

acaaaacact tttatgatga agcattcttt gcagccgcta gcaaacaacc gcttttcatt 660

aacattggcc gtggtccgtc agttgatatg gctgctttga cgcaggcatt gaaaaacaag 720

caaatcagtg ctgctgcctt ggatgtggtg gatccggaac cactgccgca agactcacca 780

ttatggggca tgacgaacgt tttgctcacg ccgcatattt cgggcacggt gccgcaatta 840

cgcgacaaag tttttaaaat atttaatgat aacctcaaaa ccttgatatc aagcggccaa 900

ttggcaagcc atcaagttga tctcacgcgc ggatactga 939

<210> 4

<211> 951

<212> DNA

<213> PPR-made

<400> 4

ggatccatga tgaagatcct gaatagtttc agtctgaaac cggaacagcg tcagaccctg 60

gaagcagcag gtcataccgt tattgatgcc gataaactgg atgatgccac cgcccagcag 120

attgatgtgg tgtatggttg gaatgcagcc gcaacccgcg tgaattttga tcgcctgcag 180

tttgttcagg caatgagcgc cggtgttgat tatctgccgc tggcagaact ggcaaaacat 240

catgtgctgc tggcaaatac cagtggcatt catgcagaac cgattgcaga atatgtgctg 300

ggtgcactgt ttaccattag ccgcggcatt ctgccggcaa ttcgtgccga tcgtgatatg 360

tggaccctgc gccaggaacg tccgccgatg accctgctga aaggtaaaac cgcagttatt 420

tttggtaccg gtcatattgg tagtaccatt gccaccaaac tgcaggcact gggcctgcat 480

accattggtg tgagtgccca tggtcgtccg gccgcaggtt ttgatcaggt tatgaccgat 540

gttgccaccc atgaagcagc cggccgcgca gatgttgtga ttaatgcact gccgctgacc 600

ccggatacca aacattttta tgatgaagca ttcttcgcag cagccagcaa acagccgctg 660

tttattaata ttggtcgtgg cccgagtgtt gatatggccg cactgaccca ggcactgaaa 720

aataagcaga ttagcgcagc cgccctggat gttgttgatc cggaaccgct gccgcaggat 780

agcccgctgt ggggtatgac caatgttctg ctgaccccgc atattagcgg taccgttccg 840

cagctgcgtg ataaagtttt taaaattttc aacgacaacc tgaagaccct gattagtagt 900

ggtcagctgg ccagccatca ggtggatctg acccgcggct attaactcga g 951

<210> 5

<211> 786

<212> DNA

<213> GDH

<400> 5

atgtataaag atttagaagg aaaagtagtg gtcataacag gttcatctac aggtttggga 60

aaatcaatgg cgattcgttt tgcgacagaa aaagccaaag tagttgtgaa ctatcgttct 120

aaggaggacg aagctaacag cgttttagaa gaaattaaaa aagttggcgg agaagcaatt 180

gctgtcaaag gtgatgtaac agttgagtct gacgttatca atttagttca atctgctatt 240

aaagagtttg gaaagctaga cgttatgatt aacaacgcag ggttagaaaa tccggtttca 300

tctcatgaaa tgtctttaag cgattggaat aaagtcattg atacgaactt aacgggagct 360

tttttaggca gccgtgaagc gattaaatat tttgtggaaa atgatattaa gggaacagtt 420

attaacatgt cgagtgttca cgagaaaatt ccttggccat tatttgttca ttatgcagca 480

agtaaaggcg gtatgaagct tatgactgaa acactggcat tagaatacgc tccaaaaggt 540

attcgtgtaa ataacattgg accaggagcg attaatacac cgattaacgc tgagaaattt 600

gctgatcctg agcagcgtgc agatgtagaa agcatgattc caatgggata catcggagag 660

ccggaagaaa ttgcagcagt tgctgcatgg ctagcttctt cagaggcgag ttatgtaaca 720

ggaattacgc tctttgctga cggcggtatg acacagtacc catcattcca agcaggacgc 780

ggataa 786

<210> 6

<211> 798

<212> DNA

<213> GDH-made

<400> 6

ggatccatgt acaaggatct ggaaggcaaa gttgtggtta ttaccggtag tagtaccggt 60

ctgggcaaaa gcatggcaat tcgctttgcc accgaaaaag caaaagtggt ggtgaattat 120

cgcagtaaag aagatgaagc aaatagcgtt ctggaagaaa ttaagaaagt tggtggtgaa 180

gccattgccg ttaaaggcga tgtgaccgtg gaaagcgatg ttattaatct ggttcagagc 240

gcaattaagg aatttggcaa actggatgtg atgattaata atgcaggtct ggaaaatccg 300

gttagcagcc atgaaatgag cctgagtgat tggaataagg ttattgatac caatctgacc 360

ggtgcctttc tgggcagtcg cgaagccatt aagtattttg tggaaaatga tatcaagggc 420

accgtgatta atatgagcag tgtgcatgaa aaaatcccgt ggccgctgtt tgttcattat 480

gcagccagca aaggtggtat gaaactgatg accgaaaccc tggcactgga atatgccccg 540

aaaggtattc gtgttaataa tattggtccg ggtgcaatta ataccccgat taatgcagaa 600

aaattcgccg atccggaaca gcgcgccgat gttgaaagta tgattccgat gggttatatt 660

ggcgaaccgg aagaaattgc cgcagtggcc gcatggctgg ccagtagtga agcaagctat 720

gttaccggca ttaccctgtt tgcagatggc ggcatgaccc agtatccgag ctttcaggca 780

ggccgcggtt aactcgag 798

Claims

1. A method for synthesizing tanshinol by an enzyme method is characterized in that levodopa is used as a substrate, and the tanshinol is produced by catalysis of tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase; the method specifically comprises the following steps:

(1) heterologous expression of tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase: respectively constructing gene engineering bacteria of tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase, and respectively preparing thalli for expressing the tyrosine ammonia lyase, the phenylpyruvate reductase and the glucose dehydrogenase by fermentation: tyrosine ammonia lyase wet thalli, phenylpyruvate reductase wet thalli and glucose dehydrogenase wet thalli;

(2) then 0.5-1.5mol of levodopa, 10-30g/L of wet tyrosine ammonia lyase, 10-30g/L of wet phenylpyruvate reductase and 10-30g/L of wet glucose dehydrogenase are placed in a reactor and uniformly mixed, the pH value is adjusted to 7.0, the temperature is 30 ℃, and the catalytic reaction is carried out for 16-20h, thus obtaining the danshensu.

2. The method for enzymatically synthesizing danshensu according to claim 1, wherein 15-20g/L of wet bacteria of tyrosine ammonia lyase, 15-20g/L of wet bacteria of phenylpyruvate reductase, and 15-20g/L of wet bacteria of glucose dehydrogenase are required for catalyzing 0.5-1.5mol of levodopa as a substrate.

3. The method for the enzymatic synthesis of tanshinol according to claim 1, wherein said tyrosine ammonia lyase is selected from rhodobacter sphaeroides.

4. The method for the enzymatic synthesis of salvianic acid A according to claim 1, wherein the phenylpyruvate reductase is selected from Lactobacillus casei.

5. The method for the enzymatic synthesis of salvianic acid A according to claim 1, wherein said glucose dehydrogenase is selected from Bacillus megaterium.

6. The enzymatic method of salvianic acid A according to claim 1, wherein said step (1) is specifically as follows:

(a) constructing a tyrosine ammonia lyase vector:

the TAL-made gene and the pET28a vector are subjected to double enzyme digestion by utilizing BamHI and XhoI respectively, and then the BamHI and the pET28a vector are connected to construct a plasmid pET28a-TAL-made for recombining and expressing tyrosine ammonia lyase;

(b) constructing a phenylpyruvate reductase vector:

carrying out double enzyme digestion on the PPR-made gene and the pET28a vector by using BamHI and XhoI respectively, and then connecting the two to construct a plasmid pET28a-PPR-made for recombining and expressing phenylpyruvic acid reductase;

(c) construction of glucose dehydrogenase vector:

carrying out double enzyme digestion on a GDH-made gene and a pET28a vector by using BamHI and XhoI respectively, and then connecting the two to construct a plasmid pET28a-GDH-made for recombining and expressing glucose dehydrogenase;

(d) respectively constructing gene engineering bacteria of tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase;

plasmids pET28a-TAL-made, pET28a-PPR-made and pET28a-GDH-made are respectively transferred into E.coli BL21 to obtain strains BL21-TAL, BL21-PPR and BL21-GDH which respectively express tyrosine ammonia lyase, phenylpyruvate reductase and glucose dehydrogenase in a recombinant mode;

(e) culturing a strain BL21-TAL in a fermentation culture medium, adding 0.1-0.5% of lactose, carrying out induction expression for 16h at 25-30 ℃, and centrifugally collecting thalli to obtain the tyrosine ammonia lyase expression thalli;

culturing a strain BL21-PPR in a fermentation culture medium, adding 0.1-0.5% of lactose, carrying out induction expression for 16h at 25-30 ℃, and centrifugally collecting thalli to obtain the phenylpyruvate reductase expressed thalli;

culturing the strain BL21-GDH in a fermentation culture medium, adding 0.1-0.5% lactose, performing induced expression for 16h at 25-30 ℃, and centrifugally collecting thalli to obtain the glucose dehydrogenase expression thalli.

7. The method for enzymatically synthesizing tanshinol according to claim 6, wherein the fermentation medium formula is: 2% of glucose, 0.3% of ammonium sulfate, 0.5% of peptone, 2% of yeast extract powder and KH₂PO₄1.25％、MgSO₄0.1%, citric acid 0.15% and lactose 0.1-0.5%, and the fermentation conditions are as follows: the pH value is 7.0, the temperature is 37 ℃, the temperature is reduced to 25-30 ℃ after induction, and the thalli are stored for standby after the fermentation is finished and centrifuged at 6000rpm for 10min at minus 20 ℃.