A biosynthetic method of medipterostilbene

Abstract

The present invention discloses a method for heterologously synthesizing a natural high-value active compound medipterocarp in Saccharomyces cerevisiae using synthetic biology technology. The present invention uses bioinformatics analysis, mining, in vivo and in vitro experimental identification, and verifies 12 key enzyme genes related to the biosynthesis of medipterocarp - cinnamic acid 4-hydroxylase, coumaroyl-CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavone synthase, 2-hydroxyisoflavone dehydratase, isoflavone 4′-O-methyltransferase, isoflavone 2′-hydroxylase, isoflavone reductase, estrogen reductase, and pterostilbene synthase. These key enzyme genes are used as expression elements of medipterocarp, and a gene expression module is constructed in a Saccharomyces cerevisiae cell factory to obtain a high-value active compound medipterocarp. The synthesis method of the present invention is economical, efficient, green and environmentally friendly, and the synthesized medipterocarp has high purity and has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically, the present invention relates to a method for biosynthesizing meditartin in the microorganism Saccharomyces cerevisiae. Background Art

[0002] Medipterocarpin is a rare flavonoid compound found in leguminous plants such as licorice, goldenseal, Astragalus membranaceus, S. serrata, and alfalfa. Studies have shown that Medipterocarpin has good anti-tumor activity. Currently, the main methods for obtaining Medipterocarpin are direct extraction from plants or chemical synthesis. Obtaining Medipterocarpin from plants requires the consumption of a large amount of original plants. Excessive mining damages the ecological environment and causes a serious shortage of plant resources. The method of chemically synthesizing Medipterocarpin has problems such as cumbersome steps, low yield, and environmental unfriendliness. Therefore, it is urgent to find a new method for obtaining Medipterocarpin in a reasonable and efficient way.

[0003] In recent years, synthetic biology has developed rapidly. The use of microbial yeast cell factories to synthesize rare and high-value pharmaceutical ingredients has become one of the important ways to produce drugs and develop new drugs. Microorganisms have a short growth cycle and can be mass-produced using a variety of sugar raw materials. Compared with plant cultivation extraction and chemical synthesis, the production of medipterostilbene by microbial fermentation is an economical and efficient way and a new green biomanufacturing model. Therefore, the use of synthetic biology methods to construct a brewer's yeast microbial cell factory for medipterostilbene will effectively solve the problem of sustainable resource utilization of medipterostilbene.

[0004] The present invention utilizes bioinformatics analysis and mining, and in vitro and in vivo experimental identification to verify 12 key enzyme genes related to the biosynthesis of medipterocarpin, namely, cinnamic acid-4-hydroxylase (C4H), coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), chalcone isomerase (CHI), 2-hydroxyisoflavone synthase (2-HIS), 2-hydroxyisoflavone dehydratase (HID), isoflavone-4′-O-methyltransferase (HI4′OMT), isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR), and pterostilbene synthase (PTS). These key enzyme genes are used as expression elements of medipterocarpin to construct a saccharomyces cerevisiae cell factory for heterologous synthesis of medipterocarpin. Summary of the invention

[0005] The purpose of the present invention is to screen out an economical, efficient, green and environmentally friendly method based on the existing technology, that is, to use brewer's yeast cell factory to heterologously synthesize a compound with anti-cancer activity, namely, meditartin.

[0006] The present invention application found that the expression and optimization of heterologous biosynthetic pathways in Saccharomyces cerevisiae require the introduction of multiple (continuous) genes at multiple different sites, and the application of the CRISPR-Cas9 system can accelerate the construction of Saccharomyces cerevisiae strains. CRISPR-Cas9 plasmids cut at 8 Saccharomyces cerevisiae integration sites. The gene expression module 1 containing three chalcone synthesis-related enzymes, CHS, CHI, and CHR, and the gene expression module 2 containing IFR4 and I′2H5 were successfully transformed into Saccharomyces cerevisiae IMX581 in sequence, and Saccharomyces cerevisiae strains DW-01 and DW-02 were obtained respectively. Using the DW-02 strain as the starting strain, the expression module 3 containing the PTS and VR genes was transformed into the corresponding Saccharomyces cerevisiae integration site to obtain the strain DW-03 (Table 1), and the DW-03 bacteria were cultured. After breaking the bacteria, crude protein was extracted, and the gene expression products were detected by UPLC using formononetin as the substrate. The experiment found that mediptertin was produced. Then, the DW-03 strain was used as the starting strain, and the expression module 4 containing 2-HIS and HID genes and the gene expression module 5 containing C4H and 4CL were transformed into the corresponding Saccharomyces cerevisiae integration sites to obtain strains DW-04 and DW-05 (Table 1). DW-04 was fed with liquiritigenin as a substrate, and its gene expression products were detected by UPLC, and daidzein was found to be produced. In order to increase the expression intensity of P450 enzymes I′2H5 and 2-HIS, the copy number of I′2H5 and 2-HIS and the redox partner CPR of P450 enzymes were increased on the basis of DW-04, and strain DW-06 was successfully constructed. Next, DW-06 was used as the chassis, and the exogenous module 7 containing HI4′OMT was transferred to verify that liquiritigenin was used as a substrate, and after the catalytic expression of 2-HIS, HI4′OMT, and HID key enzymes, formononetin was synthesized, and then meditartin was synthesized.

[0007] A biosynthetic method of medipterocarpin, characterized in that it comprises the following steps:

[0008] (1) Yeast strain DW-01 (see Table 1) was constructed. Isoliquiritigenin was subjected to the action of chalcone isomerase heterologously expressed in the yeast strain to obtain compound 1, liquiritigenin, with the structural formula as follows:

[0009]

[0010] (2) Yeast strain DW-04 (see Table 1) was constructed. Liquorice root can be used to obtain compound 2-daidzein under the action of 2-hydroxyisoflavone synthase and 2-hydroxyisoflavone dehydratase heterologously expressed in the yeast strain. The structural formula is as follows:

[0011]

[0012] (3) Yeast strain DW-07 (see Table 1) was constructed, and daidzein was used to obtain compound 3, formononetin, under the action of isoflavone-4′-O-methyltransferase heterologously expressed in the yeast strain. The structural formula is as follows:

[0013]

[0014] (4) Yeast strains DW-02, DW-03 and DW-06 (see Table 1) were constructed, and the compound meditartin was obtained by the action of isoflavone-2′-hydroxylase, isoflavone reductase, estrogen reductase and pterocarpansynthase heterologously expressed in these yeast strains. The structural formula of the compound is as follows:

[0015]

[0016] The method for constructing the Saccharomyces cerevisiae strain related to the synthesis of medipterostilbene provided by the present invention is shown in Table 1 below:

[0017] Table 1 Construction of Saccharomyces cerevisiae strains related to the synthesis of medipterostilbene

[0018]

[0019]

[0020] Note: The genes in the table are: CHS (chalcone synthase); CHI (chalcone isomerase); CHR (chalcone reductase); I2'H5 (isoflavone 2'-hydroxylase); IFR4 (isoflavone reductase); GePTS (Glycyrrhiza echinata pterocarpan synthase); VR (vestitone reductase); 2-HIS (2-hydroxyisoflavanone); HID (2-hydroxyisoflavanone dehydratase); C4H (cinnamate 4-hydroxylase); 4CL (4-coumarate CoA ligase); GmCPR (soybean cytochrome P450 reductase) cytochrome P450 cyclase; HI4′OMT (isoflavone 4′-O-methyltransferase) isoflavone-4′-O-methyltransferase.

[0021] As a preferred embodiment, the above-mentioned method for biosynthesis of medipterostilbene comprises the following steps:

[0022] (1) Isoliquiritigenin is isomerized to licorice under the catalysis of chalcone isomerase

[0023]

[0024] The 300μL enzyme reaction system is: 15μL HEPES (1M / L) buffer, 7.7μL isoliquiritigenin (10mg / mL) substrate, 30μL CHI (49.81mg / mL), and ultrapure water to 300μL. After mixing, react in a water bath overnight, extract with ethyl acetate, combine the extracts and concentrate to dryness by centrifugation, then add methanol to dissolve, and filter through a 0.22μm microporous filter membrane to obtain liquiritigenin;

[0025] (2) Liquorice is converted into daidzein under the catalysis of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID)

[0026]

[0027] 1mL enzyme reaction system is: enzyme reaction solution (0.2M K2HPO4, 1.25mM GSH, 50% sucrose, 1mM NADPH) 400μL, substrate liquiritigenin (10mg / mL) 2.5μL, NADH (70mM / L) 15μL, MgCl2 (1M / L) 5μL, 2-HIS crude enzyme solution 430μL and HID crude enzyme solution 50μL, ultrapure water to 300μL. After mixing, react in a water bath overnight, extract with ethyl acetate, combine the extracts and centrifuge to dryness, then add methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain daidzein;

[0028] (3) Daidzein is converted into formononetin under the catalysis of isoflavone-4′-O-methyltransferase (HI4′OMT)

[0029]

[0030] The 300μL enzyme reaction system is: Tris-HCl (pH 7.5, 1M / L) 60μL, β-mercaptoethanol (55mM / L) 76μL, EDTA (0.5M / L) 3μL, NADPH (10mM / L) 30μL, substrate daidzein (10mg / mL) 1.2μL, S-adenosylmethionine (SAM) (10mM / L) 14μL, HI4′OMT 30μL, ultrapure water to 300μL. After mixing, react in a water bath overnight, extract with ethyl acetate, combine the extracts and centrifuge to dryness, then add methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain formononetin;

[0031] (4) Formononetin is converted into meditartin under the catalysis of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS)

[0032]

[0033] 1mL enzyme reaction system: K3PO4 (1M / L) 100μL, sucrose (2M / L) 192.5μL, GSH (1M / L) 0.5μL, NADPH (10mM / L) 200μL, NADH (10mM / L) 200μL, MgCl2 (1M / L) 5μL, substrate formononetin (10mg / mL) 1.2μL, crude enzyme solution 250μL, ultrapure water to 300μL. Mix well and react in a water bath overnight. Formononetin can generate the intermediate product vestitone in the presence of isoflavone-2′-hydroxylase (I2′H5) and isoflavone reductase (IFR4), and then the intermediate product vestitone generates medipterocarpan under the catalysis of estrogen reductase (VR) and pterocarpan synthase (PTS). It is extracted three times with ethyl acetate, and the extracts are combined and concentrated to dryness by centrifugation, then re-dissolved in methanol, and filtered through a 0.22μm microporous filter membrane to obtain medipterocarpan.

[0034] As a preferred embodiment, the above-mentioned method for biosynthesis of medipterostilbene comprises the following steps:

[0035] (1) Isoliquiritigenin is isomerized to licorice under the catalysis of chalcone isomerase

[0036]

[0037] The 300μL enzyme reaction system is: 15μL HEPES (1M / L) buffer, 7.7μL substrate isoliquiritigenin (10mg / mL), 30μL CHI (49.81mg / mL), and ultrapure water to 300μL. After mixing, react in a water bath overnight, extract with 3 times the amount of ethyl acetate 3 times, combine the extracts and concentrate to dryness by centrifugation, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain liquiritigenin;

[0038] (2) Liquorice is converted into daidzein under the catalysis of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID)

[0039]

[0040] 1mL enzyme reaction system is: enzyme reaction solution (0.2M K2HPO4, 1.25mM GSH, 50% sucrose, 1mM NADPH) 400μL, substrate liquiritigenin (10mg / mL) 2.5μL, NADH (70mM / L) 15μL, MgCl2 (1M / L) 5μL, 2-HIS crude enzyme solution 430μL and HID crude enzyme solution 50μL, ultrapure water to 300μL. After mixing, react overnight in a 30℃ water bath, extract three times with 300μL ethyl acetate, combine the extracts and centrifuge to dryness, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain daidzein;

[0041] (3) Daidzein is converted into formononetin under the catalysis of isoflavone-4′-O-methyltransferase (HI4′OMT)

[0042]

[0043] 300μL enzyme reaction system: Tris-HCl (pH 7.5, 1M / L) 60μL, β-mercaptoethanol (55mM / L) 76μL, EDTA (0.5M / L) 3μL, NADPH (10mM / L) 30μL, substrate daidzein (10mg / mL) 1.2μL, S-adenosylmethionine (SAM) (10mM / L) 14μL, HI4′OMT 30μL, ultrapure water to 300μL. After mixing, react overnight in a 30℃ water bath, extract three times with 300μL ethyl acetate, combine the extracts and centrifuge to dryness, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain formononetin;

[0044] (4) Formononetin is converted into meditartin under the catalysis of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS)

[0045]

[0046] 1mL enzyme reaction system: K3PO4 (1M / L) 100μL, sucrose (2M / L) 192.5μL, GSH (1M / L) 0.5μL, NADPH (10mM / L) 200μL, NADH (10mM / L) 200μL, MgCl2 (1M / L) 5μL, substrate formononetin (10mg / mL) 1.2μL, crude enzyme solution 250μL, ultrapure water to 300μL. Mix well and react overnight in a 30℃ water bath. Formononetin can generate the intermediate product vestitone in the presence of isoflavone-2′-hydroxylase (I2′H5) and isoflavone reductase (IFR4), and then the intermediate product vestitone generates medipterocarpan under the catalysis of estrogen reductase (VR) and pterocarpan synthase (PTS). The product was extracted three times with 300 μL of ethyl acetate, and the extracts were combined and concentrated to dryness by centrifugation. Then, 200 μL of methanol was added for re-dissolution, and the product was filtered through a 0.22 μm microporous filter membrane to obtain medipterocarpan.

[0047] Beneficial effects:

[0048] The present invention screens out 12 key enzyme genes through a large number of experiments, namely, cinnamic acid-4-hydroxylase (C4H), coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), chalcone isomerase (CHI), 2-hydroxyisoflavone synthase (2-HIS), 2-hydroxyisoflavone dehydratase (HID), isoflavone-4′-O-methyltransferase (HI4′OMT), isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR), and pterostilbene synthase (PTS) as the expression elements of medi-pterostilbene, and constructs a brewer's yeast cell factory to heterologously synthesize medi-pterostilbene. The synthesis method of the present invention is economical, efficient, green and environmentally friendly, and the synthesized medi-pterostilbene has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Medipterocarpin biosynthetic pathway;

[0050] Figure 2 This is the HPLC chart of the conversion of isoliquiritigenin into liquiritigenin in Example 1.

[0051] Figure 3 This is the HPLC chart of the conversion of liquiritigenin into daidzein in Example 2.

[0052] Figure 4 This is the mass spectrum of the conversion of liquiritigenin into daidzein in Example 2.

[0053] Figure 5 This is the HPLC chart of the conversion of daidzein into formononetin in Example 3.

[0054] Figure 6 This is the mass spectrum of the conversion of daidzein into formononetin in Example 3.

[0055] Figure 7 This is the HPLC chart of Example 4 in which formononetin is converted into the intermediate product vestitone and then into meditartin.

[0056] Figure 8 This is the mass spectrum of the intermediate product vestitone in Example 4.

[0057] Fig. 9 This is the mass spectrum of formononetin in Example 4. DETAILED DESCRIPTION

[0058] The present invention is constructed by gene modules and introduced into yeast cells for heterologous expression. It is found that strain DW-01 can produce liquiritigenin, strain DW-05 can produce daidzein, strain DW-07 can produce formononetin, and strain DW-03 can produce meditartin. The present invention is further described below in conjunction with specific implementations, but these implementations should not be construed as limiting the present invention.

[0059] Example 1 Identification of chalcone isomerase (CHI) activity in strain DW-01

[0060] Analysis of the effect of chalcone isomerase (CHI) on the substrate isoliquiritigenin

[0061] Chalcone isomerase (CHI) catalyzes the enzymatic reaction of the substrate isoliquiritigenin as follows:

[0062]

[0063] Isoliquiritigenin can be isomerized into liquiritigenin under the catalysis of chalcone isomerase (CHI), and the enzymatic activity of chalcone isomerase (CHI) can be identified by detecting the product liquiritigenin by ultra-performance liquid chromatography (UPLC).

[0064] The test method is as follows: 300μL enzyme reaction system: buffer HEPES (1M / L) 15μL, substrate isoliquiritigenin (10mg / mL) 7.7μL, CHI (49.81mg / mL) 30μL, ultrapure water to 300μL. Mix well and react overnight in a water bath, extract with 3 times the amount of ethyl acetate, combine the extracts and centrifuge to dryness, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain liquiritigenin, take the filtrate into UPLC, and the liquid phase results are shown in Figure 2 .

[0065] Example 2 Identification of Enzyme Activities of 2-Hydroxyisoflavone Synthase (2-HIS) and 2-Hydroxyisoflavone Dehydratase (HID) in Strain DW-04

[0066] The effects of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID) on the substrate liquiritigenin were analyzed.

[0067] The enzyme reaction of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID) catalyzing the substrate liquiritigenin is as follows:

[0068]

[0069] Liquorice root can generate the intermediate product daidzein under the catalysis of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID). The enzyme activity of 2-hydroxyisoflavone synthase (2-HIS) and 2-hydroxyisoflavone dehydratase (HID) can be identified by detecting the intermediate product daidzein by ultra-performance liquid chromatography.

[0070] The test method is as follows: 1mL enzyme reaction system: enzyme reaction solution (0.2M K2HPO4, 1.25mM GSH, 50% sucrose, 1mM NADPH) 400μL, substrate glycyrrhizin (10mg / mL) 2.5μL, NADH (70mM / L) 15μL, MgCl2 (1M / L) 5μL, 2-HIS crude enzyme solution 430μL and HID crude enzyme solution 50μL, ultrapure water to 300μL. After mixing, react overnight in a 30℃ water bath, extract three times with 300μL ethyl acetate, combine the extracts and centrifuge to dryness, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain daidzein, take the filtrate and enter UPLC. The results are shown in the figure. Figure 3 The molecular weight of the product was determined by LC-MS. Figure 4 .

[0071] Example 3 Identification of the enzyme activity of isoflavone-4′-O-methyltransferase (HI4′OMT) in strain DW-07

[0072] Analysis of the effect of isoflavone-4′-O-methyltransferase (HI4′OMT) on the intermediate product daidzein

[0073] The enzyme reaction catalyzed by isoflavone-4′-O-methyltransferase (HI4′OMT) to form the intermediate daidzein is as follows:

[0074]

[0075] The intermediate product daidzein obtained from strain DW-05 is used as a substrate of isoflavone-4′-O-methyltransferase (HI4′OMT) for the next catalysis to generate the product formononetin. The product formononetin can be detected by ultra-high performance liquid chromatography to identify the enzyme activity of isoflavone-4′-O-methyltransferase (HI4′OMT).

[0076] The test method is as follows: 300μL enzyme reaction system: Tris-HCl (pH7.5, 1M / L) 60μL, β-mercaptoethanol (55mM / L) 76μL, EDTA (0.5M / L) 3μL, NADPH (10mM / L) 30μL, substrate daidzein (10mg / mL) 1.2μL, S-adenosylmethionine (SAM) (10mM / L) 14μL, HI4′OMT 30μL, ultrapure water to 300μL. After mixing, react overnight in a 30℃ water bath, extract three times with 300μL ethyl acetate, combine the extracts and centrifuge to dryness, then add 200μL methanol to dissolve, filter through a 0.22μm microporous filter membrane to obtain formononetin, take the filtrate into UPLC, the results are shown in Figure 5 The molecular weight of the product was determined by LC-MS. Figure 6 .

[0077] Example 4 Identification of the enzyme activities of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS) in strains DW-02, DW-03 and DW-06

[0078] The effects of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS) on the substrate formononetin were analyzed.

[0079] The enzyme reactions of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS) catalyzing the substrate formononetin are as follows:

[0080]

[0081] Formononetin can generate the intermediate product vestitone through isoflavone-2′-hydroxylase (I2′H5) and isoflavone reductase (IFR4), and then generate medipterocarpan under the catalysis of estrogen reductase (VR) and pterocarpan synthase (PTS). The product medipterocarpan can be detected by ultra-performance liquid chromatography to identify the enzyme activities of isoflavone-2′-hydroxylase (I2′H5), isoflavone reductase (IFR4), estrogen reductase (VR) and pterocarpan synthase (PTS).

[0082] The test method is as follows: 1mL enzyme reaction system is: K3PO4 (1M / L) 100μL, sucrose (2M / L) 192.5μL, GSH (1M / L) 0.5μL, NADPH (10mM / L) 200μL, NADH (10mM / L) 200μL, MgCl2 (1M / L) 5μL, substrate formononetin (10mg / mL) 1.2μL, crude enzyme solution 250μL, ultrapure water to 300μL. After mixing, react in a 30℃ water bath overnight. Formononetin can generate the intermediate product vestitone in isoflavone-2′-hydroxylase (I2′H5) and isoflavone reductase (IFR4), and the mass spectrum is shown in the following figure. Figure 8 Then, the intermediate product vestitone is catalyzed by estrogen reductase (VR) and pterocarpan synthase (PTS) to generate meditartin, which is extracted three times with 300 μL ethyl acetate. The combined extracts are centrifuged and concentrated to dryness, and then re-dissolved with 200 μL methanol and filtered through a 0.22 μm microporous filter membrane to obtain meditartin. The filtrate is taken into UPLC as Figure 7 The molecular weight of the product was determined by LC-MS. Fig. 9 .

[0083] Example 5 Application of Saccharomyces cerevisiae strains producing mediptertin in industry

[0084] 1. Application in food, medicine and health care products

[0085] (1) Application in the food industry

[0086] Meditartin, a product obtained by fermentation in a Saccharomyces cerevisiae cell factory, can be used in food production due to its antioxidant properties, stability and biological activity.

[0087] (2) Application in the fields of medicine and health care products

[0088] Medipterocarpus has anti-cancer activity and antioxidant properties, and can be used in the production of medicines and health products.

[0089] 2. Antioxidant Application

[0090] Due to its antioxidant and stability, Mediparotin can be used as an antioxidant in food, health products, cosmetics, daily necessities, chemical and other industries.

[0091] 3. Biological agents

[0092] Medipterocarpin, heterologously synthesized by Saccharomyces cerevisiae, can be used as a biological agent in biochemical component standards, food additives, health care capsules, powders, tablets or liquid product raw materials, etc.

[0093] The above description is only a preferred embodiment of the present invention and should not be regarded as limiting the scope of the present invention. For ordinary technicians in this technical field, various possible equivalent changes or modifications can be made based on the technical solution of the present invention and the description of its preferred embodiments without departing from the principle of the present invention, and these changes and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A biosynthetic method of medipterocarpin, characterized in that: The following steps are involved: (1) Isoliquiritigenin is isomerized to licorice under the catalysis of chalcone isomerase The enzyme reaction system is: 15 μL of 1M / L buffer HEPES, 7.7 μL of 10 mg / mL substrate isoliquiritigenin, 30 μL of 49.81 mg / mL chalcone isomerase, and ultrapure water is added to 300 μL, and mixed evenly; after reacting overnight in a water bath, it is extracted with ethyl acetate, the extracts are combined and concentrated to dryness by centrifugation, and then methanol is added to re-dissolve, and filtered through a 0.22 μm microporous filter membrane to obtain liquiritigenin; (2) Liquorice root is converted into daidzein under the catalysis of 2-hydroxyisoflavone synthase and 2-hydroxyisoflavone dehydratase The enzyme reaction system is as follows: 400 μL of enzyme reaction solution composed of 0.2M K2HPO4, 1.25mM GSH, 50% sucrose, 1mM reduced coenzyme II, 2.5 μL of 10mg / mL substrate licorice, 15 μL of 70mM / L reduced coenzyme I, 5 μL of 1M / L MgCl2, 430 μL of 2-hydroxyisoflavone synthase and 50 μL of 2-hydroxyisoflavone dehydratase, and ultrapure water is added to 300 μL, and mixed; after reacting overnight in a water bath, it is extracted with ethyl acetate, the extracts are combined and concentrated to dryness by centrifugation, and then re-dissolved with methanol, and filtered through a 0.22 μm microporous filter membrane to obtain daidzein; (3) Daidzein is converted into formononetin under the catalysis of isoflavone-4′-O-methyltransferase The enzyme reaction system is as follows: pH 7.5, 60 μL of 1M / L Tris-HCl, 76 μL of 55mM / L β-mercaptoethanol, 3 μL of 0.5M / L EDTA, 30 μL of 10mM / L reduced coenzyme II, 1.2 μL of 10mg / mL substrate daidzein, 14 μL of 10mM / L S-adenosylmethionine, 30 μL of isoflavone-4′-O-methyltransferase, and ultrapure water is added to 300 μL, and mixed evenly; after reacting overnight in a water bath, the mixture is extracted with ethyl acetate, the extracts are combined and concentrated to dryness by centrifugation, and then re-dissolved with methanol, and filtered through a 0.22 μm microporous filter membrane to obtain formononetin; (4) Formononetin is converted into meditartin under the catalysis of isoflavone-2′-hydroxylase, isoflavone reductase, estrogen reductase and pterocarpansynthase The enzyme reaction system is: 100 μL of 1M / L K3PO4, 192.5 μL of 2M / L sucrose, 0.5 μL of 1M / L GSH, 200 μL of 10mM / L reduced coenzyme II, 200 μL of 10mM / L reduced coenzyme I, 5 μL of 1M / L MgCl2, 1.2 μL of 10mg / mL substrate formononetin, 250 μL of crude enzyme solution, and ultrapure water to 300 μL; mix well and react overnight in a water bath; formononetin can generate the intermediate product vestitone in isoflavone-2′-hydroxylase and isoflavone reductase, and then the intermediate product vestitone is reacted with estrogen reductase and pterocarpan synthase to generate medipterocarpin, which was extracted three times with ethyl acetate, and the extracts were combined and concentrated to dryness by centrifugation, and then re-dissolved in methanol and filtered through a 0.22 μm microporous filter membrane to obtain medipterocarpin.

2. The biosynthesis method of medipterocarpin according to claim 1, characterized in that: The following steps are involved: (1) Isoliquiritigenin is isomerized to licorice under the catalysis of chalcone isomerase The 300 μL enzyme reaction system is: 15 μL 1M / L buffer HEPES, 7.7 μL 10 mg / mL substrate isoliquiritigenin, 30 μL 49.81 mg / mL chalcone isomerase, and ultrapure water is added to 300 μL. After mixing, react in a 30°C water bath overnight, extract with 3 times the amount of ethyl acetate for 3 times, combine the extracts and concentrate to dryness by centrifugation, then add 200 μL methanol to dissolve, and filter through a 0.22 μm microporous filter membrane to obtain liquiritigenin. (2) Liquorice root is converted into daidzein under the catalysis of 2-hydroxyisoflavone synthase and 2-hydroxyisoflavone dehydratase The enzyme reaction system is as follows: 400 μL of enzyme reaction solution composed of 0.2M K2HPO4, 1.25mM GSH, 50% sucrose, and 1mM reduced coenzyme II, 2.5 μL of 10mg / mL substrate licorice, 15 μL of 70mM / L reduced coenzyme I, 5 μL of 1M / L MgCl2, 430 μL of 2-hydroxyisoflavone synthase and 50 μL of 2-hydroxyisoflavone dehydratase, and ultrapure water is added to 300 μL, and mixed; after mixing, react in a 30°C water bath overnight, extract three times with 300 μL of ethyl acetate, combine the extracts, centrifuge and concentrate to dryness, then add 200 μL of methanol to dissolve, and filter through a 0.22 μm microporous filter membrane to obtain daidzein; (3) Daidzein is converted into formononetin under the catalysis of isoflavone-4′-O-methyltransferase The enzyme reaction system is as follows: pH 7.5, 60 μL of 1M / L Tris-HCl, 76 μL of 55mM / L β-mercaptoethanol, 3 μL of 0.5M / L EDTA, 30 μL of 10mM / L reduced coenzyme II, 1.2 μL of 10mg / mL substrate daidzein, 14 μL of 10mM / L S-adenosylmethionine, 30 μL of isoflavone-4′-O-methyltransferase, and ultrapure water to 300 μL, and mixed; after mixing, react in a 30°C water bath overnight, extract three times with 300 μL of ethyl acetate, combine the extracts, centrifuge and concentrate to dryness, then add 200 μL of methanol to dissolve, and filter through a 0.22 μm microporous filter membrane to obtain formononetin; (4) Formononetin is converted into meditartin under the catalysis of isoflavone-2′-hydroxylase, isoflavone reductase, estrogen reductase and pterocarpansynthase The enzyme reaction system is: 100μL of 1M / L K3PO4, 192.5μL of 2M / L sucrose, 0.5μL of 1M / L GSH, 200μL of 10mM / L reduced coenzyme II, 200μL of 10mM / L reduced coenzyme I, 5μL of 1M / L MgCl2, 1.2μL of 10mg / mL substrate formononetin, 250μL of crude enzyme solution, and ultrapure water to 300μL; after mixing, react in a 30℃ water bath overnight; formononetin can generate the intermediate product vestitone in isoflavone-2′-hydroxylase and isoflavone reductase, and then the intermediate product vestitone is reacted with estrogen reductase and pterocarpan Medipterocarpin was generated under the catalysis of synthase, extracted three times with 300 μL ethyl acetate, the extracts were combined and concentrated to dryness by centrifugation, then re-dissolved with 200 μL methanol, and filtered through a 0.22 μm microporous filter membrane to obtain Medipterocarpin.

Images