Chemical enzyme synthesis method of papaverine and derivative thereof

Abstract

The invention discloses a chemical enzyme synthesis method of papaverine and derivatives thereof, and belongs to the technical field of bioengineering. The (S)-tetrahydropapaverine and the derivative thereof are efficiently synthesized by utilizing artificially designed multi-enzyme cascade reaction, and the papaverine and the derivative thereof are further synthesized by a chemical method. According to the method disclosed by the invention, a catalytic reaction can be carried out through a biological enzyme or microbial cells for expressing the enzyme, so that the biosynthesis yield of the (S)-tetrahydropapaverine reaches 3.33 g / L, and the separation yield of the papaverine reaches 86%.

Description

Technical Field

[0001] This invention relates to a chemical enzyme synthesis method for papaverine and its derivatives, belonging to the field of bioengineering technology. Background Technology

[0002] Papaverine, also known as papaverine, is a benzylisoquinoline alkaloid extracted from the poppy plant. Papaverine hydrochloride is a widely used vasodilator in clinical practice. It induces nonspecific dilation of the pulmonary artery, sinus artery, and large blood vessels, relaxes smooth muscle, and has no side effects on the central nervous system. Papaverine hydrochloride injection is a common clinical drug used to relieve vasospasm and is a life-saving medication for patients undergoing finger replantation surgery. In 2018, Nicholas et al. found that papaverine can inhibit the activity of complex I in the oxidative respiratory chain of the mitochondrial inner membrane, reduce the OCR of cancer cells, alleviate tumor cell hypoxia, and improve the efficacy of radiotherapy. Furthermore, papaverine hydrochloride can also be used as an adjunct therapy for glaucoma, coronary heart disease, ischemic bowel disease, and liver cancer.

[0003] The content of papaverine in poppies is low, and as an annual, biennial, or perennial herbaceous plant, the low content and long growth cycle cannot meet the demand. Furthermore, poppy plants contain a large number of addictive substances, including morphine, cocaine, and noscapine, which further limits the clinical use of plant-derived papaverine. Chemical synthesis of papaverine requires the use of organic reagents such as acyl chlorides and phosphorus oxychloride, which is environmentally unfriendly. In terms of microbial synthesis, semi-synthesis of papaverine has been achieved using Saccharomyces cerevisiae, but the yield of its precursor, tetrahydropapaverine, is only 121 μg / L, and the synthesis from tetrahydropapaverine to papaverine is achieved using hydrogen peroxide oxidation. Therefore, there is a need to develop green and environmentally friendly methods for synthesizing papaverine. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a novel route for the synthesis of papaverine and its derivatives using readily available phenylacetic acid derivatives and dopamine as raw materials via a chemical enzymatic method. Figure 1 ).

[0005] The present invention provides a recombinant cell BM1, which expresses the following proteins based on the starting strain: carboxylic acid reductase, phosphoproteopantothenate thioethylamine transferase and norcodonine synthase; the starting strain is Escherichia coli or Bacillus subtilis.

[0006] In one embodiment, the Escherichia coli is Escherichia coli BL21(DE3).

[0007] In one embodiment, the amino acid sequence of the carboxylic acid reductase (TpCAR) is shown in SEQ ID NO.1; the amino acid sequence of the phosptophanyl thioethylamine transferase (BsSfp) is shown in SEQ ID NO.2; and the amino acid sequence of the norcodonol alkaloid synthase (TfNCS) is shown in SEQ ID NO.3.

[0008] In one embodiment, the vector used for the overexpression includes, but is not limited to, pET series plasmids, or pET28a plasmid, preferably pET28a.

[0009] The present invention also provides the application of the recombinant cell BM1 in the synthesis of papaverine or its derivatives.

[0010] In one implementation, the application includes the following steps:

[0011] (1) Inoculate recombinant cells BM1 into the culture medium, culture the strain to the logarithmic growth phase, induce, and collect the cells;

[0012] (2) Mix the recombinant cells BM1 obtained in step (1) with the substrate and react them;

[0013] The substrates include phenylacetic acid derivatives and dopamine.

[0014] In one embodiment, step (1) includes: inoculating recombinant cells into LB medium and inducing protein expression using IPTG, and collecting the cells after induction.

[0015] In one embodiment, the reaction temperature is 20–40°C, and the pH of the reaction is 4.0–11.

[0016] In one embodiment, the amount of phenylacetic acid derivative and dopamine added is 5-14 mM.

[0017] In one embodiment, the amount of recombinant cell BM1 added is 10-100 g / L.

[0018] The present invention also provides recombinant cell BM2 expressing oxygen methyltransferase (PsOMT2), methionine adenosine transferase (MAT) and homocysteine ​​hydrolase (SAHH).

[0019] In one embodiment, the recombinant cells express the amino acid sequence PsOMT2 as shown in SEQ ID NO.4 using pBR332 plasmid and pET series plasmids as vectors; the pET series plasmids include, but are not limited to, pET28a and pET21b, preferably pET28a.

[0020] In one embodiment, the amino acid sequence of the methionine adenosine transferase is shown in SEQ ID NO.7; and the amino acid sequence encoding the homocysteine ​​hydrolase is shown in SEQ ID NO.6.

[0021] The present invention also provides the application of the recombinant cells BM1 and BM2 in the synthesis of papaverine or its derivatives; the application includes the following steps:

[0022] (1) Cell culture: Recombinant cells BM1 and BM2 were inoculated into the culture medium, cultured, and protein expression was induced by IPTG. The cells were then collected.

[0023] (2) The recombinant cells BM1 obtained in step (1) are mixed with the substrate and reacted, and the reaction supernatant is collected; the substrate includes phenylacetic acid derivatives and dopamine;

[0024] (3) Mix the recombinant cells BM2 obtained in step (1) with the supernatant obtained in step (2) and react them.

[0025] In one embodiment, step (1) includes: inoculating recombinant cells BM1 or BM2 into LB medium, culturing the strain to the logarithmic growth phase, inducing, and collecting the cells.

[0026] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 4.0-11.

[0027] In one embodiment, the amount of recombinant cell BM1 added is 10-100 g / L; the amount of recombinant cell BM2 added is 10-100 g / L, preferably 20-50 g / L.

[0028] The present invention also provides recombinant cell BM3 expressing methyltransferase (Ps7OMT), methionine adenosine transferase (MAT) and homocysteine ​​hydrolase (SAHH).

[0029] In one embodiment, the recombinant cell BM3 expresses the methyltransferase gene Ps7OMT, the methionine adenosine transferase gene MAT, and the homocysteine ​​hydrolase gene SAHH using pET series plasmids or pBR332 plasmid as vectors; the pET series plasmids include, but are not limited to, pET28a and pET21b, preferably pET28a.

[0030] In one embodiment, the cells are Escherichia coli cells.

[0031] The present invention also provides the application of the recombinant cells BM1, BM2 and BM3 in the synthesis of papaverine or its derivatives.

[0032] In one implementation, the application includes the following steps:

[0033] (1) Cell culture: Recombinant cells BM1, BM2 and BM3 were inoculated into the culture medium, cultured to the logarithmic growth phase, induced, and the cells were collected;

[0034] (2) The recombinant cells BM1 obtained in step (1) are mixed with the substrate and reacted, and the reaction supernatant is collected; the substrate includes phenylacetic acid derivatives and dopamine;

[0035] (3) Mix the recombinant cells BM2 obtained in step (1) with the supernatant obtained in step (2), react them, and collect the reaction supernatant;

[0036] (4) The recombinant cells BM3 obtained in step (1) are mixed with the supernatant obtained in step (3) and reacted to obtain (S)-tetrahydropoppyrine or its derivatives.

[0037] In one embodiment, step (1) includes: seeding recombinant cells BM1, BM2, or BM3 into LB medium and culturing them at 37 degrees Celsius until OD. 600 After the pH reaches approximately 0.6, add 0.1 mM IPTG for induction, and collect the bacteria after induction.

[0038] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 4.0-11.

[0039] In one embodiment, the amount of phenylacetic acid derivative and dopamine added is 1-14 mM.

[0040] In one embodiment, the amount of recombinant cell BM1 added is 10-100 g / L; the amount of recombinant cell BM2 added is 30-60 g / L, preferably 30-40 g / L.

[0041] The present invention also provides a method for synthesizing (S)-tetrahydropaspaine or its derivatives via a one-pot, three-step enzyme cascade reaction, the method comprising the following steps:

[0042] (1) First step reaction: The reaction system includes: carboxyl reductase, norepinephrine synthase, glucose dehydrogenase, dopamine and phenylacetic acid or its derivatives, the reaction temperature is 20-40℃, and the pH of the reaction system is 4-11;

[0043] (2) Heat to deactivate the enzyme in step (1) and collect the supernatant;

[0044] (3) Add oxygen methyltransferase (PsOMT2), methyltransferase (PsN7OMT) and S-adenosylmethionine to the supernatant obtained in step (2) and react at a temperature of 20-40℃ to obtain (S)-tetrahydropopapaverine or its derivatives.

[0045] In one embodiment, the concentrations of the substances in the system of step (1) are as follows: 5 μM carboxyl reductase, 1-15 μM norcodonine synthase, 5 μM glucose dehydrogenase, 2-10 mM substrate dopamine, and 2-10 mM phenylacetic acid derivative.

[0046] In one embodiment, step (3) includes the addition of 5 μM oxymethyltransferase (PsOMT2), 5 μM oxymethyltransferase (PsN7OMT) and 5 mM S-adenosylmethionine.

[0047] The present invention also provides a method for synthesizing (S)-tetrahydropaspaine or its derivatives using the above-mentioned recombinant strain, the method comprising:

[0048] (1) Cell culture: Recombinant cells BM1, BM2 and BM3 were inoculated into the culture medium, cultured to the logarithmic growth phase, induced, and the cells were collected;

[0049] (2) The recombinant cells BM1 obtained in step (1) are mixed with the substrate and reacted, and the reaction supernatant is collected; the substrate includes phenylacetic acid derivatives and dopamine;

[0050] (3) Mix the recombinant cells BM2 obtained in step (1) with the supernatant obtained in step (2), react them, and collect the reaction supernatant;

[0051] (4) The recombinant cells BM3 obtained in step (1) are mixed with the supernatant obtained in step (3) and reacted to obtain (S)-tetrahydropoppyrine or its derivatives.

[0052] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 4.0-11.

[0053] In one embodiment, the amount of phenylacetic acid derivative and dopamine added is 1-14 mM.

[0054] In one embodiment, the amount of recombinant cell BM1 added is 10-100 g / L; the amount of recombinant cell BM2 added is 30-60 g / L, preferably 30-40 g / L; and the amount of recombinant cell BM3 added is 10-100 g / L.

[0055] This invention also provides a method for converting (S)-tetrahydropaspaine or its derivatives to papaverine and its derivatives using Pd / C as a catalyst and acetonitrile as a solvent, the method comprising the following steps:

[0056] (1) The (S)-tetrahydropapaverine or its derivatives obtained by biotransformation were extracted and concentrated, and then purified by silica gel column chromatography.

[0057] (2) Place (S)-tetrahydropapaverine or its derivatives and a catalyst in a solvent and heat in an oil bath;

[0058] (3) After the reaction is completed, the reaction solution is filtered while hot and concentrated under vacuum to obtain the crude product;

[0059] (4) The crude product was subjected to silica gel column chromatography to obtain papaverine and its derivatives.

[0060] In one embodiment, the solvent used in step (2) of the scheme includes xylene, methanol, acetonitrile and benzene, preferably xylene and acetonitrile.

[0061] In one embodiment, the catalyst used in step (2) of the scheme includes hydrogen peroxide, Raney nickel, palladium on carbon, preferably palladium on carbon.

[0062] In one embodiment, the structural formula of the phenylacetic acid or its derivative includes at least one of 1a-1h:

[0063]

[0064] In one embodiment, the structural formula of the papaverine or its derivative is shown in any of the following 6a-6h:

[0065]

[0066] The present invention also provides the use of the recombinant cells, or the method, in the preparation of (S)-tetrahydropaspaine or its derivatives.

[0067] Beneficial effects:

[0068] (1) This invention constructed a recombinant strain BM1, which can efficiently convert substrates dopamine and phenylacetic acid derivatives into the benzyl isoquinoline skeleton ((S)-3a-(S)-3h), see [link to relevant documentation]. Figure 1 );

[0069] (2) This invention constructed a recombinant strain BM2, which is capable of methylation at the 6-position hydroxyl group of substrates (S)-3a to (S)-3h. Figure 1 );

[0070] (3) This invention constructed a recombinant strain BM3, which is capable of methylation at the 7-position hydroxyl group of the substrate (S)-4a-(S)-4h. Figure 1 );

[0071] (4) The present invention also provides a method for synthesizing (S)-tetrahydropaspaine or its derivatives using phenylacetic acid derivatives and dopamine as raw materials. Using the aforementioned recombinant strain or its expressed enzyme as a catalyst, the yields of (S)-5a-(S)-5h are 3.03 g / L, 2.95 g / L, 3.51 g / L, 2.78 g / L, 2.56 g / L, 2.46 g / L, 2.45 g / L and 2.75 g / L, respectively, with ee values ​​of 98%, >99%, 98%, 98%, 98%, 98%, >99% and >99%, respectively.

[0072] (5) The present invention also provides a method for oxidizing tetrahydropapain obtained by application to papaverine, which can achieve a separation yield of (S)-6a of up to 86%. Attached Figure Description

[0073] Figure 1 This describes the synthetic routes for papaverine and its derivatives.

[0074] Figure 2 HPLC detection of (S)-3a-(S)-5a synthesized in vitro.

[0075] Figure 3 MS identification of (S)-3a synthesized in vitro.

[0076] Figure 4 MS identification of in vitro synthesized (S)-4a.

[0077] Figure 5 The standard curve for (S)-tetrahydropapaverine.

[0078] Figure 6 The 1H spectrum of (S)-tetrahydropaspaline in deuterated chloroform.

[0079] Figure 7 MS identification of (S)-tetrahydropapaverine.

[0080] Figure 8 The ee value of (S)-tetrahydropaspaline was determined.

[0081] Figure 9 The HCl spectrum, C-chromatogram, and MS spectra of papaverine in deuterated chloroform were used for identification. Detailed Implementation

[0082] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0083] (I) Reagents and Materials

[0084] Sources of reagents and materials: The antibiotics such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate used in this invention are all from Shanghai Sangon Biotech; the PCR enzymes and homologous recombinant enzymes involved in the molecular experiments in this invention are all purchased from Takara Bio Engineering (Dalian) Co., Ltd.; the chemical reagents used in this invention are all purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Titan Technology Co., Ltd.

[0085] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0086] The structural formulas of phenylacetic acid or its derivatives involved in the specific embodiments are as follows:

[0087]

[0088] The structural formulas of papaverine or its derivatives are shown in 6a-6h below:

[0089]

[0090] (II) Strains and Sequences

[0091] The primers involved in the specific implementation are shown in Table 1.

[0092] Table 1 Primer sequences for constructing recombinant strains

[0093]

[0094] The strains and their characteristics constructed in the specific implementation are shown in Table 2.

[0095] Table 2. Strains constructed in this invention

[0096]

[0097]

[0098] (III) Testing Methods

[0099] High-performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature 30℃, detection wavelength 280nm. The detection method employed a gradient separation using a two-phase solvent system of acetonitrile (containing 0.1% (v / v) trifluoroacetic acid) and double-distilled water (containing 0.1% (v / v) trifluoroacetic acid), with a mobile phase flow rate of 1 mL / min. The linear gradient was as follows: isocratic elution with 5% acetonitrile for 1 min, followed by gradient elution with 5%–60% acetonitrile for 20 min.

[0100] Protein purification method: The collected bacterial cells were resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH=8), and then the resuspended solution was homogenized using a high-pressure homogenizer. The homogenized resuspended solution was centrifuged at low temperature and high speed (4°C, 10,000 rpm for 30 min) to obtain crude enzyme solution. The solution was then desalted by nickel affinity chromatography and a Histrap™ 5 mL desalting column to obtain the purified protein.

[0101] Example 1: Construction and verification of an artificial cascade reaction pathway for (S)-tetrahydropapaverine

[0102] Establish as Figure 1 The reaction pathway shown first uses 3,4-dimethoxyphenylacetic acid (1a) as a model substrate, which is reduced to the corresponding aldehyde (1a) by carboxyl reductase (TpCAR) activated by phosphoproteopantolate thioethylamine transferase (BsSfp) (PsSfp is responsible for activating carboxyl reductase, which catalyzes the reduction of carboxylic acid). Subsequently, the substrate dopamine is condensed with the generated aldehyde by norcodonol alkaloid synthase (TfNCS) to generate the skeleton (S)-3a of benzylisoquinoline. In the second step, intermediate (S)-3a is converted to (S)-4a by 6-oxomethyltransferase (PsOMT2) and cofactor SAM. In the third step, intermediate (S)-4a is converted to the final product (S)-5a by 7-oxomethyltransferase (PsN7OMT) and cofactor SAM. Methionine adenosine transferase (EcMAT) catalyzes the reaction of L-methionine with ATP to synthesize the cofactor S-adenosine methionine (SAM). The demethylated product of SAM, SAH, is then hydrolyzed by homocysteine ​​hydrolase (MmSAHH). The specific steps are as follows:

[0103] (1): Add 5 μM carboxylic acid reductase, 5 μM norcodonine synthase, 5 μM glucose dehydrogenase, 2 mM substrate dopamine and 2 mM phenylacetic acid derivative to a 1.5 ml EP tube. The reaction system is carried out in 0.1 M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer at pH 7.5. The final reaction volume is 200 μL.

[0104] (2) Place the reaction tube from step (1) in a shaking reactor at 30°C and 800 rpm for 4 hours. After the reaction is complete, heat at 95°C for 5 minutes to inactivate the enzyme in step (1), and then centrifuge at high speed to obtain the supernatant.

[0105] (3) Second step reaction: Add 5 μM 6-oxymethyltransferase, 5 μM 7-oxymethyltransferase and 5 mM S-adenosylmethionine (pH 7.5) to the supernatant of step (2) above. The reaction system was placed in a shaking reactor at 30°C and 800 rpm for 4 h.

[0106] (4) After the reaction is complete, add 4 times the volume of acetonitrile to the above reaction solution to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5min, take 200μL of sample and load it onto the chromatographic column, and detect the product by liquid chromatography.

[0107] Example 2: Construction, protein expression, and product identification of engineered strain TpCAR-BsSfp-TfNCS-BL21 (BM1)

[0108] The target gene CAR derived from *Tsukamurella paurometabola* was codon-optimized to obtain the gene sequence shown in SEQ ID NO.8. This gene was synthesized and ligated into the multiple cloning site (MCS) of the pET28a vector to obtain the plasmid pET28a-TpCAR.

[0109] The Sfp gene fragment (nucleotide sequence shown in SEQ ID NO.9) was amplified from the genome of Bacillus subtilis and ligated into the pET28a vector to obtain the plasmid pET28a-BsSfp.

[0110] The target gene NCS from Thalictrum flavum was codon optimized to obtain the gene sequence shown in SEQ ID NO. 10. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET28a vector to obtain the plasmid pET28a-TfNCS.

[0111] After obtaining the three plasmids mentioned above, the gene fragment Sfp and fragment NCS were integrated into pET28a-TpCAR using homologous recombination technology to obtain the recombinant plasmid pET28a-T7-TpCAR-BsSfp-T7-TfNCS. The recombinant plasmid was then transformed into the engineered strain BL21 to obtain the recombinant strain TpCAR-BsSfp-TfNCS-BL21. The primers used for plasmid construction are shown in Table 1.

[0112] Single-clone strain TpCAR-BsSfp-TfNCS-BL21 was selected and transferred to 5 ml of LB medium, and cultured at 250 rpm and 37°C for 10–12 h. Subsequently, it was transferred to 500 mL of LB medium and cultured at 120 rpm and 37°C. Then, 0.1 mM IPTG was added, and the culture was induced at 25°C for 20 h. The bacterial cells were collected after centrifugation. The cells were washed three times with a buffer containing 50 mM HPEPS (pH 7.5) and 200 mM KCl, and then centrifuged to obtain the bacterial cells.

[0113] The collected bacterial cells were used to verify the synthesis of product (S)-3a in a 5 mL reaction system containing: 50 mM HPEPS buffer (pH 7.5), 30 g / L bacterial cells, 20 mM glucose, 5% DMSO, 15 mM substrate 1a, and 15 mM substrate dopamine. The reaction was carried out in a shaker at 30 °C. After 10 h, 200 μL of the sample was taken and the reaction was quenched with 800 μL of acetonitrile.

[0114] The synthesis of product (S)-3a was detected using an HPLC system. For example... Figure 2 As shown, the compound obtained by whole-cell catalysis had the same elution time as the standard, and the formation of product (S)-3a was further determined by high-resolution mass spectrometry. Figure 3 ).

[0115] Example 3: Construction, protein expression, and product identification of engineered strain EcMAT-PsOMT2-MmSAHH-BL21(BM2)

[0116] The target gene PsOMT2, derived from poppy *Papaver somniferum*, was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 11. This gene was synthesized and ligated into the NdeI and XhoI sites of the pET28a vector to obtain the plasmid pET28a-PsOMT2.

[0117] The MAT gene fragment (SEQ ID NO.14) was amplified from the Escherichia coli genome and ligated into the pET28a vector to obtain the plasmid pET28a-EcMAT.

[0118] The target gene SAHH from *Mus musculus* was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 13. This gene was synthesized and ligated into the NdeI and XhoI sites of the pET28a vector to obtain the plasmid pET28a-MmSAHH. After obtaining the above three plasmids, the gene fragments EcMAT, PsOMT2, and MmSAHH were integrated into the MCS of the pET28a plasmid using homologous recombination technology to obtain the plasmid pET28a-T7-EcMAT-PsOMT2-T7-MmSAHH. The recombinant plasmid was transformed into the engineered strain BL21 to obtain the recombinant strain EcMAT-PsOMT2-MmSAHH-BL21. The primers used for plasmid construction are shown in Table 1.

[0119] The recombinant strain EcMAT-PsOMT2-MmSAHH-BL21 was cultured using the same method as in Example 2, and bacterial cells were obtained by centrifugation. Subsequently, the recombinant strain EcMAT-PsOMT2-MmSAHH-BL21 was resuspended in the supernatant prepared in Example 2 and reacted at 30°C and 250 rpm for 6 h. The product (S)-4a was detected using the same sample post-processing method and liquid chromatography detection conditions as in Example 2. The results are as follows: Figure 2 and Figure 4 As shown.

[0120] Example 4: Construction, protein expression, and product identification of engineered strain PsN7OMT-MmSAHH-EcMAT-BL21(BM3)

[0121] The target gene PsN7OMT from poppy (P. somniferum) was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 12. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET28a vector to obtain the plasmid pET28a-PsN7OMT.

[0122] Gene fragments EcMAT, PsN7OMT, and MmSAHH were integrated into two MCS of the pET28a plasmid using homologous recombination technology to obtain the plasmid pET28a-T7-PSN7OMT-MmSAHH-T7-EcMAT. The recombinant plasmid was transformed into the engineered strain BL21 to obtain the recombinant strain PsN7OMT-MmSAHH-EcMAT-BL21. The primers used for plasmid construction are shown in Table 1.

[0123] The recombinant strain PsN7OMT-MmSAHH-EcMAT-BL21 was cultured using the same method as in Example 2, and bacterial cells were obtained by centrifugation. Subsequently, the pH of the supernatant prepared in Example 3 was adjusted to 7.5, and the recombinant strain PsN7OMT-MmSAHH-EcMAT-BL21 was resuspended to a final bacterial concentration of 30 g / L. The reaction was carried out at 30°C and 250 rpm for 6 h. The product (S)-5a was detected using the same sample post-processing method and liquid chromatography detection conditions as in Example 2, and the formation of the product was further confirmed by high-resolution mass spectrometry. All strains constructed in this invention are shown in Table 2.

[0124] Example 5: Large-scale preparation of (S)-tetrahydropaspaline in a 300 mL reaction system

[0125] The bacterial strain culture and induction methods were the same as in Examples 2-4. (S)-tetrahydropaspaine was prepared on a large scale in a 300 mL reaction system using a one-pot, three-step reaction method. The specific steps are as follows:

[0126] (a) Recombinant strains BM1, BM2 and BM3 were cultured using the same method as in Examples 2 to 4, and the cells were obtained by centrifugation.

[0127] (b) Prepare a 300 ml reaction mixture containing (by final concentration): 50 mM HPEPS buffer (pH 7.5), 30 g / L BM1 cells, 20 mM glucose, 15 mM 3,4-dimethoxyphenylacetic acid, and dopamine. Incubate the reaction mixture at 30 °C in a shaker for 8 h, then centrifuge and collect the supernatant.

[0128] (c) Resuspend the bacterial cells BM2 in the supernatant obtained in step (b) to a final concentration of 30 g / L, and react at 30 °C and 250 rpm for 8 h. After the reaction is complete, collect the supernatant by centrifugation and adjust the pH of the supernatant to 9.0 with 5 M NaOH for the next step of the reaction.

[0129] (d) Resuspend the bacterial cells BM3 in the supernatant obtained in step (c) to a final concentration of 30 g / L. Add 5 mM methionine to the resuspended solution and react at 30 °C and 250 rpm for 7 h. Perform HPLC detection and quantify (S)-5a using the product standard curve. Figure 5 ), and verified the correctness of (S)-5a through NMR and mass spectrometry. Figure 6 and Figure 7 The ee value of (S)-5a was determined to be 98% by normal phase chromatography. Figure 8 ).

[0130] Example 6: Substrate Spectrum Expansion

[0131] The bacterial strain culture and induction methods are the same as in Examples 2-4. A one-pot, three-step reaction method is used, with phenylacetic acid and its derivatives (1a-1c) containing different substituents and dopamine as substrates, added to the whole-cell catalytic system to expand the substrate spectrum. The specific steps are as follows:

[0132] (a) Recombinant strains BM1, BM2 and BM3 were cultured using the same method as in Examples 2 to 4, and the cells were obtained by centrifugation.

[0133] (b) Prepare a 300 mL reaction system containing (by final concentration): 50 mM HPEPS buffer (pH 7.5), 30 g / L BM1 bacterial cells, 5% DMSO, 15 mM 3,4-dimethoxyphenylacetic acid, and dopamine. The reaction was carried out at 30 °C in a shaker for 8 h. After the reaction, the mixture was centrifuged, and the supernatant was collected.

[0134] (c) Resuspend the bacterial cells BM2 in the supernatant obtained in step (b) to a final concentration of 30 g / L, and add 5 mM L-Met to the resuspended solution. React at 30 °C and 250 rpm for 8 h. Centrifuge the reaction solution at 7000 rpm for 10 min to collect the supernatant, and adjust the pH of the supernatant to 8.5 with 5 M NaOH for use in the next step of the reaction.

[0135] (d) The bacterial cells BM3 were resuspended in the supernatant obtained in step (c) to a final concentration of 30 g / L, and 5 mM methionine was added. The mixture was reacted at 30 °C and 250 rpm for 6 h. The reaction was performed by HPLC, and the product standard curve was used to quantify (S)-5a-(S)-5h.

[0136] The results showed that all substrates (1a-1h) could be converted into the corresponding product (S)-5a-(S)-5h, and the specific yields are shown in Table 3.

[0137] Table 3. Conversion rates of phenylacetic acid derivatives with different substituents and corresponding ee values ​​of (S)-tetrahydropaspaline and its derivatives.

[0138]

[0139] Example 7: Chemical catalytic oxidation of (S)-tetrahydropapain to papaverine

[0140] To achieve the conversion of (S)-tetrahydropapain to papaverine, the synthesis of papaverine was first catalyzed in 5 mM Tris-HCl buffer at 70 °C using hydrogen peroxide as a catalyst. The reaction system contained: 5 mM Tris-HCl buffer, 0.1 mM (S)-tetrahydropapain, 5% H₂O₂ (by volume), pH 9.5, and reacted at 70 °C for 5 h. The results showed that the yield was only 5%.

[0141] Using commercially available Pd / C as a catalyst, the results of adjusting different solvents, reaction temperatures, and catalyst dosages are shown in Table 4. The reaction system contained 0.1 mM (S)-tetrahydropaspaline, Pd / C, and acetonitrile or xylene as the solvent. The results showed that the highest yield, reaching 86%, was achieved when the solvent was acetonitrile, the reaction temperature was 80 °C, the catalyst was Pd / C, and the catalyst dosage was 10% [Pd] by molar percentage.

[0142] Table 4. Screening of catalysts and reaction conditions for the oxidation of (S)-tetrahydropaspaine to papaverine.

[0143]

[0144]

[0145] The specific reaction process is as follows:

[0146] 0.1 mM (S)-tetrahydropapaverine, 10% [Pd], and 3 mL of acetonitrile were added to a 50 mL two-necked flask. The mixture was refluxed at 80 °C. After reacting for 24 h, Pd / C was removed by filtration, and the crude product was obtained by concentration under reduced pressure. The crude product was then purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluents. Analysis of the product showed that the yield of (S)-6a (Papaverine) was 86%. Figure 9 The structural identification results are as follows:

[0147] 1 H NMR(400MHz,Chloroform-d)δ6.83(d,J=8.1Hz,1H),6.80(dd,J=8.0,1.5Hz,1H),6.76(d,J=1.3Hz,1H),6.67(s,1H),6.60(s,1H),4.12–4.0 9(m,1H),3.87(s,3H),3.86(s,6H),3.84(s,3H),3.19(ddd,J=14.2,8.9,5.1Hz,2H),2.94–2.83(m,2H),2.80–2.65(m,2H),1.77(s,1H)ppm;

[0148] 13 C NMR(151MHz,Chloroform-d)δ148.9,147.6,147.4,146.9,131.4,130.5,127.5,121 .4,112.3,111.8,111.2,109.3,56.9,56.0,55.9,55.8,55.8,42.2,41.0,29.6ppm.

[0149] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Recombinant Escherichia coli, characterized in that, For any one of (a) to (c): (a) Recombinant cells BM1: using Escherichia coli BL21 as the host, expressing carboxylic acid reductase TpCAR, phosptophanate thioethylamine transferase BsSfp and norcodonine synthase TfNCS; (b) Recombinant cells BM2: using Escherichia coli BL21 as the host, expressing 6-O-methyltransferase PsOMT2, methionine adenosine transferase MAT and homocysteine ​​hydrolase SAHH; (c) Recombinant cells BM3: Using Escherichia coli BL21 as the host, expressing 7-O-methyltransferase Ps7OMT, methionine adenosine transferase MAT and homocysteine ​​hydrolase SAHH.

2. The recombinant Escherichia coli according to claim 1, characterized in that, pET series plasmids were used as expression vectors.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that, The amino acid sequence of the carboxylic acid reductase is shown in SEQ ID NO.1; the amino acid sequence of the phosphoproteopantothenate thioethylamine transferase is shown in SEQ ID NO.2; the amino acid sequence of the norcodonine synthase is shown in SEQ ID NO.3; the amino acid sequence of the methionine adenosine transferase is shown in SEQ ID NO.7; the amino acid sequence of the homocysteine ​​hydrolase is shown in SEQ ID NO.6; the amino acid sequence of the 6-oxomethyltransferase is shown in SEQ ID NO.4; and the amino acid sequence of the 7-oxomethyltransferase is shown in SEQ ID NO.

5.

4. A cell catalyst containing any one of the recombinant Escherichia coli according to claims 1 to 3.

5. The use of the recombinant Escherichia coli according to any one of claims 1 to 3, or the cell catalyst according to claim 4, in the synthesis of papaverine or its derivatives.

6. A method for synthesizing papaverine or its derivatives, characterized in that, The method includes: Culture the recombinant Escherichia coli according to claim 1 and collect bacterial cells; the recombinant Escherichia coli cells include the recombinant cells BM1, BM2 and / or BM3. The bacterial cells are reacted with a substrate, which includes phenylacetic acid or a derivative thereof, and dopamine.

7. The method according to claim 5, characterized in that, The recombinant cells BM1 were reacted with the substrate at 20℃ to 40℃; the concentration of phenylacetic acid or its derivatives in the reaction system was 5 to 14 mM, and the concentration of dopamine was 5 to 14 mM.

8. The method according to claim 7, characterized in that, Collect the supernatant after the reaction according to claim 7, and react it with recombinant cells BM2 at 20℃~40℃.

9. The method according to claim 8, characterized in that, Collect the supernatant after the reaction according to claim 8, and react it with recombinant cells BM3 at 20℃~40℃.

10. The method according to any one of claims 6 to 9, characterized in that, The phenylacetic acid or its derivatives include at least one of 1a to 1h:

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