Method for synthesizing Rotudinine and derivatives thereof and construction of whole-cell catalytic system

Abstract

The invention discloses a method for synthesizing Rotudinine and a derivative thereof and construction of a whole-cell catalytic system. According to the invention, the Rotudinine and the derivative thereof can be efficiently synthesized by utilizing an artificially designed multi-enzyme cascade reaction method. According to the invention, enzymes of a synthetic route are respectively constructed into escherichia coli chassis cells to obtain a whole-cell catalyst comprising M1a, M2a, M3a and M4a, the whole-cell catalyst is used for synthesizing Rotudinine and a derivative thereof, and the yield of Rotudinine can reach 2.44 g / L.

Description

Technical Field

[0001] The invention relates to a method for synthesizing Rotundine and its derivatives and the construction of a whole-cell catalytic system, belonging to the technical field of bioengineering. Background Art

[0002] Alkaloids are a class of nitrogen-containing organic compounds that are widely found in nature, and more than 27,000 structures have been reported. As an important secondary metabolite, alkaloids are often known for their unique pharmacological activities, such as poppy used for vasodilation in clinical surgery; morphine, a powerful analgesic; berberine with extremely strong antibacterial activity; colchicine used to treat acute gout, etc. The powerful pharmacological activity of alkaloids has brought great market potential, but their complex stereochemical structure is a difficulty in chemical synthesis. Synthesis usually has low yields and requires the intervention of heavy metal catalysts, which is contrary to the concept of contemporary green chemistry. Therefore, the acquisition of most medicinal alkaloids still depends on plant extraction, but plants have a long growth cycle and complex components. Extraction from them often faces problems such as low efficiency, long time consumption, and high cost. Some secondary metabolites with low content are even more difficult to obtain, which greatly restricts the development and use of related drugs. With the rapid development of synthetic biology, and in order to meet the huge gap in the market for medicinal alkaloids, expression systems such as Escherichia coli, yeast, and tobacco are widely used for the de novo synthesis of alkaloids. In 2011, researchers developed an engineered Escherichia coli for the efficient synthesis of (S)-Reticuline, an important precursor of benzylisoquinoline alkaloids; in 2015, the Keasling team performed 56 gene editing in yeast and successfully achieved a 31-step heterologous total synthesis of vinblastine; in 2021, with the excavation and reporting of the complete synthesis pathway of colchicine in plants, the Sattely team achieved the total synthesis of colchicine in tobacco through a transient expression system. However, due to the complex structure of alkaloids and the lengthy metabolic pathways, the catalytic enzymes involved have a wide selectivity for alkaloid intermediates in the main metabolic pathway, resulting in problems such as uncontrollable metabolic flow and low yield of target compounds; at the same time, most alkaloids are toxic, and high concentrations of target products can cause toxic effects on cells, etc., and the de novo synthesis yield of alkaloids often cannot meet market demand. Therefore, strategies such as multi-enzyme cascade catalysis and resting cells began to gain attention.

[0003] Levo-tetrahydropalmatine, also known as levo-tetrahydropalmatine, and its drug name is Rotundine. It is a tetrahydroberberine isoquinoline alkaloid with a chiral center. It can usually be extracted from Stephania tetraphylla and Corydalis yanhusuo, and is a CFDA-approved alkaloid monomer analgesic preparation. Natural corydalis often exists in the form of racemates, among which the levo-tetrahydropalmatine (Rotundine) is considered to be the main component that exerts pharmacological effects. In recent years, the pharmacological activity of Rotundine has been widely studied, especially its analgesic, anti-addiction, anti-inflammatory, neuroprotective and anti-cancer activities. These studies have shown that Rotundine is a promising compound that can be used to treat dysmenorrhea, drug addiction, inflammatory diseases, neuropathic pain, cancer, cerebral edema, and acute global cerebral ischemia-reperfusion injury. At present, the main source of L-tetrahydropalmatine is still plant extraction, which is limited by its extremely low content in plants, the extraction efficiency is less than 0.4‰, and there are problems such as long time consumption and high cost. In order to improve the efficiency of Rotundine acquisition, Tang Pei's team used a fully mixed flow reactor in the synthesis of the protoberberine skeleton to achieve efficient synthesis of Rotundine. The ee value of the final product was above 90% and the yield was about 65%, but the use of heavy metal catalyst Ir did not conform to the concept of green chemistry. In order to achieve the synthesis of Rotundine more efficiently and environmentally friendly, heterologous biosynthesis has become the focus of researchers, but the natural synthesis pathway of Rotundine in plants is still unknown, which brings difficulties to the heterologous reconstruction of its natural pathway. Therefore, the Smolke team reconstructed the natural biosynthetic pathway of (S)-Scoulerine, an important intermediate of berberine alkaloids, and the oxygen methyltransferase TfS9OMT discovered in yellow thrush into Saccharomyces cerevisiae. Based on the structural modification of TfS9OMT, dopamine feeding and condition optimization during fermentation, the biosynthesis of Rotundine was achieved for the first time, with a yield of 3.60μg / L; in 2021, the Liu Jihua and Yu Boyang teams optimized the expression of berberine bridge enzyme BBE, and achieved the active expression of the key enzyme BBE in Escherichia coli through strategies such as optimizing gene sources, expression tags and expression vectors, increasing the yield of Rotundine to 1.19mg / L. Recently, the research group of Professor Huang Luqi constructed an engineered Saccharomyces cerevisiae through metabolic engineering and achieved a yield of 68.6mg / L of Rotundine, but the current biosynthesis yield is still at a low level. Summary of the invention

[0004] In order to solve the above technical problems, the present invention designs a new route for synthesizing Rotundine and its derivatives by using simple and readily available phenylacetic acid derivatives and dopamine as raw materials and utilizing a multi-enzyme cascade reaction.

[0005] The present invention provides an oxygen methyltransferase mutant, wherein the oxygen methyltransferase mutant has an oxygen methyltransferase with an amino acid sequence as shown in SEQ ID NO.8 as a parent and has one and / or more mutations in (a) to (c):

[0006] (a) mutating the serine at position 16 of the parent to alanine, glycine or leucine;

[0007] (b) mutating the threonine at position 115 of the parent to alanine, glycine or leucine;

[0008] (c) The proline at position 301 of the parent gene is mutated to alanine, glycine or leucine.

[0009] In one embodiment, the oxygen methyltransferase mutant has an oxygen methyltransferase with an amino acid sequence as shown in SEQ ID NO.8 as a parent;

[0010] Mutating the serine at position 16 of the parent to glycine, and mutating the proline at position 301 of the parent to alanine; or

[0011] Mutating the serine at position 16 of the parent to glycine, and mutating the proline at position 301 of the parent to glycine; or

[0012] The serine at position 16 of the parent is mutated to glycine, and the threonine at position 115 is mutated to alanine; or

[0013] The serine at position 16 of the parent was mutated to glycine, the threonine at position 115 was mutated to alanine, and the proline at position 301 of the parent was mutated to alanine.

[0014] The present invention also provides a gene encoding the oxygen methyltransferase mutant.

[0015] The present invention also provides an expression vector carrying the gene of the oxygen methyltransferase mutant.

[0016] In one embodiment, the vector used for the overexpression includes but is not limited to pET series plasmids, or pRSFDuet-1 plasmid, or pCDFDuet-1 plasmid, or pETDuet-1 plasmid, or pACYCDuet-1 plasmid, preferably pETDuet-1.

[0017] The present invention also provides a recombinant cell expressing the mutant or the gene, or carrying the expression vector.

[0018] In one embodiment, the recombinant cell uses Escherichia coli BL21 (DE3) as a chassis cell and overexpresses the oxygen methyltransferase mutant, methionine adenosyltransferase (MAT) and homocysteine ​​hydrolase (SAHH).

[0019] In one embodiment, the amino acid sequence of the oxygen methyltransferase is shown in SEQ ID NO.8, or the oxygen methyltransferase is the above-mentioned nitrogen methyltransferase mutant; the amino acid sequence of the methionine adenosyltransferase is shown in Genbank: AAN81976.1; the amino acid sequence of the homocysteine ​​hydrolase is shown in Genbank accession number: EDL06108.1.

[0020] The present invention also provides the use of the oxygen methyltransferase mutant, or the gene encoding the oxygen methyltransferase mutant, or the recombinant cell M4a in synthesizing Rotundine or its derivatives.

[0021] In one embodiment, the knockout and construction methods of Escherichia coli alcohol dehydrogenase (yqhD, yahK, yjgB), aldehyde-keto reductase (dkgB, yeaE, dkgA) and transcription factor (yqhC) genes are referenced in Kunjapur AM, Tarasova Y, Prather KL J. Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli [J]. Journal of the American Chemical Society, 2014, 136 (33): 11644-54.

[0022] The present invention provides a whole cell catalyst for catalytically preparing Rotundine and its derivatives, comprising recombinant cells (a) to (d):

[0023] (a) Recombinant cell M1a: Based on the starting strain IAA, the following proteins are expressed: carboxylic acid reductase, phosphopantethein transferase and higenamine synthase; the starting strain is IAA, which has been disclosed in the paper "Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids";

[0024] (b) Recombinant cell M2a: Using Escherichia coli BL21 (DE3) as the chassis cell, overexpressing oxygen methyltransferase (Ps6OMT), nitrogen methyltransferase (CNMT), methionine adenosyltransferase (MAT) and homocysteine ​​hydrolase (SAHH);

[0025] (c) Recombinant cell M3a: Using Escherichia coli BL21 (DE3) as the chassis cell, berberine bridge enzyme (EcBBE), riboflavin synthase (RibH), riboflavin synthase (RibC) and bifunctional riboflavin kinase (RibF) and analytical chaperone Gro7 were overexpressed;

[0026] (d) Recombinant cell M4a: Using Escherichia coli BL21 (DE3) as the chassis cell, the oxygen methyltransferase mutant, methionine adenosyltransferase (MAT) and homocysteine ​​hydrolase (SAHH) were overexpressed.

[0027] In one embodiment, the amino acid sequence of the carboxylate reductase (TpCAR) is as shown in Genbank: WP_013126039.1; the amino acid sequence of the phosphopanthocyanate transferase (BsSfp) is as shown in Genbank: CP137756.1; the amino acid sequence of the norcoclaurine synthase (TfNCS) is as shown in Genbank: ACO90248.13.

[0028] In one embodiment, the amino acid sequence of the oxygen methyltransferase (Ps6OMT) is shown in Genbank: NP_001413547.1; the amino acid sequence of the nitrogen methyltransferase (CNMT) is shown in Genbank: BAB71802.1; the amino acid sequence of the methionine adenosyltransferase is shown in Genbank: AAN81976.1; and the amino acid sequence of the homocysteine ​​hydrolase is shown in Genbank: EDL06108.1.

[0029] In one embodiment, the amino acid sequence of the berberine bridge enzyme (EcBBE) is shown in PDB ID: 3D2D_A; the amino acid sequence of the riboflavin synthase (RibH) is shown in Genbank Accession No.: CAD6020726.1; the amino acid sequence of the riboflavin synthase (RibC) is shown in Genbank Accession No.: CAD6006425.1; the amino acid sequence of the bifunctional riboflavin kinase (RibF) is shown in Genbank Accession No.: ADD75835.1.

[0030] In one embodiment, the amino acid sequence of the oxygen methyltransferase (T'fS9OMT) is shown in PDB ID: 6NEJ; the amino acid sequence of the methionine adenosyltransferase is shown in Genbank: AAN81976.1; and the amino acid sequence of the homocysteine ​​hydrolase is shown in Genbank: EDL06108.1.

[0031] The present invention also provides the use of the recombinant cells M1a-M4a in synthesizing Rotundine or its derivatives.

[0032] In one embodiment, the use comprises: mixing the recombinant cell M1a with a substrate for reaction; the substrate comprises a phenylacetic acid derivative and dopamine.

[0033] In one embodiment, the reaction system contains 20-30 mM ascorbate, preferably sodium ascorbate.

[0034] In one embodiment, the recombinant cells (M1a, M2a, M3a or M4a) are prepared as follows: the strain is inoculated into LB medium, cultured at 20-40°C for 10-12h, transferred to 2YT medium, and cultured at 20-40°C until OD 600 When the value reaches about 0.6-0.8, cool down to 15-25℃, add IPTG with a final concentration of 0.05-0.5mM to induce for 10-20h, and collect the bacteria.

[0035] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.5-8.5.

[0036] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0037] In one embodiment, the added amount of the phenylacetic acid derivative is 10-20 mM; the added amount of dopamine is 20-30 mM.

[0038] In one embodiment, the application includes: using the supernatant after the reaction of the recombinant cell M1a as a reaction system, and reacting the recombinant cell M2a in the reaction system; the reaction contains 20-30mM ascorbate, 30-40mM polyphosphate or ATP, and 30-40mM L-methionine; the ascorbate is preferably sodium ascorbate; the polyphosphate is preferably a metal salt of ATP.

[0039] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.5-8.5.

[0040] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0041] In one embodiment, the amount of the recombinant cell M1a added is 50-60 g / L; the amount of the recombinant cell M2a added is 30-120 g / L, preferably 80-90 g / L.

[0042] In one embodiment, the application includes: using the supernatant after the reaction of the recombinant cell M2a as a reaction system, and reacting the recombinant cell M3a in the reaction system.

[0043] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.5-9.5.

[0044] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0045] In one embodiment, the application includes: using the supernatant after the reaction of the recombinant cell M3a as a reaction system, and reacting the recombinant cell M4a in the reaction system; the reaction system contains 30-40mM polyphosphate or ATP, and 30-40mM L-methionine, and the polyphosphate is preferably a metal salt of ATP.

[0046] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.5-9.5.

[0047] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0048] The present invention also provides a method for synthesizing Rotundine or its derivatives by realizing an in vitro enzyme cascade reaction in a one-pot four-step method.

[0049] In one embodiment, the method is catalyzed by an enzyme and comprises the following steps:

[0050] (1) First step reaction: the reaction system includes: carboxylic acid reductase, higenamine synthase, glucose dehydrogenase, dopamine and phenylacetic acid or its derivatives, the reaction temperature is 20-40°C, the reaction system pH is 6-8, and the reaction time is 2-8h;

[0051] (2) heating to inactivate the enzyme in step (1) and collecting the supernatant;

[0052] (3) adding oxygen methyltransferase, nitrogen methyltransferase and S-adenosylmethionine to the supernatant obtained in step (2), the reaction temperature is 20-40° C., and the reaction time is 2-8 h;

[0053] (4) heating to inactivate the enzyme in step (3) and collecting the supernatant;

[0054] (5) adding berberine bridge enzyme and flavin adenine dinucleotide to the supernatant obtained in step (4), the reaction temperature is 20-40° C., and the reaction time is 2-8 h;

[0055] (6) heating to inactivate the enzyme in step (5) and collecting the supernatant;

[0056] (7) Adding oxygen methyltransferase and S-adenosylmethionine to the supernatant obtained in step (6) at a reaction temperature of 20-40° C. for a reaction time of 2-8 h to obtain Rotundine or a derivative thereof.

[0057] In one embodiment, the step (1) further comprises 5-10 mM magnesium ions, 5-10 mM polyphosphate or ATP, 1-3 mM NADP + , 1-20 mM glucose, 1-5 mM ascorbic acid or ascorbate.

[0058] In one embodiment, the concentrations of the substances in the system of step (1) are: 5-10 μM carboxylic acid reductase, 5-10 μM higenamine synthase, 5-10 μM glucose dehydrogenase, 1-10 mM substrate dopamine, and 1-10 mM phenylacetic acid derivative.

[0059] In one embodiment, 5-10 μM oxygen methyltransferase, 5-10 μM nitrogen methyltransferase and 5-10 mM S-adenosylmethionine are added in step (3).

[0060] In one embodiment, 5-10 μM berberine bridge enzyme and 5-10 mM flavin adenine dinucleotide are added in step (5).

[0061] In one embodiment, 5-10 μM oxygen methyltransferase and 5-10 mM S-adenosylmethionine are added in step (7).

[0062] In one embodiment, the method uses a cell expressing an enzyme as a catalyst to perform a catalytic reaction, and the method comprises:

[0063] (1) mixing the recombinant cell M1a with a substrate for reaction, and collecting the reaction supernatant; the substrate includes a phenylacetic acid derivative and dopamine;

[0064] (2) mixing the recombinant cell M2a with the supernatant obtained in step (1), reacting the mixture, and collecting the reaction supernatant;

[0065] (3) mixing the recombinant cell M3a with the supernatant obtained in step (2), reacting the mixture, and collecting the reaction supernatant;

[0066] (4) Mixing the recombinant cell M4a with the supernatant obtained in step (3) and reacting to obtain Rotundine or its derivatives.

[0067] In one embodiment, the reaction in step (1) contains 20-30 mM ascorbate, preferably sodium ascorbate.

[0068] In one embodiment, the reaction system of step (2) contains 30-40 mM polyphosphate or ATP, and 30-40 mM L-methionine, and the polyphosphate is preferably a metal salt of ATP.

[0069] In one embodiment, the reaction system of step (4) contains 30-40 mM polyphosphate or ATP, and 30-40 mM L-methionine, and the polyphosphate is preferably a metal salt of ATP.

[0070] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.5-9.5.

[0071] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0072] In one embodiment, the added amount of the phenylacetic acid derivative is 10-20 mM; the added amount of dopamine is 20-30 mM.

[0073] In one embodiment, the added amount of the recombinant cell M1a is 50-60 g / L; the added amount of the recombinant cell M2a is 80-90 g / L; the added amount of the recombinant cell M3a is 80-90 g / L; and the added amount of the recombinant cell M4a is 70-80 g / L.

[0074] In one embodiment, the structural formula of the phenylacetic acid derivative is as follows:

[0075]

[0076] In one embodiment, the structural formula of the phenylacetic acid or its derivatives includes at least one of 1a-1g:

[0077]

[0078] In one embodiment, the structural formula of Rotundine is:

[0079] In one embodiment, the structural formula of Rotundine or its derivative is as shown in any one of (S)-8a to (S)-8g below:

[0080]

[0081] The present invention also provides the use of the mutant, the recombinant cell, or the method in preparing Rotundine or its derivatives.

[0082] Beneficial effects:

[0083] (1) The present invention provides an oxygen methyltransferase mutant, wherein the enzyme activity of the mutant S16G / P301A is increased by 19 times compared with the wild-type enzyme; the enzyme activity of the mutant S16G is increased by 11 times compared with the wild-type enzyme; and the enzyme activity of the mutant P301A is increased by 13 times compared with the wild-type enzyme;

[0084] (2) The present invention provides a recombinant strain M1a, which can efficiently convert substrates dopamine and phenylacetic acid derivatives into benzylisoquinoline skeletons ((S)-3a-(S)-3g, see Figure 1 ), the conversion rate is 95%, and the ee value is >99%;

[0085] (3) The present invention provides a recombinant strain M2a, which is capable of synthesizing S-adenosylmethionine from L-methionine and ATP and is capable of synthesizing S-adenosylmethionine from the substrate (S)-3a-(S)-3g (see Figure 1 ) is methylated at the 6-hydroxyl group and at the imino group to generate (S)-5a-(S)-5g;

[0086] (4) The present invention provides a recombinant strain M3a, which can increase the production of intracellular FAD and can also produce (S)-5a-(S)-5g (see Figure 1 ) to achieve oxidative carbon-carbon coupling;

[0087] (5) The present invention provides a recombinant strain M4a, which is capable of synthesizing S-adenosylmethionine from L-methionine and ATP and is capable of synthesizing S-adenosylmethionine from the substrate (S)-6a-(S)-6g (see Figure 1 ) are methylated at the 2nd and 9th hydroxyl groups;

[0088] (6) The present invention also provides a method for synthesizing Rotundine or its derivatives using phenylacetic acid derivatives and dopamine as raw materials. Through multi-enzyme cascade catalysis or whole-cell catalysis by recombinant strains, the yield of Rotundine can reach 2.44 g / L, and the yield of (S)-8b can reach 1.01 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The invention provides a synthetic route for Rotundine and its derivatives.

[0090] Figure 2 To transform TfS9OMT M Efficient synthesis of Rotundine; TfS9OMT M The relative activity of the mutants towards substrate (S)-6a, the catalytic activity of the parent towards substrate (S)-6a was set as 1.

[0091] Figure 3 This is the standard curve of the product Rotundine.

[0092] Figure 4 This is the standard curve of product (S)-8f.

[0093] Figure 5 HPLC identification of in vitro multi-enzyme cascade catalysis.

[0094] Figure 6 HPLC and MS identification of (S)-3a synthesized by the recombinant strain TpCAR-BsSfp-TfNCS (strain M1a).

[0095] Figure 7 HPLC and MS characterization of (S)-5a synthesized by the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT (strain M2a).

[0096] Figure 8 HPLC and MS identification of (S)-6a synthesized by the recombinant strain EcBBE#-RibH-RibC-RibF;Gro7 (strain M3a).

[0097] Fig. 9 HPLC and MS characterization of (S)-8a synthesized by the recombinant strain TfS9OMT#-MmSAHH-EcMAT (strain M4a).

[0098] Fig.10 To expand the substrate spectrum using four recombinant strains M1a, M2a, M3a, and M4a.

[0099] Fig.11 This is the H spectrum of Rotundine in deuterated chloroform.

[0100] Fig.12 The time curves of rotundine synthesis by strains M1a, M2a, M3a, and M4a are shown respectively. DETAILED DESCRIPTION

[0101] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0102] (1) Reagents and materials

[0103] Purchase sources of reagents and materials: The antibiotics such as ampicillin sodium, kanamycin sulfate, streptomycin sulfate, chloramphenicol, etc. used in the present invention are all from Shanghai Bioengineering; the cofactors ATP sodium salt, NADPH, SAM, etc. used in the present invention are all from San Chemical Technology (Shanghai) Co., Ltd.; the PCR enzyme and homologous recombination enzyme involved in the molecular experiments in the present invention are all purchased from Bao Biotechnology (Dalian) Co., Ltd.; the chemical reagents used in the present invention are all purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Titan Technology Co., Ltd.

[0104] (2) Culture medium

[0105] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0106] 2YT medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L.

[0107] (3) Detection method

[0108] Activity detection of O-methyltransferase (TfS9OMT) and its mutants: 200 μL reaction system includes 100 mM Tris-HCl buffer (pH 8.0), 5 μM TfS9OMT, 10 mM MgCl2, 2 mM (S)-6a and 8 mM SAM (pH 8.0). The whole system is carried out in a reaction shaker at a reaction temperature of 37 ° C for 4 h. After the reaction is completed, 4 volumes of methanol are added to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5 min, take 200 μL sample and load it on the chromatographic column, according to the standard curve of product (S)-8a (see Figure 3 ) were used to analyze the yield of each mutant.

[0109] High performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature of 30°C, detection wavelength of 280nm. The detection method uses 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) for gradient separation, and the mobile phase flow rate is 1mL min -1 The linear gradient was as follows: isocratic elution with 5% acetonitrile for 1 min, followed by gradient elution with 5%-50% acetonitrile for 25 min.

[0110] Protein purification method: Resuspend the collected bacteria in lysis buffer (50mmol / L Tris-HCl, 300mmol / L NaCl, 20mM imidazole, pH=8), and then crush the resuspension with a high-pressure homogenizer. The crushed resuspension is centrifuged at low temperature and high speed, 4°C, 10000rpm for 30min to obtain a crude enzyme solution. Desalt the solution by nickel column affinity chromatography and desalting column (HistrapTM 5mL Desalting) to finally obtain the purified protein.

[0111] Table 1 Strains constructed in some embodiments of the present invention

[0112]

[0113] Note: Strain IAA was disclosed in the paper "Modular assembly of an artificially concisebiocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids".

[0114] Table 2 Primer sequences for constructing recombinant strains

[0115]

[0116]

[0117] Example 1: Construction and verification of artificial cascade reaction pathways of model products

[0118] like Figure 1 As shown in the figure, the first step is to use 3-hydroxy, 4-methoxyphenylacetic acid (1) as the model substrate, through the carboxylic acid reductase (TpCAR) activated by phosphopantothenate thioethylamine transferase (BsSfp), the metal ion magnesium ion (Mg 2+), cofactors ATP and NADPH, is reduced to the corresponding aldehyde (1a), and then another substrate dopamine is condensed with the generated aldehyde under the action of norcoclaurine synthase (TfNCS) to generate the skeleton of phenethylisoquinoline (S)-1-(3-hydroxy-4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol ((S)-3a). In the second step, the intermediate (S)-3a is converted into (S)-5a under the action of oxygen methyltransferase (Ps6OMT) and nitrogen methyltransferase (CNMT) and cofactor SAM. In the third step, the intermediate (S)-5a is converted into the final product (S)-6a under the action of berberine bridge enzyme (EcBBE) and cofactor FAD. In the fourth step, the intermediate (S)-6a is converted into the final product (S)-8a (Rotundine) under the action of oxygen methyltransferase (TfS9OMT) and cofactor SAM. The cofactor SAM is synthesized by methionine adenosyltransferase (EcMAT), and the product SAH after SAM demethylation is hydrolyzed by homocysteine ​​hydrolase (MmSAHH).

[0119] The specific steps are as follows:

[0120] First step reaction: add 5-10 μM carboxylic acid reductase, 5-10 μM higenamine synthase, 5-10 μM glucose dehydrogenase, 5-10 mM magnesium chloride, 5-10 mM ATP, 1-3 mM NADP to a 1.5 ml EP tube. + , 10 mM glucose, 2 mM sodium ascorbate, 2 mM substrate dopamine and 2 mM phenylacetic acid derivative, the reaction system was carried out in 0.1 M tris (hydroxymethylaminomethane) (Tris) buffer, pH 8.0, and the final reaction volume was 200 μL.

[0121] (2) The reaction tube in step (1) was placed in a shaking reactor at 30°C and 800 rpm for 4 hours. After the reaction was completed, the tube was heated at 95°C for 5 minutes to inactivate the enzyme in step (1), and the supernatant was obtained by high-speed centrifugation.

[0122] (3) Second step reaction: Add 5-10 μM oxygen methyltransferase, 5-10 μM nitrogen methyltransferase and 5-10 mM S-adenosylmethionine (SAM, pH 8.0) to the supernatant of step (2).

[0123] (4) The reaction tube in step (3) was placed in a shaking reactor at 30°C and 800 rpm for 4 hours. After the reaction was completed, the reaction tube was heated at 95°C for 5 minutes to inactivate the enzyme in step (1), and the supernatant was obtained by high-speed centrifugation.

[0124] (5) Step 3: Add 5-10 μM berberine bridge enzyme and 5-10 μM flavin adenine dinucleotide to the supernatant of step (4) above, and adjust the pH of the reaction system to 9.0.

[0125] (6) The reaction tube in step (5) was placed in a shaking reactor at 30°C and 800 rpm for 4 hours. After the reaction was completed, the reaction tube was heated at 95°C for 5 minutes to inactivate the enzyme in step (1), and the supernatant was obtained by high-speed centrifugation.

[0126] (7) Step 4: Add 5-10 μM oxygen methyltransferase and 5-10 mM S-adenosylmethionine (SAM, pH 8.0) to the supernatant of step (6). Place the reaction system in a shaking reactor at 30° C. and 800 rpm for 4 h.

[0127] (8) After the reaction is completed, 4 volumes of methanol are added to the reaction solution to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5 min, take 200 μL of sample and load it on the chromatographic column, and detect the product by liquid chromatography.

[0128] The reaction results are as follows Figure 5 As shown, in the experimental group, a new peak appeared at the same position as the standard product, which was preliminarily determined to be the product (S)-8a (Rotundine).

[0129] Example 2: Key rate-limiting enzyme in the artificial cascade reaction pathway (oxygen methyltransferase TfS9OMT M )

[0130] In order to increase the yield of product (S)-8a, the rate-limiting enzyme TfS9OMT in the cascade reaction pathway was M (shown in SEQ ID NO.8) was modified. First, the substrate (S)-7a was docked to TfS9OMT using molecular docking software (Discovery Studio). M Next, we selected the substrate (S)-7a around the D-loop. All amino acids in the range were mutated to alanine, glycine or leucine. The gene shown in SEQ ID NO.9 was used as the starting sequence, and the primers for introducing mutations were shown in Table 3. The mutants were expressed in Escherichia coli BL21 (DE3) using pET21b (+) as the expression vector. The recombinant Escherichia coli expressing the mutants were cultured, the bacterial cells were collected, and the mutant proteins were obtained by purification after being crushed.

[0131] The relative yield of the product (S)-8a (Rotundine) represents the relative activity of different mutants. The specific steps are: add 5-10 μM oxygen methyltransferase and 5-10 mM S-adenosylmethionine to the reaction system containing 2 mM (S)-6a, react in an oscillating reactor at 30°C and 800 rpm for 4 hours, and measure the content of the product (S)-8a.

[0132] like Figure 2 As shown, four single mutants with higher activity were obtained, TfS9OMT M -S16G,TfS9OMT M -T115A,TfS9OMT M -P301G and TfS9OMT M Secondly, a semi-rational design and combined mutation strategy were used to obtain the optimal mutant TfS9OMT M -S16G / P301A. Compared with wild-type TfS9OMT M , mutant TfS9OMT M -S16G / P301A activity was increased 19-fold.

[0133] Table 3 Primer sequences of CNMT mutants

[0134]

[0135]

[0136] Table 4 Nitrogen methyltransferase TfS9OMT M Relative enzyme activity of mutants

[0137] Mutants Relative activity (WT activity is about 1) <![CDATA[TfS9OMT M ]]> 1 <![CDATA[TfS9OMT M -S16G]]> 11 <![CDATA[TfS9OMT M -T115A]]> 5 <![CDATA[TfS9OMT M -P301G]]> 12 <![CDATA[TfS9OMT M -P301A]]> 13 <![CDATA[TfS9OMT M -S16G / P301A]]> 19

[0138] Note: Relative activity = mutant enzyme activity ÷ parent enzyme activity × 100%.

[0139] TfS9OMT M -S16G / P301A was reacted according to the multi-enzyme cascade reaction described in Example 1, except that the oxygen methyltransferase TfS9OMT was replaced M TfS9OMT M -S16G / P301A, the results are as follows Figure 5 shown.

[0140] Example 3: Construction of engineered strain TpCAR-BsSfp-TfNCS (M1a), protein expression and product identification

[0141] The target gene CAR (Genbank accession number WP_013126039.1) from Tsukamurella paurometabola was codon optimized to obtain the gene sequence shown in SEQ ID NO.1, which was synthesized and ligated to the first multiple cloning site (MCS1) of the pRSFDuet vector to obtain the plasmid pRSFDuet-TpCAR.

[0142] The Sfp gene fragment (Genbank: CP137756.1) was amplified from the genome of Bacillus subtilis and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b-BsSfp.

[0143] The target gene NCS (Genbank accession number ACO90248.1) from Thalictrum flavum was codon optimized to obtain the gene sequence shown in SEQ ID NO.3, which was synthesized and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-TfNCS.

[0144] After obtaining the above three plasmids, the gene fragments Sfp and NCS were integrated into MCS1 and MCS2 of pRSFDuet-TpCAR respectively by homologous recombination technology to obtain the plasmid pRSFDuet-T7-rbs-TpCAR-rbs-BsSfp-T7-rbs-TfNCS. The recombinant plasmid was transformed into the engineered strain IAA (the strain was disclosed in the paper "Modular assembly of anartificially concise biocatalytic cascade for the manufacture ofphenethylisoquinoline alkaloids") to obtain the recombinant strain TpCAR-BsSfp-TfNCS (M1a). The primers used for plasmid construction are shown in Table 3.

[0145] The monoclonal strain TpCAR-BsSfp-TfNCS (M1a) was picked and transferred to 5 ml LB medium and cultured at 250 rpm and 37°C for 10-12 h. Subsequently, it was transferred to 500 ml 2YT medium and cultured at 220 rpm and 37°C. When the bacterial OD 600When the value reaches about 0.6-0.8, cool to 18℃, culture for 30min, add IPTG with a final concentration of 0.2mM, induce culture at 18℃ for 16h, collect the cells after centrifugation, wash the cells three times with buffer containing 100mM Tris (pH 8.0) and 200mM NaCl, and obtain the cells by centrifugation.

[0146] Subsequently, the synthesis of product (S)-3a was verified by a 5 ml reaction system, the reaction system was: 100 mM Tris buffer (pH 8.0), 200 mM NaCl, the final bacterial concentration OD 600 The reaction was carried out in a shaker at 30°C. After 6 hours, 200 μL of the sample was taken and 800 μL of methanol was added to quench the reaction. The sample was centrifuged at 20000 g for 5 minutes and filtered with a 0.22 μm filter membrane.

[0147] The synthesis of product (S)-3a was detected by HPLC system. Figure 6 As shown, the compound obtained by whole-cell catalysis had the same peak time as the standard, and the generation of the product was further confirmed by high-resolution mass spectrometry. The yield of (S)-3a was 3.84 g / L, with a yield of 98.0%.

[0148] Example 4: Construction of engineered strain Ps6OMT-MmSAHH-CNMT-EcMAT (M2a), protein expression and product identification

[0149] The target gene 6OMT (Genbank accession number: NP_001413547.1) from poppy Papaver somniferum was codon optimized to obtain the gene sequence shown in SEQ ID NO.4, which was synthesized and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-Ps6OMT.

[0150] The target gene CNMT (Genbank accession number: BAB71802.1) from Coptis japonica was codon optimized to obtain the gene sequence shown in SEQ ID NO.5, which was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-CNMT.

[0151] The MAT gene fragment (Genbank accession number: AAN81976.1) was amplified from the Escherichia coli genome and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcMAT.

[0152] The target gene SAHH from house mouse Mus musculus was codon optimized to obtain the gene sequence shown in SEQ ID NO.6, which was synthesized and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-MmSAHH.

[0153] After obtaining the above four plasmids, the gene fragments CNMT, EcMAT, Ps6OMT and MmSAHH were integrated into the two MCSs of the pCDFDuet plasmid using homologous recombination technology to obtain the plasmid MmSAHH--EcMATpCDFDuet-T7-rbs-Ps6OMT-rbsMmSAHH-T7-rbs-CNMT-EcMAT. The recombinant plasmid was transformed into the engineering strain BL21 to obtain the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT (M2a).

[0154] The recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT (M2a) was cultured using the same method as in Example 3, and the cells were obtained by centrifugation. Subsequently, the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT was resuspended in the supernatant after the whole cell reaction of the recombinant strain TpCAR-BsSfp-TfNCS in Example 3, so that the final cell concentration OD 600 The final concentration of 40 mM L-Met and 40 mM ATP was added to the above resuspension and the mixture was reacted at 30°C and 250 rpm for 15 h. The product (S)-5a was detected using the same sample post-treatment method and liquid phase detection conditions as in Example 3. Figure 7 As shown, the compound obtained by whole-cell catalysis had the same peak time as the standard, and the generation of the product was further confirmed by high-resolution mass spectrometry. The yield of (S)-5a was 3.99 g / L, with a yield of 93.3%.

[0155] Example 5: Construction of the engineered strain EcBBE#-RibH-RibC-RibF; Gro7(M3a), protein expression and product identification

[0156] The 354th position of EcBBE from Eschscholzia californica (PDB ID: 3D2D_A) was mutated to tryptophan (R354W), and codons were optimized to obtain the gene sequence shown in SEQ ID NO.7. The sequence was synthesized and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcBBE R354W .

[0157] The RibH gene fragment (Genbank: CAD6020726.1) was amplified from the Escherichia coli genome and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibH.

[0158] The RibC gene fragment (Genbank: CAD6006425.1) was amplified from the Escherichia coli genome and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibC.

[0159] The RibF gene fragment (Genbank: ADD75835.1) was amplified from the Escherichia coli genome and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibF.

[0160] Truncated EcBBE R354W The 22 amino acids at the N-terminus were fused with an MBP tag at the N-terminus (the sequence encoding MBP is shown in SEQ ID NO.11), and then connected to the first multiple cloning site (MCS) of the pCDFDuet vector using homologous recombination technology, and the promoter T7 of the first multiple cloning site was replaced with the endogenous promoter ssrA of Escherichia coli (the sequence of ssrA is shown in SEQ ID NO.10), obtaining the plasmid pCDFDuet-TssrA-MBP-EcBBE R354W (NΔ22AA), MBP-EcBBE R354W (NΔ22AA) was named EcBBE#.

[0161] After obtaining the above plasmid, the gene fragment RibC was integrated into pCDFDuet-TssrA-MBP-EcBBE using homologous recombination technology. R354W (NΔ22AA) at the MCS1 site and RibF and RibH were integrated into pCDFDuet-TssrA-MBP-EcBBE R354WThe MCS2 site of (NΔ22AA) was inserted into the ribosome to obtain the plasmid pCDFDuet-TssrA-rbs-EcBBE#-rbs-RibC-T7-rbs-RibH-rbs-RibF (wherein the rbs sequence is AAGGAG). The recombinant plasmid was co-transformed with the plasmid pGro7 of the molecular chaperone Gro7 into Escherichia coli BL21 (DE3) to obtain the recombinant strain EcBBE#-RibH-RibC-RibF; Gro7 (M3a).

[0162] The recombinant strain EcBBE#-RibH-RibC-RibF;Gro7(M3a) was cultured in the same manner as in Example 3, and the cells were obtained by centrifugation. Subsequently, the supernatant of the whole cell reaction of the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT(M2a) was prepared according to the method in Example 4, the pH was adjusted to 9.0, and the recombinant strain EcBBE#-RibH-RibC-RibF;Gro7(M3a) was resuspended in the supernatant to a final cell concentration of OD 600 The reaction was carried out at 21°C, 30°C and 250 rpm for 9 hours. The product (S)-1d was detected by the same sample post-treatment method and liquid phase detection conditions as in Example 3, and the formation of the product was further confirmed by high-resolution mass spectrometry. Figure 8 As shown, the yield was 3.19 g / L and the yield was 75.1%.

[0163] Example 6: Construction of engineered strain TfS9OMT#-MmSAHH-EcMAT, protein expression and product identification

[0164] TfS9OMT was constructed according to the method of Example 2 M-S16G / P301A (named as TfS9OMT#), the gene encoding the mutant (shown in SEQ ID NO.9) was connected to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-TfS9OMT#.

[0165] The MAT gene fragment (Genbank accession number: AAN81976.1) was amplified from the Escherichia coli genome and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcMAT.

[0166] The target gene SAHH from house mouse Mus musculus was codon optimized to obtain the gene sequence shown in SEQ ID No.6, which was synthesized and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-MmSAHH.

[0167] The gene fragments TfS9OMT#, EcMAT and MmSAHH were integrated into the two MCSs of the pETDuet plasmid using homologous recombination technology to obtain the plasmid pETDuet-T7-rbs-TfS9OMT#-T7-rbs-MmSAHH-rbs-EcMAT. The recombinant plasmid was transformed into the engineering strain BL21 to obtain the recombinant strain TfS9OMT#-MmSAHH-EcMAT (M4a).

[0168] The recombinant strain TfS9OMT#-MmSAHH-EcMAT(M4a) was cultured according to the method of Example 3, and the cells were obtained by centrifugation. Subsequently, the pH of the supernatant after the whole cell reaction of the recombinant strain EcBBE#-RibH-RibC-RibF;Gro7(M3a) obtained in Example 5 was adjusted to 8.5, and then the recombinant strain TfS9OMT#-MmSAHH-EcMAT(M4a) was resuspended in the supernatant of the reaction solution with pH 8.5, so that the final cell concentration OD 600 The reaction was carried out at 21°C, 40°C and 250 rpm for 11 h. The product (S)-8a (Rotundine) was detected using the same sample post-treatment method and liquid phase detection conditions as in Example 3, and the formation of the product was further confirmed by high-resolution mass spectrometry. Fig. 9 As shown, the yield was 2.44 g / L and the yield was 52.8%.

[0169] Example 7: Substrate spectrum expansion

[0170] In a one-pot four-step reaction mode, phenylacetic acid and its derivatives (ag) and dopamine with different substituents were added to the whole-cell catalytic system to expand the substrate spectrum. Phenylacetic acid derivatives with different substituents.

[0171] The reaction conditions of the one-pot four-step method are as follows:

[0172] (a) The bacterial cells of the recombinant strains M1a, M2a, M3a and M4a were prepared according to the methods of Examples 3 to 6.

[0173] (b) Prepare 5 ml of reaction system for substrate conversion. The reaction system is: 100 mM Tris buffer (pH 8.0), the final concentration of bacterial M1a is OD 600 15, 20 mM MgCl2, 20 mM glucose, 5% DMSO, 6 mM substrate 1, 8.4 mM substrate dopamine and 20 mM sodium ascorbate; the reaction was carried out in an oscillator at 30°C for 6 hours, after which the supernatant was collected by centrifugation at 7000 rpm for 10 minutes and used for the next reaction;

[0174] (c) Resuspend the M2a cells in the supernatant obtained by centrifugation in step (b) to a final concentration of OD600 21, and add 20mM L-Met and 20mM ATP (pH 7.5) to the above resuspension, react at 30°C, 250rpm for 15h; after the reaction, centrifuge at 7000rpm for 10min to collect the supernatant, and adjust the pH of the supernatant to 9.0 with 5M NaOH for the next reaction;

[0175] (d) Resuspend the M3a cells in the supernatant obtained by centrifugation in step (c) to a final concentration of OD 600 The mixture was reacted at 21°C, 30°C, and 250 rpm for 9 hours. After 9 hours of reaction, the supernatant was collected by centrifugation at 7000 rpm for 10 minutes, and the pH of the supernatant was adjusted to 8.5 with 5 M NaOH for the next reaction.

[0176] (e) Resuspend the M4a cells in the supernatant obtained in step (d) to a final concentration of OD 600 21, and 20 mM L-Met and 20 mM ATP (pH 7.5) were added to the above resuspension, and the reaction was carried out at 40°C and 250 rpm for 11 hours. After 11 hours, HPLC detection was performed, and (S)-8b-(S)-8g was qualitatively analyzed by LC-MS.

[0177] The results showed that all substrates ( Fig.10 All of the above products (S)-8a and (S)-8g were converted into the corresponding products (S)-8b-(S)-8g. The yield of product (S)-8b obtained by whole cell catalysis was 1.01 g / L.

[0178] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An oxygen methyltransferase mutant, wherein the oxygen methyltransferase mutant has an oxygen methyltransferase with an amino acid sequence as shown in SEQ ID NO.8 as a parent and has one and / or more mutations in (a) to (c): (a) mutating the serine at position 16 of the parent to alanine, glycine or leucine; (b) mutating the threonine at position 115 of the parent to alanine, glycine or leucine; (c) Mutating the proline at position 301 of the parent to alanine, glycine or leucine.

2. Biomaterial, characterized in that For (a) to (c): (a) a gene encoding the oxygen methyltransferase mutant according to claim 1; (b) a vector carrying the gene described in (a); (c) A recombinant cell expressing the mutant according to claim 1, or the gene according to (a), or containing the vector according to (b).

3. Recombinant Escherichia coli, characterized in that Using Escherichia coli BL21 (DE3) as a chassis cell, the oxygen methyltransferase mutant, methionine adenosyltransferase and homocysteine ​​hydrolase described in claim 1 are overexpressed; the amino acid sequence of the methionine adenosyltransferase is shown in Genbank accession number: AAN81976.1; the amino acid sequence of the homocysteine ​​hydrolase is shown in Genbank accession number: EDL06108.

1.

4. A whole-cell catalyst, characterized in that A recombinant Escherichia coli comprising the recombinant Escherichia coli according to claim 3, and the recombinant cells of (a) to (c): (a) Recombinant cell M1a: Based on the starting strain, the following proteins are expressed: carboxylic acid reductase, phosphopantothenate mercaptoethylamine transferase and higenamine synthase; the starting strain is IAA; (b) Recombinant cell M2a: Using Escherichia coli BL21 (DE3) as the chassis cell, overexpressing oxygen methyltransferase, nitrogen methyltransferase, methionine adenosyltransferase and homocysteine ​​hydrolase; (c) Recombinant cell M3a: Using Escherichia coli BL21 (DE3) as the chassis cell, berberine bridge enzyme, riboflavin synthase, riboflavin synthase and bifunctional riboflavin kinase and analytical chaperone were overexpressed.

5. The whole-cell catalyst according to claim 4, characterized in that The preparation method of the recombinant cell is as follows: the recombinant cell is cultured in LB medium at 20-40° C. for 10-12 hours, transferred to 2YT medium, and cultured at 20-40° C. until OD 600 When the value reaches 0.6-0.8, cool down to 15-25℃, induce with IPTG at a final concentration of 0.05-0.5mM for 10-20h, and collect the bacterial cells.

6. A method for synthesizing Rotundine or its derivatives, characterized in that: The following steps are involved: (1) mixing the recombinant cell M1a with a substrate for reaction, and collecting the reaction supernatant; the substrate includes phenylacetic acid or a phenylacetic acid derivative, and dopamine; (2) mixing the recombinant cell M2a with the supernatant obtained in step (1), reacting the mixture, and collecting the reaction supernatant; (3) mixing the recombinant cell M3a with the supernatant obtained in step (2), reacting the mixture, and collecting the reaction supernatant; (4) mixing the recombinant cell M4a with the supernatant obtained in step (3), reacting the mixture, and collecting the reaction supernatant; The recombinant cell M1a expresses carboxylic acid reductase, phosphopanthocyanate transferase and higenamine synthase on the basis of the starting strain; The recombinant cell M2a is based on Escherichia coli BL21 (DE3) and expresses: oxygen methyltransferase, nitrogen methyltransferase, methionine adenosyltransferase and homocysteine ​​hydrolase; The recombinant cell M3a is based on E. coli BL21 (DE3) and expresses: berberine bridge enzyme, riboflavin synthase RibC, riboflavin synthase RibF, bifunctional riboflavin kinase RibH, molecular chaperone Gro7; The recombinant cell M4a is expressed on the basis of Escherichia coli BL21 (DE3): the oxygen methyltransferase mutant according to claim 1, methionine adenosyltransferase and homocysteine ​​hydrolase; The starting strain is Escherichia coli in which the alcohol dehydrogenase, aldehyde-keto reductase and transcription factor yqhC genes are knocked out; the alcohol dehydrogenase includes genes yqhD, yahK, and yjgB, and the aldehyde-keto reductase includes genes dkgB, yeaE, and dkgA.

7. The method according to claim 6, characterized in that The addition amount of the recombinant cell M1a is 50-60 g / L; the addition amount of the recombinant cell M2a is 80-90 g / L; the addition amount of the recombinant cell M3a is 80-90 g / L; and the addition amount of the recombinant cell M4a is 70-80 g / L.

8. The method according to claim 6, characterized in that The reaction temperature is 20-40°C, and the reaction pH is 7.5-8.

5.

9. The method according to claim 6, characterized in that The structural formula of Rotundine or its derivative is shown in any one of (S)-8a to (S)-8g below:

10. Use of the oxygen methyltransferase mutant according to claim 1, or the biomaterial according to claim 2, or the recombinant Escherichia coli according to claim 3, or the whole cell catalyst according to any one of claims 4 to 5, or the method according to any one of claims 6 to 9 in the synthesis of Rotundine or its derivatives.

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