Method for synthesis of rotundine and derivative thereof, and construction of whole-cell catalytic system

Abstract

The present invention belongs to the technical field of bioengineering. Provided are a method for the synthesis of rotundine and a derivative thereof, and the construction of a whole-cell catalytic system. A method for multi-enzyme cascade synthesis of rotundine and a derivative thereof is artificially designed, and an O-methyltransferase and a berberine bridge enzyme involved in the reaction are mutated to improve the catalytic capacity of enzymes. Enzymes involved in the synthetic route are further respectively expressed in recombinant Escherichia coli to obtain a whole-cell catalyst comprising M1a, M2a, M3a and M4a, and the whole-cell catalyst is used to synthesize rotundine and a derivative thereof. The synthesized rotundine yield can be up to 2.44 g / L.

Description

A method for synthesizing Rotundine and its derivatives and the construction of a whole-cell catalytic system Technical Field

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

[0002] Alkaloids are a class of nitrogen-containing organic compounds widely found in nature, with over 27,000 structures reported to date. As important secondary metabolites, alkaloids are often known for their unique pharmacological activities. Examples include poppy alkaloids used for vasodilation in clinical surgery; morphine, a potent analgesic; berberine, with strong antibacterial activity; and colchicine, used to treat acute gout. The powerful pharmacological activity of alkaloids brings enormous market potential, but their complex stereochemical structures pose a challenge to chemical synthesis. Synthesis typically results in low yields and requires the intervention of heavy metal catalysts, which contradicts the principles of modern green chemistry. Therefore, the acquisition of most medicinal alkaloids still relies on plant extraction. However, plants have long growth cycles and complex compositions, making extraction often inefficient, time-consuming, and costly. Furthermore, some low-content secondary metabolites are difficult to obtain, significantly hindering the development and use of related drugs. With the rapid development of synthetic biology and to meet the huge market demand for medicinal alkaloids, expression systems from Escherichia coli, yeast, and tobacco have been widely used for the de novo synthesis of alkaloids. In 2011, researchers developed an engineered E. coli strain for the efficient synthesis of (S)-Reticuline, an important precursor of the benzylisoquinoline alkaloid. In 2015, Keasling's team successfully achieved the heterologous total synthesis of vinblastine in 31 steps through 56 gene edits in yeast. In 2021, with the discovery and reporting of the complete synthetic pathway of colchicine in plants, Sattely's team achieved the total synthesis of colchicine in tobacco using a transient expression system. However, due to the complex structure of alkaloids, the lengthy metabolic pathways, and the broad selectivity of the catalytic enzymes involved for alkaloid intermediates in the main metabolic pathway, problems such as uncontrollable metabolic flux and low yield of the target compound arise. Furthermore, most alkaloids are toxic, and high concentrations of the target product can cause cellular toxicity. Therefore, the yield of de novo alkaloid synthesis often cannot meet market demand. Therefore, strategies such as multi-enzyme cascade catalysis and resting cells have begun to receive attention.

[0003] Levo-tetrahydropalmatine, also known as levo-tetrahydropalmatine, is a tetrahydroberberine isoquinoline alkaloid with a single chiral center. It is typically extracted from plants such as *Corydalis yanhusuo* and *Corydalis*, and is a CFDA-approved monomeric analgesic alkaloid. Natural corydalis yanhusuo often exists in racemic form, with the levorotatory isoform, levo-tetrahydropalmatine (rotundine), considered the main component responsible for its pharmacological effects. In recent years, the pharmacological activities of rotundine have been extensively studied, particularly its analgesic, anti-addictive, anti-inflammatory, neuroprotective, and anticancer activities. These studies indicate that rotundine is a promising compound for treating dysmenorrhea, drug addiction, inflammatory diseases, neuropathic pain, cancer, cerebral edema, and acute global cerebral ischemia-reperfusion injury. Currently, the main source of levo-tetrahydropalmatine remains plant extraction, limited by its extremely low content in plants, resulting in an extraction efficiency of <0.4‰, and also suffering from problems such as long processing time and high cost. To improve the efficiency of rotundine acquisition, Tang Pei's team used a fully mixed-flow reactor in the original berberine skeleton synthesis, achieving highly efficient rotundine synthesis with end product ee values ​​all above 90% and yields around 65%. However, the use of the heavy metal catalyst Ir does not conform to the principles of green chemistry. To achieve more efficient and environmentally friendly rotundine synthesis, heterologous biosynthesis has become a focus of researchers. However, the natural synthetic pathway of rotundine in plants remains unknown, posing a challenge to the heterologous reconstruction of its natural pathway. Therefore, Smolke's team simultaneously reconstructed the natural biosynthetic pathway of (S)-Scoulerine, an important intermediate in berberine alkaloids, and the oxygen methyltransferase TfS9OMT discovered in *Hemiberlea spp.* into *Saccharomyces cerevisiae*. Based on the structural modification of TfS9OMT, dopamine feeding during fermentation, and condition optimization, they achieved the first biosynthesis of Rotundine, with a yield of 3.60 μg / L. In 2021, Liu Jihua and Yu Boyang's team, based on the expression optimization of the berberine brining enzyme BBE, achieved the active expression of the key enzyme BBE in *E. coli* by optimizing gene source, expression tag, and expression vector, increasing the Rotundine yield to 1.19 mg / L. Recently, Professor Huang Luqi's research group constructed engineered *Saccharomyces cerevisiae* through metabolic engineering, achieving a Rotundine yield of 68.6 mg / L, but the current biosynthetic yield remains at a low level. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a novel route for synthesizing Rotundine and its derivatives using readily available phenylacetic acid derivatives and dopamine as raw materials and through a multi-enzyme cascade reaction.

[0005] This invention provides an oxymethyltransferase mutant, wherein the oxymethyltransferase mutant uses the oxymethyltransferase with the amino acid sequence shown in SEQ ID NO.12 as the parent, and has one and / or more mutations in (a) to (c):

[0006] (a) Mutate the 16th serine residue of the parent to alanine, glycine, or leucine;

[0007] (b) Mutate the threonine at position 115 of the parental line to alanine, glycine, or leucine;

[0008] (c) Mutate the 301st proline in the parent to alanine, glycine, or leucine.

[0009] In one embodiment, the oxygen methyltransferase mutant uses the oxygen methyltransferase with the amino acid sequence shown in SEQ ID NO.12 as the parent.

[0010] The parental serine at position 16 was mutated to glycine, and the parental proline at position 301 was mutated to alanine; or

[0011] Mutate the 16th serine residue of the parental stem to glycine, and mutate the 301st proline residue of the parental stem to glycine; or

[0012] The parental serine at position 16 was mutated to glycine, and the threonine at position 115 was mutated to alanine; or

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

[0014] The present invention also provides a gene encoding the above-mentioned oxymethyltransferase mutant.

[0015] The present invention also provides an expression vector carrying the gene of the said oxymethyltransferase 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 described above.

[0018] In one embodiment, the recombinant cells are based on Escherichia coli BL21(DE3) as chassis cells and overexpress the O-methyltransferase mutant, methionine adenosine transferase (MAT), and homocysteine ​​hydrolase (SAHH).

[0019] In one embodiment, the amino acid sequence of the oxygen methyltransferase is as shown in SEQ ID NO.12, or the oxygen methyltransferase is a mutant of the above-mentioned nitrogen methyltransferase; the amino acid sequence of the methionine adenosine transferase is as shown in Genbank: AAN81976.1; and the amino acid sequence of the homocysteine ​​hydrolase is as shown in EDL06108.1.

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

[0021] In one embodiment, the knockout and construction method of Escherichia coli alcohol dehydrogenase (yqhD, yahK, yjgB), aldehyde-ketone reductase (dkgB, yeaE, dkgA) and transcription factor (yqhC) genes is described in the reference 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] This invention provides a whole-cell catalyst for the catalytic preparation of Rotundine and its derivatives, comprising recombinant cells containing (a) to (d):

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

[0024] (b) Recombinant cells M2a: Escherichia coli BL21(DE3) as chassis cells, overexpressed with oxygen methyltransferase (Ps6OMT), nitrogen methyltransferase (CNMT), methionine adenosine transferase (MAT) and homocysteine ​​hydrolase (SAHH).

[0025] (c) Recombinant cells M3a: Escherichia coli BL21(DE3) as chassis cells, overexpressing berberine briningase (EcBBE / EcBBE) R354W ), riboflavin synthase (RibH), riboflavin synthase (RibC), and bifunctional riboflavin kinase (RibF) and analysis companion Gro7;

[0026] (d) Recombinant cells M4a: Escherichia coli BL21(DE3) was used as the chassis cells and the O-methyltransferase mutant, methionine adenosine transferase (MAT) and homocysteine ​​hydrolase (SAHH) were overexpressed.

[0027] In one embodiment, the amino acid sequence of the carboxylic acid reductase (TpCAR) is shown in Genbank: WP_013126039.1; the amino acid sequence of the phosptophanyl thioethylamine transferase (BsSfp) is shown in Genbank: CP137756.1; and the amino acid sequence of the norcodonol alkaloid synthase (TfNCS) is 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 adenosine transferase is shown in Genbank accession number: AAN81976.1; and the amino acid sequence of the homocysteine ​​hydrolase is shown in Genbank: EDL06108.1.

[0029] In one embodiment, the berberine brining enzyme (EcBBE) has the amino acid sequence shown in (a) or (b):

[0030] (a) The amino acid sequence as shown in PDB ID: 3D2D_A;

[0031] (b) Based on (a), mutate arginine at position 354 to phenylalanine or tryptophan; or mutate tryptophan at position 165 to glycine; or mutate alanine at position 421 to phenylalanine or tryptophan.

[0032] In one embodiment, the amino acid sequence of the riboflavin synthase (RibH) is shown in Genbank: CAD6020726.1; the amino acid sequence of the riboflavin synthase (RibC) is shown in Genbank: CAD6006425.1; and the amino acid sequence of the bifunctional riboflavin kinase (RibF) is shown in Genbank: ADD75835.1.

[0033] In one embodiment, the amino acid sequence of the oxygen methyltransferase (T'fS9OMT) is shown as PDB ID: 6NEJ; the amino acid sequence of the methionine adenosine transferase is shown as Genbank accession number: AAN81976.1; and the amino acid sequence of the homocysteine ​​hydrolase is shown as Genbank: EDL06108.1.

[0034] The present invention also provides the application of the above-mentioned recombinant cells M1a to M4a in the synthesis of Rotundine or its derivatives.

[0035] In one embodiment, the application includes: mixing recombinant cells M1a with a substrate; the substrate includes a phenylacetic acid derivative and dopamine.

[0036] In one embodiment, the reaction system contains 20-30 mM ascorbate, preferably sodium ascorbate.

[0037] In one embodiment, the recombinant cells (M1a, M2a, M3a, or M4a) are prepared as follows: the strain is inoculated into LB medium and cultured at 20-40°C for 10-12 hours, then transferred to 2YT medium and cultured at 20-40°C until OD. 600 When the pH reaches approximately 0.6-0.8, cool the temperature to 15-25℃, add IPTG at a final concentration of 0.05-0.5mM to induce induction for 10-20 hours, and then collect the bacterial cells.

[0038] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 7.5-8.5.

[0039] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0040] In one embodiment, the amount of the phenylacetic acid derivative added is 10-20 mM; the amount of dopamine added is 20-30 mM.

[0041] In one embodiment, the application includes: using the supernatant of the recombinant cells M1a after reaction as a reaction system, and reacting the recombinant cells M2a in the reaction system; the reaction contains 20-30 mM ascorbate, 30-40 mM polyphosphate or ATP, and 30-40 mM L-methionine; the ascorbate is preferably sodium ascorbate; the polyphosphate is preferably a metal salt of ATP.

[0042] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 7.5-8.5.

[0043] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0044] In one embodiment, the amount of recombinant cell M1a added is 50-60 g / L; the amount of recombinant cell M2a added is 30-120 g / L, preferably 80-90 g / L.

[0045] In one embodiment, the application includes: using the supernatant of the recombinant cells M2a after reaction as a reaction system, and reacting the recombinant cells M3a in the reaction system.

[0046] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction 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] In one embodiment, the application includes: using the supernatant of the recombinant cells M3a after reaction as a reaction system, and reacting the recombinant cells M4a in the reaction system; the reaction system contains 30-40 mM polyphosphate or ATP, and 30-40 mM L-methionine, wherein the polyphosphate is preferably a metal salt of ATP.

[0049] In one embodiment, the reaction temperature is 20-40°C, and the pH of the reaction is 7.5-9.5.

[0050] In one embodiment, the reaction time is 6-16 hours, or the reaction time is not less than 6 hours.

[0051] The present invention also provides a method for synthesizing Rotundine or its derivatives by an in vitro enzyme cascade reaction using a one-pot, four-step process.

[0052] In one embodiment, the method uses an enzyme as a catalyst for catalysis and includes the following steps:

[0053] (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℃, the pH of the reaction system is 6-8, and the reaction time is 2-8h;

[0054] (2) Heat to deactivate the enzyme in step (1) and collect the supernatant;

[0055] (3) Add oxymethyltransferase, nitrogen methyltransferase and S-adenosylmethionine to the supernatant obtained in step (2), and the reaction temperature is 20-40℃ and the reaction time is 2-8h.

[0056] (4) Heat to deactivate the enzyme in step (3) and collect the supernatant;

[0057] (5) Add berberine brining enzyme and flavin adenine dinucleotide to the supernatant obtained in step (4), and the reaction temperature is 20-40℃ and the reaction time is 2-8h;

[0058] (6) Heat to deactivate the enzyme in step (5) and collect the supernatant;

[0059] (7) Add oxymethyltransferase and S-adenosylmethionine to the supernatant obtained in step (6), and the reaction temperature is 20-40℃ and the reaction time is 2-8h to obtain Rotundine or its derivatives.

[0060] In one embodiment, step (1) further includes 5-10 mM magnesium ions, 5-10 mM polyphosphate or ATP, and 1-3 mM NADP. + 1-20mM glucose, 1-5mM ascorbic acid or ascorbate.

[0061] In one embodiment, the concentrations of the substances in the system of step (1) are as follows: 5-10 μM carboxyl reductase, 5-10 μM norcodonine synthase, 5-10 μM glucose dehydrogenase, 1-10 mM substrate dopamine, and 1-10 mM phenylacetic acid derivative.

[0062] In one embodiment, 5-10 μM oxymethyltransferase, 5-10 μM nitrogen methyltransferase and 5-10 mM S-adenosylmethionine were added in step (3).

[0063] In one embodiment, 5-10 μM berberine brining enzyme and 5-10 mM flavin adenine dinucleotide were added in step (5).

[0064] In one embodiment, 5-10 μM oxymethyltransferase and 5-10 mM S-adenosylmethionine are added in step (7).

[0065] In one embodiment, the method uses the recombinant cells M1a to M4a as a catalyst to carry out the catalytic reaction.

[0066] In one embodiment, the structural formula of the phenylacetic acid derivative is shown below:

[0067] In one embodiment, the structural formula of the phenylacetic acid or its derivative includes at least one of 1a-1g:

[0068] In one embodiment, the structural formula of the Rotundine is:

[0069] In one embodiment, the structural formula of the Rotundine or its derivatives is shown in any of the following (S)-8a to (S)-8g:

[0070] The present invention also provides the use of the mutant, or the recombinant cell, or the method in the preparation of Rotundine or its derivatives. Beneficial effects:

[0071] (1) The present invention provides an oxymethyltransferase mutant. The mutant S16G / P301A has an enzyme activity that is 19 times higher than that of the wild-type enzyme; the mutant S16G has an enzyme activity that is 11 times higher than that of the wild-type enzyme; and the mutant P301A has an enzyme activity that is 13 times higher than that of the wild-type enzyme.

[0072] (2) The present invention provides a recombinant strain M1a, which can efficiently convert substrates dopamine and phenylacetic acid derivatives into a b-benzylisoquinoline skeleton with a conversion rate of 95% and an ee value >99%.

[0073] (3) The present invention provides a recombinant strain M2a, which can synthesize S-adenosylmethionine using L-methionine and ATP, and can also achieve methylation at the 6th hydroxyl group and imino group of the substrate (S)-3a-(S)-3g to generate (S)-5a-(S)-5g.

[0074] (4) The present invention provides a recombinant strain M3a, which expresses a berberine brining enzyme mutant with enhanced catalytic ability and the molecular chaperone Gro7, which can increase the production of intracellular FAD and achieve carbon-oxidative coupling on the substrate (S)-5a-(S)-5g.

[0075] (5) The present invention provides a recombinant strain M4a expressing the oxygen methyltransferase mutant, which can synthesize S-adenosylmethionine using L-methionine and ATP, and can achieve methylation at the 2 and 9 hydroxyl positions of the substrate (S)-6a-(S)-6g.

[0076] (6) The present invention also provides a method for synthesizing Rotundine or its derivatives using phenylacetic acid derivatives and dopamine as raw materials. Under the catalysis of the recombinant strains M1a to M4a, the yield of Rotundine is 2.44 g / L and the yield of (S)-8b is 1.01 g / L. Attached Figure Description

[0077] Figure 1 shows the synthetic routes of Rotundine and its derivatives.

[0078] Figure 2 shows the modified TfS9OMT. M Efficient synthesis of Rotundine. TfS9OMT M The relative activity of the mutant to substrate (S)-6a was set to 1, and the catalytic activity of the parent to substrate (S)-6a was set to 1.

[0079] Figure 3 shows the standard curve of the product Rotundine.

[0080] Figure 4 shows the standard curve of product (S)-8f.

[0081] Figure 5 shows the HPLC identification of in vitro multi-enzyme cascade catalysis.

[0082] Figure 6 shows the HPLC and MS identification of (S)-3a synthesized by the recombinant strain TpCAR-BsSfp-TfNCS (strain M1a).

[0083] Figure 7 shows the HPLC and MS identification of (S)-5a synthesized by the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT (strain M2a).

[0084] Figure 8 shows the HPLC and MS identification of (S)-6a synthesized by the recombinant strain EcBBE#-RibH-RibC-RibF;Gro7 (strain M3a).

[0085] Figure 9 shows the HPLC and MS identification of (S)-8a synthesized by the recombinant strain TfS9OMT#-MmSAHH-EcMAT (strain M4a).

[0086] Figure 10 shows the substrate spectrum expansion using four recombinant strains M1a, M2a, M3a, and M4a.

[0087] Figure 11 shows the H spectrum of Rotundine in deuterated chloroform.

[0088] Figure 12 shows the time curves for the synthesis of Rotundine using strains M1a, M2a, M3a, and M4a. Detailed Implementation

[0089] 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.

[0090] (1) Reagents and materials

[0091] Sources of reagents and materials: The antibiotics ampicillin sodium, kanamycin sulfate, streptomycin sulfate, chloramphenicol, etc. used in this invention are all from Shanghai Sangon Biotech; the cofactors ATP sodium salt, NADPH, SAM, etc. used in this invention are all from Saen Chemical Technology (Shanghai) Co., Ltd.; the PCR enzymes and homologous recombinases 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.

[0092] (2) Culture medium

[0093] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0094] 2YT medium: tryptone 16g / L, yeast extract 10g / L, sodium chloride 5g / L.

[0095] (3) Detection method

[0096] Enzyme activity assay of TfS9OMT and its mutants: A 200 μL reaction system consisted of 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 entire system was run in a shaker at 37 °C for 4 h. After the reaction, 4 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20000 g for 5 min. 200 μL of the sample was loaded onto a chromatographic column, and the yield of each mutant was analyzed based on the standard curve of product (S)-8a (Figure 3).

[0097] 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. -1 The linear gradient is as follows: first, elute isocratically with 5% acetonitrile for 1 min, then elute with a gradient of 5%-50% acetonitrile for 25 min.

[0098] Berberine-bridged enzyme (EcBBE) activity assay: A 200 μL reaction system consisted of 50 mM Tris-HCl buffer (pH 9.0), 5 μM EcBBE enzyme or mutant, 2 mM (S)-Reticuline, and 10 mM sodium ascorbate. The entire system was run in a shaker at 30 °C for 4 h. After the reaction, 4 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20,000 g for 5 min. 200 μL of the sample was then loaded onto the chromatographic column.

[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. -1 The linear gradient was as follows: first, elution was performed isocratically with 5% acetonitrile for 1 min, followed by elution with a gradient of 5%–50% acetonitrile for 25 min. The yield of each mutant was analyzed based on the standard curve of product (S)-8f (Figure 4).

[0100] (4) 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℃, 10000 rpm for 30 min) to obtain crude enzyme solution. The solution was desalted by nickel affinity chromatography and desalting column (Histrap™ 5 mL Desalting) to finally obtain the purified protein.

[0101] (5) Strains and sequences

[0102] Table 1. Strains constructed in some embodiments of the present invention. Note: Strain IAA was published in the paper "Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids".

[0103] Table 2 Primer sequences for constructing recombinant strains

[0104] Example 1: Design of Artificial Cascade Reaction Pathways

[0105] The reaction route for the synthesis of Rotundine and its derivatives is established as shown in Figure 1. The first step uses 3-hydroxy,4-methoxyphenylacetic acid (1) as a model substrate, and involves the activation of carboxylic acid reductase (TpCAR) by phosphate pantothenate thioethylamine transferase (BsSfp), followed by the reaction of magnesium ions (Mg...). 2+ The intermediate (S) is reduced to the corresponding aldehyde (1a) under the action of cofactors ATP and NADPH. Then, under the action of norcodonol synthase (TfNCS), the aldehyde condenses with another substrate, dopamine, to form the skeleton of phenethyl isoquinoline (S)-1-(3-hydroxy-4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol ((S)-3a). In the second step, intermediate (S)-3a is converted to (S)-5a under the action of oxygen methyltransferase (Ps6OMT) and nitrogen methyltransferase (CNMT) and cofactor SAM. In the third step, intermediate (S)-5a is converted to the final product (S)-6a under the action of berberine bridging enzyme (EcBBE) and cofactor FAD. In the fourth step, intermediate (S)-6a is converted to the final product (S)-8a (Rotundine) under the action of oxygen methyltransferase (TfS9OMT) and cofactor SAM. The cofactor SAM is synthesized by methionine adenosine transferase (EcMAT), and the product SAH after SAM demethylation is hydrolyzed by homocysteine ​​hydrolase (MmSAHH).

[0106] The specific steps are as follows:

[0107] (1): Add 5-10 μM carboxyl reductase, 5-10 μM norepinephrine synthase, 5-10 μM glucose dehydrogenase, 5-10 mM magnesium chloride, 5-10 mM ATP, and 1-3 mM NADP to a 1.5 ml EP tube. +The reaction mixture consisted of 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(hydroxymethyl)aminomethane (Tris) buffer at pH 8.0, with a final reaction volume of 200 μL.

[0108] (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.

[0109] (3) Second step reaction: Add 5-10 μM oxymethyltransferase, 5-10 μM nitrogen methyltransferase and 5-10 mM S-adenosylmethionine (SAM, pH 8.0) to the supernatant of step (2) above.

[0110] (4) Place the reaction tube from step (3) 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.

[0111] (5) Third step reaction: Add 5-10 μM berberine brining 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.

[0112] (6) Place the reaction tube from step (5) 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.

[0113] (7) Fourth step reaction: Add 5-10 μM oxymethyltransferase and 5-10 mM S-adenosylmethionine (SAM, pH 8.0) to the supernatant of step (6) above. The reaction system was placed in a shaking reactor at 30°C and 800 rpm for 4 h.

[0114] (8) After the reaction is complete, add 4 times the volume of methanol 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.

[0115] Example 2: Modification of key rate-limiting enzymes in artificial cascade reaction pathways

[0116] (1) Oxymethyltransferase TfS9OMT M Transformation

[0117] To increase the yield of product (S)-8a, the rate-limiting enzyme TfS9OMT (nucleotide sequence shown in SEQ ID NO.9) in the cascade reaction pathway was modified. First, substrate (S)-7a was docked to TfS9OMT using molecular docking software (Discovery Studio). M (In the substrate pocket of a protein with a mutant N191D / F205S / M111L on the starting sequence of Thalictrum flavum) Next, the D loop of substrate (S)-7a was selected. Mutate all amino acids within the specified range to alanine, glycine, or leucine. TfS9OMT M The primers used for mutant design are shown in Table 3. Using the gene shown in SEQ ID NO.13 as the starting sequence, the primers for introducing the mutation are shown in Table 3. The above mutant was expressed in *E. coli* BL21(DE3) using pET21b(+) as the expression vector. Recombinant *E. coli* expressing the mutant were cultured, and the bacterial cells were collected, lysed, and purified to obtain the mutant protein.

[0118] The relative activity of different mutants was expressed as the relative yield of product (S)-8a (Rotundine). The specific steps were as follows: 5-10 μM oxymethyltransferase and 5-10 mM S-adenosylmethionine were added to a reaction system containing 2 mM (S)-6a, and the reaction was carried out in a shaking reactor at 30 °C and 800 rpm for 4 h. The content of product (S)-8a was then determined.

[0119] As shown in Figure 2, four single mutants with high activity, TfS9OMT, were obtained. M -S16G,TfS9OMT M -T115A,TfS9OMT M -P301G and TfS9OMT M -P301A. Secondly, a semi-rational design and combinatorial mutation strategy was employed to obtain the optimal mutant TfS9OMT. M -S16G / P301A. Compared to wild-type TfS9OMT M mutant TfS9OMT M -S16G / P301A activity increased by 19 times.

[0120] Table 3 Primer sequences for CNMT mutants

[0121] Table 4. N-methyltransferase TfS9OMT M Mutant relative enzyme activity Note: Relative activity = mutant enzyme activity ÷ parent enzyme activity × 100%.

[0122] The mutant 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 The mutant TfS9OMT M -S16G / P301A, the result is shown in Figure 5.

[0123] (2) Modification of berberine brining enzyme BBE

[0124] Based on the previously reported complex structures containing substrate (S)-Reticuline and BBE protein, substrate (S)-Reticuline was selected. The amino acids within the target range were modified. Using the EcBBE gene shown in SEQ ID NO.16 as the starting sequence, this sequence was ligated into the vector pCDFDuet-1. Mutations were introduced using primers shown in Table 1 and Table 5. PCR was performed using the upstream and downstream primers corresponding to the mutation points, followed by ligation to construct a vector containing the target mutation point. This vector was then transformed into *E. coli* BL21 to construct a recombinant strain.

[0125] The recombinant bacteria expressing the mutant were cultured at 37°C for 2 h, followed by induction at 16°C for 12 h. Cells were collected and resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH 8). The resuspended cells were then homogenized using a high-pressure homogenizer. The homogenized resuspended cells were centrifuged at low temperature and high speed (4°C, 10,000 rpm for 30 min) to obtain the crude enzyme solution. The solution was then desalted using nickel affinity chromatography and a Histrap™ 5 mL desalting column to obtain the purified protein.

[0126] The enzyme protein was used to catalyze the preparation of (S)-Scolerine. The reaction system consisted of 50 mM Tris-HCl buffer (pH 9.0), 5 μM EcBBE mutant, 2 mM (S)-Reticuline, and 10 mM sodium ascorbate. The entire reaction was carried out in a shaker at 30 °C for 12 h. After the reaction, 4 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20,000 g for 5 min. 200 μL of the sample was then loaded onto a chromatographic column. The yield of each mutant was analyzed based on the standard curve of the (S)-Scolerine product (Figure 3). Using the starting sequence as a control, the relative activity of different mutants was represented by the relative yield of the (S)-Scolerine product after the reaction.

[0127] Table 5 Primer sequences of EcBBE mutants

[0128] As shown in Figure 2, a highly active single mutant, EcBBE, was obtained. R354W Compared to wild-type EcBBE, mutant EcBBE R354W The activity was increased by 132 times.

[0129] Table 6. Relative enzyme activities of the EcBBE mutant of N-methyltransferase.

[0130] Finally, the in vitro reaction described in Example 1 was passed (the difference being that the berberine briningase EcBBE was replaced with the mutant EcBBE). R354W The feasibility of efficiently synthesizing (S)-Scolerine was verified in vitro.

[0131] Example 4: Construction, protein expression, and product identification of engineered strain TpCAR-BsSfp-TfNCS(M1a)

[0132] The target gene CAR (Genbank accession number WP_013126039.1) derived from *Tsukamurella paurometabola* was codon-optimized to obtain the gene sequence shown in SEQ ID NO.1. This gene was synthesized and ligated into the first multiple cloning site (MCS1) of the pRSFDuet vector to obtain the plasmid pRSFDuet-TpCAR.

[0133] 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.

[0134] 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. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-TfNCS.

[0135] After obtaining the three plasmids mentioned above, the gene fragments Sfp and NCS were integrated into MCS1 and MCS2 of pRSFDuet-TpCAR, respectively, using 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 (disclosed in the paper "Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids") to obtain the recombinant strain TpCAR-BsSfp-TfNCS(M1a). The primers used for plasmid construction are shown in Table 3.

[0136] Single-clone strain TpCAR-BsSfp-TfNCS(M1a) 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 2YT medium and cultured at 220 rpm and 37°C. When the bacterial OD... 600 When the pH reaches approximately 0.6-0.8, cool the temperature to 18℃ and incubate for 30 minutes. Then add IPTG to a final concentration of 0.2 mM and induce culture at 18℃ for 16 hours. Collect the bacterial cells after centrifugation. Wash the bacterial cells three times with a buffer containing 100 mM Tris (pH 8.0) and 200 mM NaCl, and centrifuge to obtain the bacterial cells.

[0137] Subsequently, the synthesis of product (S)-3a was verified using a 5 ml reaction system. The reaction system consisted of 100 mM Tris buffer (pH 8.0), 200 mM NaCl, and a final bacterial cell concentration of OD0.0. 600 The reaction mixture consisted of 15 and 20 mM MgCl2, 20 mM glucose, 5% DMSO, 13 mM substrate 1a, 18.2 mM dopamine, and 30 mM sodium ascorbate. The reaction was carried out in a shaker at 30 °C. After 6 h, a 200 μL sample was taken and quenched with 800 μL of methanol. The sample was centrifuged at 20,000 g for 5 min and filtered through a 0.22 μm filter membrane.

[0138] The synthesis of product (S)-3a was detected using an HPLC system. As shown in Figure 6, the compound obtained by whole-cell catalysis had the same elution time as the standard, and the formation 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%.

[0139] Example 4: Construction, protein expression, and product identification of engineered strain Ps6OMT-MmSAHH-CNMT-EcMAT(M2a)

[0140] The target gene 6OMT (Genbank accession number: NP_001413547.1) derived from poppy *Papaver somniferum* was codon-optimized to obtain the gene sequence shown in SEQ ID NO.4. This gene was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-Ps6OMT.

[0141] 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. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-CNMT.

[0142] The MAT gene fragment (Genbank accession number: AAN81976.1) was amplified from the Escherichia coli genome and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcMAT.

[0143] The target gene SAHH from house mouse Mus musculus was codon optimized to obtain the gene sequence shown in SEQ ID NO.6. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-MmSAHH.

[0144] After obtaining the above four plasmids, the gene fragments CNMT, EcMAT, Ps6OMT, and MmSAHH were integrated into two MCSs of the pCDFDuet plasmid using homologous recombination technology, resulting in the plasmids MmSAHH--EcMATpCDFDuet-T7-rbs-Ps6OMT-rbs and MmSAHH-T7-rbs-CNMT-EcMAT. The recombinant plasmids were then transformed into the engineered strain BL21 to obtain the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT (M2a).

[0145] The recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT(M2a) was cultured using the same method as in Example 3, and bacterial cells were obtained by centrifugation. Subsequently, the recombinant strain Ps6OMT-MmSAHH-CNMT-EcMAT was resuspended in the supernatant after whole-cell reaction of the recombinant strain TpCAR-BsSfp-TfNCS in Example 3, and its final bacterial concentration (OD) was determined. 600 The concentration was 21, and 40 mM L-Met and 40 mM ATP were added to the resuspension at a final concentration. The reaction was carried out at 30 °C and 250 rpm for 15 h. The product (S)-5a was detected using the same sample post-processing method and liquid chromatography detection conditions as in Example 3. As shown in Figure 7, the compound obtained by whole-cell catalysis had the same peak time as the standard, and the formation 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%.

[0146] Example 5: Construction, protein expression, and product identification of engineered strain EcBBE#-RibH-RibC-RibF; Gro7(M3a).

[0147] Referring to the method in Example 2, the EcBBE code was synthesized. R354W The gene sequence was obtained and ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcBBE. R354W .

[0148] The RibH gene fragment (Genbank: CAD6020726.1) was amplified from the Escherichia coli genome and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibH.

[0149] The RibC gene fragment (Genbank: CAD6006425.1) was amplified from the Escherichia coli genome and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibC.

[0150] The RibF gene fragment (Genbank: ADD75835.1) was amplified from the Escherichia coli genome and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b(+)-RibF.

[0151] Shorten EcBBE R354WThe 22 amino acids at the N-terminus were fused with an MBP tag (the sequence encoding MBP is shown in SEQ ID NO. 15), and then ligated into the first multiple cloning site (MCS) of the pCDFDuet vector using homologous recombination technology. The promoter T7 at the first multiple cloning site was replaced with the E. coli endogenous promoter ssrA (the ssrA sequence is shown in SEQ ID NO. 14), resulting in the plasmid pCDFDuet-TssrA-MBP-EcBBE. R354W (NΔ22AA), MBP-EcBBE R354W (NΔ22AA) is named EcBBE#.

[0152] After obtaining the above plasmid, the gene fragment RibC was integrated into pCDFDuet-TssrA-MBP-EcBBE using homologous recombination technology. R354W The MCS1 site of (NΔ22AA) was used to integrate RibF and RibH into pCDFDuet-TssrA-MBP-EcBBE. R354W The plasmid pCDFDuet-TssrA-rbs-EcBBE#-rbs-RibC-T7-rbs-RibH-rbs-RibF (where the rbs sequence is AAGGAG) was obtained by targeting the MCS2 site of (NΔ22AA). The recombinant plasmid pCDFDuet-TssrA-rbs-EcBBE#-rbs-RibC-T7-rbs-RibH-rbs-RibF was co-transformed with the molecular chaperone Gro7 into the engineered strain BL21 to obtain the recombinant strain EcBBE#-RibH-RibC-RibF (M3a).

[0153] The recombinant strain EcBBE#-RibH-RibC-RibF;Gro7(M3a) was cultured using the same method 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 this supernatant to achieve a final cell concentration of OD200. 600 The reaction was carried out at 21-30°C and 250 rpm for 9 hours. The product (S)-1d was detected using the same sample post-processing method and liquid chromatography detection conditions as in Example 3, and the formation of the product was further confirmed by high-resolution mass spectrometry, as shown in Figure 8. The yield was 3.19 g / L, and the yield was 75.1%.

[0154] Example 6: Construction, protein expression, and product identification of engineered strain TfS9OMT#-MmSAHH-EcMAT

[0155] TfS9OMT was constructed according to the method in Example 2. M-S16G / P301A (Named TfS9OMT#), the gene encoding the mutant (shown in SEQ ID NO.9) was ligated to the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b(+)-TfS9OMT#.

[0156] The MAT gene fragment (Genbank accession number: AAN81976.1) was amplified from the Escherichia coli genome and ligated into the pET-21b(+) vector to obtain the plasmid pET-21b(+)-EcMAT.

[0157] The target gene SAHH from house mouse Mus musculus was codon optimized to obtain the gene sequence shown in SEQ ID NO.7. The gene was synthesized and ligated into the NdeI and XhoI sites of the pET-21b(+) vector to obtain the plasmid pET-21b-MmSAHH.

[0158] The gene fragments TfS9OMT#, EcMAT, and MmSAHH were integrated into two MCSs of the pETDuet plasmid using homologous recombination technology to obtain the plasmid pETDuet-T7-rbs-TfS9OMT#-T7-rbs-MmSAHH-rbs-EcMAT. This recombinant plasmid was then transformed into the engineered strain BL21 to obtain the recombinant strain TfS9OMT#-MmSAHH-EcMAT(M4a).

[0159] The recombinant strain TfS9OMT#-MmSAHH-EcMAT(M4a) was cultured according to the method in Example 3, and the cells were obtained by centrifugation. Subsequently, the supernatant from the whole-cell reaction of the recombinant strain EcBBE#-RibH-RibC-RibF;Gro7(M3a) obtained in Example 5 was adjusted to pH 8.5, and the recombinant strain TfS9OMT#-MmSAHH-EcMAT(M4a) was resuspended in the supernatant of the pH 8.5 reaction solution to achieve a final cell concentration OD0.05. 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-processing method and liquid chromatography detection conditions as in Example 3, and the formation of the product was further confirmed by high-resolution mass spectrometry, as shown in Figure 9. The yield was 2.44 g / L, and the yield was 52.8%.

[0160] Example 7: One-pot, four-step preparation of Rotundine

[0161] Recombinant plasmids expressing each enzyme using pET-21b(+) were constructed according to the methods described in Examples 3-6. These plasmids were then transformed into *E. coli* BL21(DE3). The resulting recombinant cells were resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH = 8), and the resuspended cells were then homogenized using a high-pressure homogenizer. The homogenized resuspended cells were centrifuged at low temperature and high speed (4°C, 10000 rpm for 30 min) to obtain a crude enzyme solution. The solution was desalted using nickel affinity chromatography and a Histrap™ 5 mL desalting column to obtain the purified protein. The enzyme protein was collected and used to prepare Rotundine according to the method described in Example 1.

[0162] The results are shown in Figure 5. In the experimental group, new peaks appeared in the reaction solution at each step, and the positions of these new peaks were consistent with those of the intermediate standard. Furthermore, the final one-pot, four-step reaction solution showed a new peak at the same position as the standard, which was preliminarily identified as the product (S)-8a (Rotundine). Mass spectrometry and NMR analysis confirmed that the final product was (S)-8a (Rotundine). Simultaneously, the in vitro reaction results demonstrated that this one-pot, four-step method can achieve complete conversion of a 2 mM substrate.

[0163] Example 8: Substrate Spectrum Expansion

[0164] Using a one-pot, four-step reaction method, phenylacetic acid and its derivatives (ag) containing different substituents and dopamine were added to a whole-cell catalytic system to expand the substrate spectrum and prepare any of the products shown below (S)-8a to (S)-8g:

[0165] The reaction conditions for the one-pot, four-step process are as follows:

[0166] (a) Recombinant bacterial cells of strains M1a, M2a, M3a and M4a were prepared according to the methods in Examples 3 to 6.

[0167] (b) Prepare a 5 ml reaction system for substrate transformation. The reaction system consists of: 100 mM Tris buffer (pH 8.0), and a final concentration of M1a cells (OD). 600 The reaction mixture consisted of 15 and 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 a shaker at 30 °C for 6 h. After 6 h, the supernatant was collected by centrifugation at 7000 rpm for 10 min and used for the next reaction.

[0168] (c) Resuspend the bacterial cells M2a in the supernatant obtained by centrifugation in step (b) to achieve a final bacterial cell concentration OD.600 The pH was set to 21, and 20 mM L-Met and 20 mM ATP (pH 7.5) were added to the resuspension. The mixture was reacted at 30°C and 250 rpm for 15 h. After 15 h, the supernatant was collected by centrifugation at 7000 rpm for 10 min, and the pH of the supernatant was adjusted to 9.0 with 5 M NaOH for use in the next reaction.

[0169] (d) Resuspend the bacterial cells M3a in the supernatant obtained by centrifugation in step (c) to achieve a final bacterial cell concentration OD. 600 The reaction was carried out at 21-30℃ and 250 rpm for 9 hours. After 9 hours, the supernatant was collected by centrifugation at 7000 rpm for 10 minutes, and the pH of the supernatant was adjusted to 8.5 with 5M NaOH for use in the next reaction.

[0170] (e) Resuspend the bacterial cells M4a in the supernatant obtained in step (d) to achieve a final bacterial cell concentration OD. 600 The concentration was 21, and 20 mM L-Met and 20 mM ATP (pH 7.5) were added to the above resuspension. The mixture was reacted at 40 °C and 250 rpm for 11 h. After 11 h, HPLC detection was performed, and (S)-8b-(S)-8g was qualitatively analyzed by LC-MS.

[0171] The results showed that all substrates (ag in Figure 10) could be converted into the corresponding product (S)-8b-(S)-8g. Among them, the yield of product (S)-8b obtained by whole-cell catalysis was 1.01 g / L.

[0172] 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. An oxygen methyltransferase mutant, wherein the oxygen methyltransferase mutant uses the oxygen methyltransferase with the amino acid sequence shown in SEQ ID NO. 12 as the parent, and has one and / or more mutations in (a) to (c): (a) Mutate the 16th serine residue of the parent to alanine, glycine or leucine; (b) Mutate the threonine at position 115 of the parental line to alanine, glycine, or leucine; (c) Mutate the 301st proline in the parent to alanine, glycine or leucine.

2. A biomaterial, characterized in that, For (a) to (c): (a) The gene encoding the oxymethyltransferase mutant of claim 1; (b) A vector carrying the gene described in (a); (c) Expressing the mutant of claim 1, or the gene of (a), or a recombinant cell containing the vector of (b).

3. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the chassis cell, the oxymethyltransferase mutant, methionine adenosyltransferase, and homocysteine ​​hydrolase described in claim 1 were 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 EDL06108.

1.

4. A whole-cell catalyst, characterized in that, Containing the recombinant Escherichia coli of claim 3, and the recombinant cells of (a) to (c): (a) Recombinant cells M1a: Based on the starting strain, the following proteins were expressed: carboxyl reductase, phosphoproteopantolate thioethylamine transferase, and norcodonine synthase; the starting strain was IAA; (b) Recombinant cells M2a: Escherichia coli BL21(DE3) as chassis cells, overexpressed with oxymethyltransferase, nitrogen methyltransferase, methionine adenosine transferase and homocysteine ​​hydrolase; (c) Recombinant cells M3a: Escherichia coli BL21(DE3) as chassis cells, overexpressing berberine briningase, riboflavin synthase, riboflavin synthase and bifunctional riboflavin kinase and analysis companion.

5. The whole-cell catalyst according to claim 4, characterized in that, The berberine brining enzyme has the amino acid sequence shown in (a) or (b): (a) The amino acid sequence as shown in PDB ID: 3D2D_A; (b) Based on (a), mutate arginine at position 354 to phenylalanine or tryptophan; or mutate tryptophan at position 165 to glycine; or mutate alanine at position 421 to phenylalanine or tryptophan.

6. The whole-cell catalyst according to claim 4 or 5, characterized in that, The recombinant cells were prepared as follows: the recombinant cells were cultured in LB medium at 20-40℃ for 10-12 hours, then transferred to 2YT medium and cultured at 20-40℃ until OD. 600 When the pH reaches 0.6-0.8, cool the temperature to 15-25℃ and induce with IPTG at a final concentration of 0.05-0.5mM for 10-20 hours, then collect the bacterial cells.

7. A method for synthesizing Rotundine or its derivatives, characterized in that, Using phenylacetic acid or phenylacetic acid derivatives as substrates, a catalytic reaction is carried out using the whole-cell catalyst or a combination of enzymes as described in any one of claims 4 to 6; the combination of enzymes includes carboxyl reductase, norcodonine synthase, glucose dehydrogenase, oxymethyltransferase, nitrogen methyltransferase and berberine bridging enzyme.

8. The method according to claim 7, characterized in that, The catalytic reaction using the whole-cell catalyst according to any one of claims 4 to 6 includes the following steps: (1) Recombinant cells M1a are mixed with a substrate and reacted, and the reaction supernatant is collected; the substrate includes phenylacetic acid or a phenylacetic acid derivative, and dopamine; (2) Mix the recombinant cells M2a with the supernatant obtained in step (1), react them, and collect the reaction supernatant; (3) Mix the recombinant cells M3a with the supernatant obtained in step (2), react, and collect the reaction supernatant; (4) Mix the recombinant cells M4a with the supernatant obtained in step (3), react them, and collect the reaction supernatant; The recombinant cell M1a expressed the following enzymes based on the starting strain: carboxyl reductase, phosphoproteopantothenate thioethylamine transferase, and norcodonine synthase. The recombinant cell M2a was based on Escherichia coli BL21(DE3) and expressed the following enzymes: oxymethyltransferase, nitrogen methyltransferase, methionine adenosine transferase, and homocysteine ​​hydrolase. The recombinant cell M3a was based on Escherichia coli BL21(DE3) and expressed the following: berberine brining enzyme, riboflavin synthase RibC, riboflavin synthase RibF, bifunctional riboflavin kinase RibH, and molecular chaperone Gro7. The recombinant cell M4a is based on Escherichia coli BL21(DE3) expressing: the oxygen methyltransferase mutant of claim 1, methionine adenosine transferase and homocysteine ​​hydrolase; The starting strain is an *E. coli* strain in which alcohol dehydrogenase, aldehyde-ketone reductase, and transcription factor yqhC genes have been knocked out; the alcohol dehydrogenase includes genes yqhD, yahK, and yjgB, and the aldehyde-ketone reductase includes genes dkgB, yeaE, and dkgA.

9. The method according to claim 8, characterized in that, The amount of recombinant cells M1a added is 50-60 g / L; the amount of recombinant cells M2a added is 80-90 g / L; the amount of recombinant cells M3a added is 80-90 g / L; and the amount of recombinant cells M4a added is 70-80 g / L.

10. The method according to claim 9, characterized in that, The reaction temperature is 20-40℃, and the pH of the reaction is 7.5-8.

5.

11. The method according to claim 7, characterized in that, Includes the following steps: (1) Carboxylic acid reductase, norepinephrine synthase and glucose dehydrogenase were reacted in a reaction system containing dopamine and phenylacetic acid or its derivatives at 20-40℃ and pH 6-8 for 2-8 hours; after the reaction was completed, the enzymes were inactivated. (2) Add oxymethyltransferase, nitrogen methyltransferase and S-adenosylmethionine to the supernatant obtained in step (1) and react at 20-40℃ for 2-8 h; after the reaction is completed, the enzymes are inactivated. (3) Add berberine brining enzyme and flavin adenine dinucleotide to the supernatant obtained in step (2) and react at 20-40℃ for 2-8 hours; after the reaction is completed, inactivate the enzyme. (4) Add oxymethyltransferase and S-adenosylmethionine to the supernatant obtained in step (3) and react at 20-40℃ for 2-8 hours to obtain Rotundine or its derivatives.

12. The method according to any one of claims 7 to 11, characterized in that, The structural formula of the Rotundine or its derivatives is shown in any one of (S)-8a to (S)-8g:

13. The use of the oxymethyltransferase mutant of claim 1, or the biomaterial of claim 2, or the recombinant Escherichia coli of claim 3, or the whole-cell catalyst of any one of claims 4 to 6, or the method of any one of claims 7 to 12 in the synthesis of Rotundine or its derivatives.

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