A system and method for the synthesis of adrenaline and its analogues by a multi-enzyme-level conjugation

Abstract

This invention provides a system and method for the multi-enzyme-based synthesis of adrenaline and its analogues. The system includes a norenaline synthesis module and several alkylation modules. Each norenaline synthesis module includes several first flow reactors and a norenaline synthase encapsulated within each flow reactor. Each alkylation module includes an alkylation cofactor reservoir, several alkylation flow reactors, and an alkylating enzyme encapsulated within each alkylation flow reactor. The alkylation cofactor reservoir and the first flow reactors are simultaneously connected to the alkylation flow reactors. This invention promotes forward reaction by modularizing the synthesis pathway of adrenaline and its analogues, reducing inter-component inhibition and improving the problem of incompatible reaction conditions. Combined with an immobilized enzyme continuous flow reactor, the enzymatic synthesis of adrenaline achieves high conversion rate, high optical purity, and high space-time yield.

Description

Technical Field

[0002] This invention belongs to the field of drug synthesis technology, specifically relating to a system and method for the synthesis of adrenaline and its analogues by multi-enzyme conjugation. Background Technology

[0003] Adrenaline is a hormone secreted by the adrenal glands. When people face stress or danger, the body triggers a series of stress responses under the influence of adrenaline. It is commonly used in emergency care, treatment of cardiac arrest, and bronchial asthma, and has shown good clinical efficacy. Compared to other commercially available anti-anaphylactic shock drugs, adrenaline has advantages such as rapid onset of action, fewer side effects, and improved survival rates. In recent years, with its increasing market share, it has gradually become the first-line drug for clinical emergency care. Secondly, due to the different physiological activities exhibited by different nitrogen substituents in adrenaline, norepinephrine, epinephrine, and isoproterenol are currently used clinically. Although all three are adrenaline-like drugs, they differ significantly in their receptors. Norepinephrine is an α-receptor agonist with a weak effect on cardiac β-receptors, primarily used for the contraction of smooth muscle in the skin and mucous membranes. Epinephrine acts on both α and β receptors, causing vasoconstriction and also affecting the myocardium and sinoatrial node, thus being the most widely used in emergency care. Isoproterenol, on the other hand, is a typical β-receptor agonist, with a stronger effect on the myocardium and liver compared to epinephrine. Therefore, the development of adrenaline analogs with different substituents is of great significance for clinical treatment.

[0004] Because adrenaline possesses a chiral center, it exists in two enantiomers: (R)-adrenaline and (S)-adrenaline. However, only (R)-adrenaline exhibits physiological activity. Therefore, chiral resolution has become a key focus in adrenaline synthesis. Currently reported methods for adrenaline synthesis, including patents CN113735720A and CN118206457, utilize large amounts of organic reagents, metal catalysts, or hydrogen to achieve high optical purity, resulting in significant resource waste and increased labor costs, contradicting the current emphasis on green chemistry. In recent years, with the development of genetic engineering, protein modification, and computer technology, multi-enzyme catalysis has shown great potential in the synthesis of fine chemicals and pharmaceuticals due to its advantages such as mild conditions, controllable reactions, and high selectivity. However, due to the compatibility issues between enzymes, systems, and compounds in multi-enzyme cascade catalysis, one-pot synthesis struggles to achieve a balance among these three elements, leading to problems such as numerous byproducts, difficulty in separating intermediates, and low yields.

[0005] Therefore, developing a safe, green, and efficient synthetic route has become a key focus in the research and development of adrenaline-based drugs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, modular segmentation of the multi-enzyme-level synthesis pathway can significantly reduce the occurrence of the aforementioned problems. Based on this, the main objective of this invention is to provide a system and method for the multi-enzyme-level synthesis of adrenaline and its analogues, which features a short reaction pathway, low raw material cost, avoidance of intermediate product separation, high degree of automation, and the ability to further improve yield.

[0007] Specifically, a system for the multi-enzyme-level synthesis of adrenaline and its analogues includes: The norepinephrine synthesis module includes several first flow reactors, a norepinephrine synthase, and a protocatechuic aldehyde-glycine storage tank that provides substrates for the first flow reactors. The norepinephrine synthase is encapsulated in several first flow reactors and consists of immobilized threonine aldolase (LTA) and tyrosine decarboxylase (TDC). A plurality of alkylation modules are provided, each alkylation module comprising an alkylation cofactor reservoir, a plurality of alkylation flow reactors, and an alkylating enzyme, wherein the alkylating enzyme is encapsulated within the plurality of the alkylation flow reactors; the alkylation cofactor reservoir and the first flow reactor are simultaneously connected to the alkylation flow reactors and respectively provide the alkylation cofactor substrate and norepinephrine to the alkylation flow reactors; the alkylating enzyme is composed of immobilized phenylethanolamine-N-methyltransferase (PNMT) and halide methyltransferase (HMT).

[0008] Preferably, the threonine aldolase is derived from the intestines of black bears. bl LTA, the tyrosine decarboxylase being derived from Enterococcus faecalis. sf TDC, the phenylethanolamine-N-methyltransferases are of human origin. h PNMT and European hare ory PNMT; the alkyltransferase is derived from *Paederia cuspidatum*. acl HMT or its mutants acl HMT-P8L / W27F / V265W.

[0009] Based on the above, the immobilized threonine aldolase and tyrosine decarboxylase are simultaneously encapsulated in the same first flow reactor or separately encapsulated in two first flow reactors; the immobilized phenylethanolamine-N-methyltransferase and halide methyltransferase are simultaneously encapsulated in the same alkylation flow reactor or separately encapsulated in two alkylation flow reactors.

[0010] Based on the above, the carriers used to immobilize the corresponding enzymes in the first flow reactor and the alkylation flow reactor are long-chain amino resins, such as amino resins with models LXTE-700, ESQ-3, ESR-3, and HA109.

[0011] Based on the above, the norepinephrine synthesis module further includes a norepinephrine storage tank connected to the first flow reactor. The protocatechuic aldehyde-glycine storage tank, the first flow reactor, and the norepinephrine storage tank are connected by pipelines to form a circulation system. A substrate supply pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the first flow reactor. An intermediate material four-way valve is installed on the pipeline between the first flow reactor and the norepinephrine storage tank. A norepinephrine circulation pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the norepinephrine storage tank.

[0012] Based on the above, the alkylation module further includes a target product storage tank connected to the alkylation flow reactor. The alkylation flow reactor is connected to the first flow reactor, the alkylation cofactor storage tank, and the target product storage tank via pipelines. An alkylation cofactor supply pump and an alkylation material four-way valve are installed on the pipeline between the alkylation flow reactor and the alkylation cofactor storage tank. The alkylation material four-way valve is connected to an intermediate material four-way valve. The target product is adrenaline or an analogue thereof. The alkylation cofactor storage tank contains S-adenosine-L-homocysteine ​​and an alkyl donor, wherein the alkyl donor is methyl p-toluenesulfonate or a haloalkane.

[0013] The system described above includes a methylation module and an ethylation module. The methylation module includes a methyl flow reactor for synthesizing adrenaline, a methyl cofactor reservoir, and an adrenaline reservoir. The methyl flow reactor, methyl cofactor reservoir, and adrenaline reservoir are connected by a pipeline. A second four-way valve and a methyl cofactor supply pump are installed on the pipeline between the methyl flow reactor and the methyl cofactor reservoir. The methyl flow reactor encapsulates immobilized phenylethanolamine-N-methyltransferase and halide methyltransferase. The methyl cofactor reservoir contains S-adenosyl-L-homocysteine ​​and methyl p-toluenesulfonate or halomethane. The second four-way valve is connected to the intermediate material four-way valve. The ethylation module includes an ethyl flow reactor for synthesizing ethyl adrenaline, an ethyl cofactor reservoir, and an ethyl adrenaline reservoir. The ethyl flow reactor, ethyl cofactor reservoir, and ethyl adrenaline reservoir are connected by a pipeline. A third four-way valve and an ethyl cofactor supply pump are installed on the pipeline between the ethyl flow reactor and the ethyl cofactor reservoir. The ethyl flow reactor encapsulates immobilized phenylethanolamine-N-methyltransferase and a halide methyltransferase mutant. The ethyl cofactor reservoir is used to hold S-adenosine-L-homocysteine ​​and haloethane. The third four-way valve is connected to the second four-way valve.

[0014] The present invention also provides a method for synthesizing adrenaline and its analogues using the above system, comprising the steps of: Synthesizing norepinephrine: Protocatechuic aldehyde and glycine are added to the protocatechuic aldehyde-glycine storage tank and transported to the first flow reactor, where norepinephrine is synthesized. The synthesized norepinephrine is then transported to the alkylation flow reactor. Synthesis of the target product: The alkylation cofactor substrate in the alkylation cofactor reservoir is transported to the alkylation flow reactor, and norepinephrine and the alkylation cofactor substrate are used to synthesize adrenaline or its analogues in the alkylation flow reactor.

[0015] Based on the method described above, when the concentration of norepinephrine synthesized in the first flow reactor does not reach the predetermined requirement, the step of synthesizing norepinephrine further includes: firstly, by controlling the intermediate material four-way valve, collecting the norepinephrine synthesized in the first flow reactor into a norepinephrine storage tank, and then transporting it to the protocatechuic aldehyde-glycine storage tank through a norepinephrine circulation pump, and continuing to repeat the cycle to synthesize norepinephrine until the concentration of synthesized norepinephrine reaches the predetermined requirement, and then switching the intermediate material four-way valve to transport it to the alkylation flow reactor.

[0016] In the step of synthesizing norepinephrine, the first flow reactor is encapsulated with bl LTA cell lysis buffer and sf TDC cell lysis fluid, the bl LTA cell lysis buffer and sf The weight ratio of the TDC cell lysis buffer is 0.5:1-8:1, the substrate supply pump flow rate is 10-200 μL / min, and the synthesis temperature is 20-40℃; thus, continuous flow synthesis of norepinephrine can be achieved. Preferably, the... bl LTA cell lysis buffer and sf The weight ratio of the TDC cell lysis solution is 1:1-5:1, and the flow rate of the substrate supply pump is 20-100 μL / min.

[0017] Based on the method described above, the step of synthesizing the target product includes: placing S-adenosine-L-homocysteine ​​and an alkyl donor in an alkylation cofactor reservoir; controlling a four-way valve for alkylation materials; and feeding S-adenosine-L-homocysteine, the alkyl donor, and norepinephrine together into the alkylation flow reactor via an alkylation cofactor supply pump and a substrate supply pump, respectively; synthesizing adrenaline or an analogue in the alkylation flow reactor; and collecting the adrenaline in the target product reservoir, wherein the alkyl donor is methyl p-toluenesulfonate or a haloalkane.

[0018] Furthermore, the alkylation flow reactor contains pure enzyme solutions of phenylethanolamine-N-methyltransferase and halide methyltransferase, with a concentration ratio of phenylethanolamine-N-methyltransferase to halide methyltransferase of 1:1 to 10:1. The synthesis temperature is 20-40°C, the flow rate of the cofactor supply pump is 10-200 μL / min, and the flow rate of the substrate supply pump is 10-200 μL / min. In this way, continuous flow synthesis of adrenaline or its analogues can be achieved.

[0019] Based on the method described above, the step of synthesizing the target product includes synthesizing adrenaline: placing S-adenosine-L-homocysteine ​​and a methyl donor in a methyl cofactor reservoir, controlling a second four-way valve to deliver S-adenosine-L-homocysteine, the methyl donor, and norepinephrine together to a methyl flow reactor via a methyl cofactor supply pump and a substrate supply pump, respectively, synthesizing adrenaline in the methyl flow reactor, and collecting it in the adrenaline reservoir, wherein the methyl donor is methyl p-toluenesulfonate or a halomethane; the methyl flow reactor is encapsulated with... ory PNMT pure enzyme solution and acl HMT pure enzyme solution, and in it ory PNMT enzyme and acl The concentration ratio of HMT enzyme is 1:1-5:1, and the flow rate of the methyl cofactor supply pump is 50-100 μL / min.

[0020] Based on the method described above, the step of synthesizing the target product includes synthesizing ethyl epinephrine: placing S-adenosyl-L-homocysteine ​​and haloethane in the ethyl cofactor reservoir, controlling the third four-way valve and the second four-way valve to deliver S-adenosyl-L-homocysteine, haloethane, and norepinephrine together to the ethyl flow reactor, synthesizing ethyl epinephrine in the ethyl flow reactor, and collecting it in the ethyl epinephrine reservoir; the ethyl flow reactor is encapsulated with... h PNMT pure enzyme solution and acl HMT-P8L / W27F / V265W pure enzyme solution, and in it h PNMT enzyme and acl The concentration ratio of HMT-P8L / W27F / V265W enzyme is 1:1-5:1, and the flow rate of the methyl cofactor supply pump is 50-100 μL / min.

[0021] Based on the method described above, the substrate supply pump and each alkylation cofactor supply pump operate at a flow rate ratio of 1:1 to 1:5 for 24-72 hours, and the product concentration is sampled and detected every 0.5-2 hours; in this way, the synthesis of substances such as norepinephrine, epinephrine and ethyl epinephrine can be achieved in a fully continuous flow.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses a modular multi-enzyme-level synthesis method for adrenaline and its analogues. A non-natural multi-enzyme-level synthesis pathway is designed, and key enzyme elements are identified and optimized. Based on factors such as product requirements, enzyme catalytic function, and differences in reaction conditions, the multi-enzyme catalytic synthesis pathway for adrenaline is decomposed into two reaction modules: a norepinephrine synthesis module composed of aldolase and decarboxylase, and an alkylation module. Through the exploration of the decomposition of these two reaction modules, the forward reaction is promoted, the inhibitory effects between components are reduced, and the problem of incompatible reaction conditions is improved. Combined with an immobilized enzyme continuous flow reactor, the enzymatic synthesis of adrenaline achieves high conversion rate, high optical purity, and high space-time yield. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the system for the multi-enzyme-level synthesis of adrenaline and analogues provided in Embodiment 1 of the present invention; Figure 2 High-performance liquid chromatogram and time curve of the norepinephrine synthesis module reaction; Figure 3 The high-performance liquid chromatogram of the product adrenaline and its change over time are shown. Figure 4 A comparison of the conversion rates of ethyl adrenaline catalyzed by phenylethanolamine-N-methyltransferase from human and European hare sources; Figure 5 for bl LTA, sf The immobilization efficiency and activity histogram of TDC, where Figure (a) shows... bl LTA immobilization efficiency and activity (b) sf TDC immobilization efficiency and activity; Figure 6 for ory PNMT acl The immobilization efficiency and activity of HMT are shown in the bar chart, where Figure (a) is an example. ory PNMT immobilization efficiency and activity (b) acl HMT immobilization efficiency and activity; Figure 7 for h PNMT acl Immobilization efficiency and activity histogram of HMT-P8L / W27F / V265W, where (a) h PNMT immobilization efficiency and activity (b) acl Immobilization efficiency and activity of HMT-P8L / W27F / V265W; Figure 8Spatiotemporal yield curves of adrenaline synthesis from multi-enzyme conjugation at different flow rates; Figure 9 Spatiotemporal yield curves for the synthesis of adrenaline from multiple enzymes at different enzyme weight ratios; Figure 10 Spatiotemporal yield curves of adrenaline synthesis from multi-enzyme stage under different flow rate ratios; Figure 11 Spatiotemporal yield curves of ethyl adrenaline synthesized from multiple enzymes at different flow rates; Figure 12 The plot shows the spatiotemporal yield changes of adrenaline and ethyl adrenaline in a continuous flow system over 48 hours of continuous operation. Figure 13 Spatiotemporal yield curves of cauliflower alkaloid and N-methylcauliflower alkaloid synthesized by multi-enzyme stage at different flow rates. Detailed Implementation

[0024] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0025] All terms used in this invention are commonly used in the relevant field. Unless otherwise specified, all raw materials, equipment, preparation processes and methods, and testing methods used are existing technologies in the relevant field. All materials used are commercially available. In this invention, it is assumed that the construction and transformation of plasmid vectors, as well as methods for E. coli gene knockout, knock-in, and gene mutation, can employ various methods commonly used in the field, which will not be elaborated upon further.

[0026] In the following examples, Escherichia coli BL21(DE3) and other commonly used Escherichia coli strains are available commercially. BL21Δmtn(DE3) is a reference to Liao, C., Seebeck, FP. S-adenosylhomocysteine ​​asa methyl transfer catalyst in biocatalytic methylation reactions. Nature Catalysis .2, 696-701. Obtained through modification; all raw materials and reagents used are common items and can be obtained commercially.

[0027] All enzymes involved in this invention are derived from commonly used substances, and the sources of these enzymes are not limited to those listed in this invention. Any enzyme with a similarity of less than 80% to the enzymes listed in this invention is within the scope of protection of this invention. In the following examples and comparative examples, in addition to the key enzymes shown in Table 1, other enzymes, such as the various enzymes used in the pathway for synthesizing shikimic acid from a carbon source, are also existing commonly used enzymes.

[0028] Table 1 Enzyme Source Table Modular multi-enzyme-level synthesis pathways can significantly improve yields and enable automated synthesis of products. This invention modularizes the synthesis of adrenaline and its analogues, primarily into a norepinephrine synthesis module consisting of threonine aldolase and tyrosine decarboxylase, and an alkylation module consisting of phenylethanolamine-N-methyltransferase and halide methyltransferase, to synthesize adrenaline and its analogues. Accordingly, this invention provides a system and method for the multi-enzyme-level synthesis of adrenaline and its analogues.

[0029] In one embodiment, a method for synthesizing adrenaline and its analogues using a multi-enzyme conjugation process specifically includes the following steps: Step 1: Screening and constructing genetically engineered bacteria containing key enzyme elements in the synthesis pathway of adrenaline and its analogues. By consulting protein databases such as NCBI, BRENDA, and UNIPROT and comparing sequence similarity, threonine aldolase, tyrosine decarboxylase, phenylethanolamine-N-methyltransferase, and halide methyltransferase from different sources were screened. Their gene sequences were codon-optimized, and the optimized sequences were synthesized by BGI Genomics. These optimized sequences were then subcloned into the pET-28(a) vector to obtain recombinant plasmids. The plasmids of threonine aldolase and tyrosine decarboxylase were electroporated into the *E. coli* expression host BL21(DE3) to obtain engineered bacteria containing threonine aldolase and tyrosine decarboxylase from different sources. Similarly, the plasmids of phenylethanolamine-N-methyltransferase and halide methyltransferase were electroporated into the SAH nucleoside enzyme-deficient *E. coli* expression host BL21Δmtn(DE3) to obtain engineered bacteria containing phenylethanolamine-N-methyltransferase and halide methyltransferase from different sources.

[0030] Step 2: Cultivate expressible genes derived from the gut of black bears. bl LTA's genetically engineered bacteria BL21(DE3) / pET28(a)- bl LTA, source of Enterococcus faecalis sf TDC's genetically engineered bacteria BL21(DE3) / pET28(a)- sf TDC, Human Origin h PNMT and European hare oryPNMT's genetically engineered bacterium BL21Δmtn(DE3) / pET28(a)- h PNMT, BL21Δmtn(DE3) / pET28(a)- ory PNMT, and Aspergillus lanceolata acl HMT's genetically engineered bacteria BL21Δmtn(DE3) / pET28(a)- acl HMT, mutant acl The genetically engineered bacterium BL21Δmtn(DE3) / pET28(a) of HMT-P8L / W27F / V265W- acl HMT-P8L / W27F / V265W Pick the genetically engineered bacteria BL21(DE3) / pET28(a) from the LB culture dish in step 1. bl LTA, BL21(DE3) / pET28(a)- sf TDC、BL21Δmtn(DE3) / pET28a- h PNMT, BL21Δmtn(DE3) / pET28(a)- ory PNMT, BL21Δmtn(DE3) / pET28(a)- acl HMT, BL21Δmtn(DE3) / pET28(a)- acl Single colonies of HMT-P8L / W27F / V265W were inoculated into 3-10 mL of LB medium containing kanamycin resistance. After incubation at 37°C on a shaker for 12-16 hours, the colonies were transferred to 300-1000 mL of LB medium for expansion culture. The colonies were cultured at 37°C on a shaker until the OD value reached 0.6-0.8. Then, IPTG was added to a final concentration of 0.05-0.5 mM, and expression was induced at 18°C ​​for 18-22 hours. The bacterial cells were then collected by centrifugation.

[0031] Step 3: Purify the genetically engineered strain BL21(DE3) / pET28a- expressing threonine aldolase. bl LTA, a genetically engineered bacterium of tyrosine decarboxylase, BL21(DE3) / pET28a- sf TDC, phenylethanolamine-N-methyltransferase BL21Δmtn(DE3) / pET28a- h PNMT, BL21Δmtn(DE3) / pET-28(a)- ory PNMT, halide methyltransferase BL21Δmtn(DE3) / pET-28(a)- acl HMT and BL21Δmtn(DE3) / pET28(a)- acl HMT-P8L / W27F / V265W The bacterial cells collected in step 2 were resuspended in 10 mM imidazole buffer at pH 8.0. The resuspended genetically engineered bacteria were then homogenized using a high-pressure homogenizer. The lysate was centrifuged at 12000 rpm and 4°C for 20 min to obtain crude enzyme solutions of threonine aldolase, tyrosine decarboxylase, phenylethanolamine-N-methyltransferase, and halide methyltransferase. Next, the crude enzyme solutions were bound to a Ni-NTA gel column for 1 h. Unbound liquid was allowed to flow out from the bottom of the column, and unbound proteins were eluted with 50 mM imidazole buffer. After the G250 protein staining solution showed no color change, the nickel column was washed with 300 mM imidazole buffer, and the proteins were collected. Finally, the proteins were concentrated and desalted by centrifugation in a 10000 kDa ultrafiltration tube to obtain the desired enzyme solution. bl LTA, sf TDC, ory PNMT acl HMT h PNMT and acl The pure enzyme solution of HMT-P8L / W27F / V265W was used as a backup enzyme solution for subsequent experiments.

[0032] Step 4: In vitro synthesis of norepinephrine, epinephrine, and ethyl epinephrine. 1) Using protocatechuic aldehyde (final concentration 30-80 mmol / L) and glycine (final concentration 0.5-1.5 mol / L) as the reaction substrate, and pyridoxal 5-phosphate (final concentration 0.3-0.8 mmol / L) as the cofactor, add the aforementioned [reaction material] at a final concentration of 1-5 g / L. bl LTA pure enzyme solution, 0.1-0.5 g / L of the aforementioned sf TDC pure enzyme solution was added, and PB buffer was added to adjust the reaction system to 0.2 mL, pH 6-8. The reaction was carried out at 20-50℃ for 1-24 h. After the catalytic reaction was completed, 0.1% trifluoroacetic acid was added in equal volume. After centrifugation, the supernatant was taken and the synthesized product was detected by HPLC as norepinephrine.

[0033] 2) Using norepinephrine at a final concentration of 1-4 mmol / L and S-adenosyl-L-homocysteine ​​as reaction substrates, and methyl p-toluenesulfonate at a final concentration of 2-8 mmol / L as a methyl donor, add [amount of a solution] at a final concentration of 5-20 μmol / L. ory PNMT pure enzyme solution, 0.1-5 μmol / L acl HMT pure enzyme solution was added, and PB buffer was added to adjust the reaction system to 0.2 mL, pH 6-8, and the reaction was carried out at 25-70℃ for 1-24 h. After the catalytic reaction was completed, 0.1% trifluoroacetic acid was added in equal volume. After centrifugation, the supernatant was taken and the synthesized product was detected by HPLC to be adrenaline.

[0034] 3) Using norepinephrine and S-adenosyl-L-homocysteine ​​at a final concentration of 0.05-2 mmol / L as the reaction substrate, and 4-15 mmol / L haloethane as the ethyl donor, add 1-4 g / L of [agent name missing] to the reaction. h PNMT pure enzyme solution, 0.5-2 g / L acl HMT-P8L / W27F / V265W pure enzyme solution was added, and PB buffer was added to adjust the reaction system to 0.2 mL, pH 6-8. The reaction was carried out at 25-70℃ for 1-24 h. After the catalytic reaction was completed, 0.1% trifluoroacetic acid was added in equal volume. The supernatant was taken and the synthesized product was detected by HPLC to be ethyl adrenaline.

[0035] Step 5: Screen different types of commercially available amino resins. First, wash the amino carrier with phosphate buffer, then treat the amino resin with glutaraldehyde aqueous solution. Add the purified enzyme solution from Step 4 to the activated amino resin. After rotating the reaction at room temperature, wash the resin to obtain the immobilized enzyme and measure its activity. Freeze-dry for later use. Specifically, the amino resin activation process involves adding 2-10 mL of 2.5% glutaraldehyde fixative to 1 g of carrier, placing it in a rotary mixer, reacting at 20-30℃ for 1-4 h, rinsing the resin carrier three times with pH 8.0 phosphate buffer, and drying it for later use; weighing 5-20 mg of different types of amino resin, adding 1-2 mg of pure enzyme solution, measuring the A280 of the supernatant, placing it on a rotary mixer, treating it at 20-30℃ for 12-24 h, measuring the A280 of the supernatant again, calculating the immobilized enzyme efficiency, and further detecting the activities of immobilized threonine aldolase, tyrosine decarboxylase, phenylethanolamine-N-methyltransferase, and halide methyltransferase, screening out LXTE-700 to immobilize phenylethanolamine-N-methyltransferase, and ESQ-3 to immobilize threonine aldolase, tyrosine decarboxylase, and halide methyltransferase. Therefore, immobilized enzymes can be used to synthesize substances such as norepinephrine, epinephrine, and ethyl epinephrine, providing a basis for the modular synthesis of epinephrine and its analogues.

[0036] Step 6: Prepare reactors for the norepinephrine synthesis module and alkylation module, and construct a continuous flow system. Among them, a multi-enzyme-level conjugation system for adrenaline and its analogues was constructed, including: The norepinephrine synthesis module includes a plurality of first flow reactors, a norepinephrine synthase composed of immobilized threonine aldolase (LTA) and tyrosine decarboxylase (TDC), and a protocatechuic aldehyde-glycine reservoir that provides substrate to the first flow reactors, wherein the norepinephrine synthase is encapsulated in the plurality of first flow reactors. A plurality of alkylation modules, each alkylation module comprising an alkylation cofactor reservoir, a plurality of alkylation flow reactors, and immobilized phenylethanolamine-N-methyltransferase (PNMT) and halide methyltransferase (HMT), wherein the immobilized enzymes are encapsulated in the plurality of the alkylation flow reactors; the alkylation cofactor reservoir and the first flow reactor are simultaneously connected to the alkylation flow reactors and respectively provide the alkylation cofactor substrate and norepinephrine to the alkylation flow reactors.

[0037] In one embodiment, the norepinephrine synthesis module further includes a norepinephrine storage tank for collecting the synthesis product from the first flow reactor. The protocatechuic aldehyde-glycine storage tank, the first flow reactor, and the norepinephrine storage tank are connected by pipelines to form a circulation system. A substrate supply pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the first flow reactor. An intermediate material four-way valve is installed on the pipeline between the first flow reactor and the norepinephrine storage tank. A norepinephrine circulation pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the norepinephrine storage tank.

[0038] In one embodiment, the alkylation module further includes a target product storage tank for collecting the synthesis products from the alkylation flow reactor. The alkylation flow reactor is connected to the first flow reactor, the alkylation cofactor storage tank, and the target product storage tank via pipelines. An alkylation cofactor supply pump and an alkylation material four-way valve are installed on the pipeline between the alkylation flow reactor and the alkylation cofactor storage tank. The alkylation material four-way valve is connected to an intermediate material four-way valve. The alkylation cofactor storage tank is used to hold S-adenosine-L-homocysteine ​​and an alkyl donor, wherein the alkyl donor is methyl p-toluenesulfonate or a haloalkane.

[0039] In one embodiment, the system for synthesizing adrenaline and its analogues via a multi-enzyme stage includes a methylation module and an ethylation module. The methylation module includes a methyl flow reactor for synthesizing adrenaline, a methyl cofactor reservoir, and an adrenaline reservoir. The methyl flow reactor, methyl cofactor reservoir, and adrenaline reservoir are connected by a pipeline. A second four-way valve and a methyl cofactor supply pump are installed on the pipeline between the methyl flow reactor and the methyl cofactor reservoir. The methyl flow reactor encapsulates immobilized phenylethanolamine-N-methyltransferase and halide methyltransferase. The methyl cofactor reservoir holds S-adenosine-L-homocysteine ​​and methyl p-toluenesulfonate or halomethane. The second four-way valve is connected to the intermediate material four-way valve. The ethylation module includes an ethyl flow reactor for synthesizing ethyl adrenaline, an ethyl cofactor reservoir, and an ethyl adrenaline reservoir. The ethyl flow reactor, ethyl cofactor reservoir, and ethyl adrenaline reservoir are connected by a pipeline. A third four-way valve and an ethyl cofactor supply pump are installed on the pipeline between the ethyl flow reactor and the ethyl cofactor reservoir. The ethyl flow reactor encapsulates immobilized phenylethanolamine-N-methyltransferase and a halide methyltransferase mutant. The ethyl cofactor reservoir is used to hold S-adenosine-L-homocysteine ​​and haloethane. The third four-way valve is connected to the second four-way valve.

[0040] Using tubular columns with hollow channels having an inner diameter of 2-10 mm and a length of 50-200 mm as modular reactors, the immobilized enzymes from step 5 were loaded into the hollow channels of different tubular columns according to the reaction type, forming norepinephrine synthesis modules, adrenaline and SAM cycling modules, and ethyl adrenaline and ethyl SAM analog synthesis modules. bl LTA@ESQ-3 and sf TDC@ESQ-3 is packaged separately in two reactors or together in one reactor to form a norepinephrine synthesis module. ory PNMT@LXTE-700 and acl HMT@ESQ-3 are co-encapsulated in a reactor to form a methylation module, and further... h PNMT@LXTE-700 and acl HMT-P8L / W27F / V265W@ESQ-3 are encapsulated together in a reactor to form an ethylation module.

[0041] A method for synthesizing adrenaline and its analogues using the above system includes the following steps: Synthesizing norepinephrine: Protocatechuic aldehyde and glycine are added to the protocatechuic aldehyde-glycine storage tank and transported to the first flow reactor, where norepinephrine is synthesized. The synthesized norepinephrine is then transported to the alkylation flow reactor. Synthesis of the target product: The alkylation cofactor substrate in the alkylation cofactor reservoir is transported to the alkylation flow reactor, and norepinephrine and the alkylation cofactor substrate are used to synthesize adrenaline or its analogues in the alkylation flow reactor.

[0042] When the concentration of norepinephrine synthesized in the first flow reactor does not reach the predetermined requirement, the step of synthesizing norepinephrine further includes: first collecting the norepinephrine synthesized in the first flow reactor into a norepinephrine storage tank by controlling the intermediate material four-way valve, and then transporting it to the protocatechuic aldehyde-glycine storage tank by the norepinephrine circulation pump, and continuing to repeat the cycle to synthesize norepinephrine until the concentration of synthesized norepinephrine reaches the predetermined requirement, and then switching the intermediate material four-way valve to transport it to the alkylation flow reactor.

[0043] The target product for synthesis includes adrenaline and / or ethyl adrenaline, and the steps for synthesizing the target product include: Synthesizing adrenaline: S-adenosine-L-homocysteine ​​and halomethane are placed in the methyl cofactor reservoir. The second four-way valve is controlled to transport S-adenosine-L-homocysteine, the halomethane, and norepinephrine together to the methyl flow reactor. Adrenaline is synthesized in the methyl flow reactor and collected in the adrenaline reservoir. Synthesis of ethyl adrenaline: S-adenosyl-L-homocysteine ​​and haloethane are placed in the ethyl cofactor reservoir. The third four-way valve and the second four-way valve are controlled to transport S-adenosyl-L-homocysteine, the haloethane, and norepinephrine together to the ethyl flow reactor. Ethyl adrenaline is synthesized in the ethyl flow reactor and collected in the ethyl adrenaline reservoir.

[0044] Step 7: Optimize the reactor module for continuous flow synthesis of adrenaline and its analogues, and determine the stability of the continuous flow system.

[0045] By adjusting the enzyme ratio of the reactor module, the flow rate of the main pump and the cofactor supply pump, the temperature, and other conditions, the yields and space yields of the products norepinephrine, epinephrine, and ethyl epinephrine were monitored. After the optimized conditions were determined, the continuous flow system was continuously run, and the product formation was measured every few hours.

[0046] Therefore, each of the norepinephrine synthesis modules encapsulates immobilized blLTA and sfTDC cell lysate, and the adrenaline and ethyl adrenaline modules encapsulate immobilized... ory PNMT and acl HMT, hPNMT and aclPure enzyme solution of HMT-P8L / W27F / V265W. In the continuous flow synthesis module for norepinephrine, the wet cell weight ratio of threonine aldolase to tyrosine decarboxylase is 0.5:1-8:1, the flow rate is 10-200 μL / min, and the temperature is 20-40℃. Preferably, the wet cell weight ratio is 1:1-5:1, and the flow rate is 20-100 μL / min. The adrenaline and ethyl adrenaline modules are synthesized at an enzyme concentration of 1:1-10:1, a flow rate of 10-200 μL / min, and a temperature of 20-40℃. Preferably, the enzyme concentration ratio is 1:1-5:1, and the flow rate is 50-100 μL / min.

[0047] The fully continuous flow system operates with the substrate supply pump as the main pump and the flow rate ratio between the main pump and each alkylation cofactor supply pump being 1:1 to 1:5 for 24-72 hours. Product concentration is sampled and detected every 0.5-2 hours.

[0048] Example 1: A system for the synthesis of adrenaline and its analogues via multi-enzyme-level conjugation. Please see Figure 1 This embodiment provides a multi-enzyme-level combined system for synthesizing adrenaline and its analogues. This system is a continuous flow system for synthesizing adrenaline and its analogues. Specifically, it includes: a first flow reactor 1 for synthesizing norepinephrine, a methyl flow reactor 2 for synthesizing adrenaline, an ethyl flow reactor 3 for synthesizing ethyl adrenaline, a protocatechuic aldehyde-glycine reservoir 4, a norepinephrine reservoir 5, a methyl cofactor reservoir 6, an ethyl cofactor reservoir 7, an ethyl adrenaline reservoir 8, and an adrenaline reservoir 9; a substrate supply pump 10; a norepinephrine circulation pump 11; a methyl cofactor supply pump 12; an ethyl cofactor supply pump 13; and three four-way valves 14.

[0049] Substrate supply pump 10 delivers protocatechuic aldehyde and glycine into the first flow reactor 1, where norepinephrine is synthesized. The effluent flows through a pipeline and is controlled by a four-way valve 14 mounted on it to the norepinephrine storage tank 5. It is then circulated back to the protocatechuic aldehyde-glycine storage tank 4 via the norepinephrine circulation pump 11, continuing the cycle of norepinephrine synthesis until the synthesized norepinephrine concentration reaches the predetermined requirement. Then, the four-way valve 14 is switched with the four-way valve 14 in the methylation synthesis module, and the reaction solution of the next-stage adrenaline synthesis module passes through a methylation cofactor. The feed pump 12 delivers the material into the main pipeline, and together with the effluent from the first flow reactor 1 in the norepinephrine module, it enters the methyl flow reactor 2 in the adrenaline synthesis module. Finally, the product adrenaline is collected by the adrenaline storage tank 9. Similarly, the four-way valve 14 in the ethylation synthesis module delivers the reaction liquid of the ethyl adrenaline synthesis module through the ethyl cofactor feed pump 13, and together with the effluent from the first flow reactor 1 in the norepinephrine module, it enters the ethyl flow reactor 3 in the ethyl adrenaline synthesis module. Finally, the product ethyl adrenaline is collected by the ethyl adrenaline storage tank 8.

[0050] Example 2: Synthesis of norepinephrine catalyzed by the cascade of threonine aldolase and tyrosine decarboxylase 1) Preparation of reaction solution: Protocatechuic aldehyde standard was dissolved in DMF to prepare a 1 M stock solution. Glycine and pyridoxal phosphate were dissolved in 0.1 M, pH 8.0 phosphate buffer to prepare 4 M and 50 mM stock solutions, respectively. The stock solutions were mixed to obtain a reaction solution containing 100 mM protocatechuic aldehyde, 2 M glycine and 1 mM pyridoxal phosphate.

[0051] 2) Catalytic reaction: The reaction system consisted of 200 μL. 100 μL of the above reaction solution was added to achieve a final concentration of protocatechuic aldehyde of 50 mM, glycine of 1 M, and pyridoxal phosphate of 0.5 mM. Then, a final concentration of 2.5 g / L of [a specific chemical compound] was added. bl LTA and 0.5 g / L sf TDC was then used to adjust the reaction system to 200 μL with phosphate buffer, pH 6.5, and the reaction was carried out at 25°C for 1-24 h.

[0052] 3) Sampling and Detection: After the reaction was completed, 200 μL of 0.1% trifluoroacetic acid was added to terminate the reaction. After centrifugation at 5000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane into a liquid chromatography (HPLC) bottle. HPLC column: Shim-pack Scepter C18-120 (4.6 × 250 mm, 5 μm); mobile phase: sodium octane sulfonate-methanol solution; UV detector: detection wavelength 280 nm; column temperature: 35℃; flow rate: 1 mL / min. After HPLC detection, the following results were obtained: Figure 2 The results shown in the figure indicate that the peak at 4.5 min is the detection peak of the product norepinephrine. The calculated product amount is 5.2 g / L, and the diastereoselectivity (de) value can reach more than 95%.

[0053] Example 3: Synthesis of adrenaline catalyzed by phenylethanolamine-N-methyltransferase and methyltransferase 1) Preparation of reaction stock solutions: Norepinephrine standard was dissolved in 0.1 M hydrochloric acid to prepare a 20 mM stock solution, S-adenosine-L-homocysteine ​​was dissolved in 0.1 M hydrochloric acid to prepare a 10 mM stock solution, and methyl p-toluenesulfonate was dissolved in tert-butanol to prepare a 400 mM stock solution.

[0054] 2) Catalytic reaction: The reaction system consisted of 200 μL containing 2 mM norepinephrine, 0.1 mM S-adenosyl-L-homocysteine, and 4 mM methyl p-toluenesulfonate, followed by the addition of [a specific ingredient] to a final concentration of 10 μM. ory PNMT, 1 μM acl HMT was then used to adjust the reaction system to 200 μL with phosphate buffer, pH 7.5, and the reaction was carried out at 30°C for 1-24 h.

[0055] 3) Sampling and Detection: After the reaction was completed, 200 μL of 0.1% trifluoroacetic acid was added to terminate the reaction. After centrifugation at 5000 rpm for 5 minutes, the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane into a liquid chromatography bottle. Detection was then performed using high-performance liquid chromatography (HPLC). The HPLC column was a ShimNex CS C18 (4.6 × 250 mm, 5 μm). The mobile phases were A: 0.1% trifluoroacetic acid, B: methanol. The gradient elution program was t0 = 0% B, t2 = 2% B, t3 = 10% B, t7 = 30% B, t9 = 50% B, t1 = 2% B, t2 = 2% B, t3 = 10% B, t7 = 30% B, t8 = 50% B, t9 = 50% B, t1 = 2% B, t2 = 2% B, t3 = 10% B, t9 = 3 ...9 = 30% B, t1 = 2% B, t2 = 2% B, t1 = 2% B, t2 = 2% B, t1 = 2% B, t2 = 2% B, t1 = 2% B, t2 = 2% B, t1 = 2% B, t2 = 2% B, t1 = 2% B, 11 =100%B,t 13 =0%B,t 15 =0%B, UV detector: detection wavelength 280 nm, column temperature 30℃, flow rate 1 mL / min. After detection by high-performance liquid chromatography, the following results were obtained: Figure 3The results shown in the figure indicate that the peak at 6.5 min is the detection peak of the product adrenaline, and the calculated yield is 80%.

[0056] Example 4: Synthesis of Ethyl Adrenaline Catalyzed by Phenylethanolamine-N-Methyltransferase and Methyltransferase 1) Preparation of reaction stock solutions: Norepinephrine standard was dissolved in 0.1 M hydrochloric acid to prepare a 20 mM stock solution, S-adenosine-L-homocysteine ​​was dissolved in 0.1 M hydrochloric acid to prepare a 10 mM stock solution, and iodoethane was dissolved in DMSO to prepare a 400 mM stock solution.

[0057] 2) Catalytic reaction: The reaction system consisted of 200 μL containing 1 mM norepinephrine, 0.5 mM S-adenosyl-L-homocysteine, and 8 mM iodoethane, with the final concentration of 2 g / L then added. h PNMT ory PNMT, 1 g / L acl HMT was then used to adjust the reaction system to 200 μL with phosphate buffer, pH 7.5, and the reaction was carried out at 30°C for 1-24 h.

[0058] 3) Sampling and Detection: After the reaction was completed, 200 μL of 0.1% trifluoroacetic acid was added to terminate the reaction. After centrifugation at 5000 rpm for 5 minutes, the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane into a liquid chromatography (HPLC) bottle. HPLC column: ShimNex CS C18 (4.6 × 250 mm, 5 μm); mobile phase: A: 0.1% trifluoroacetic acid, B: methanol; UV detector: detection wavelength 280 nm; column temperature: 30℃; flow rate: 1 mL / min; gradient elution program: same as in Example 2. After HPLC detection, the following results were obtained: Figure 4 The result is shown in the figure below. h PNMT compared to ory PNMT has high catalytic efficiency, with a norepinephrine conversion rate of up to 35%.

[0059] Example 5: Efficiency and activity of enzymes immobilized with blLTA and sfTDC Four commercially available amino resins were selected: LXTE-700, ESQ-3, ESR-3, and HA109. 10 mg of the resin carrier was weighed, washed three times with pH 8.0 phosphate buffer, and 1 mL of 2.5% glutaraldehyde fixative was added. The mixture was rotated for 2 hours at 25°C and 18 rpm / min, followed by three more washes with pH 8.0 phosphate buffer. 1 mg of [the resin] was added to each [resin]. bl LTA and sfTDC (stem cell lysis buffer) was used to determine the A280 of the supernatant. After rotational binding at 25°C for 20 h, the A280 of the supernatant was measured again. The supernatant was discarded, leaving solid particles, which were washed three times with pH 8.0 phosphate buffer. The reaction solution was added, and the product formation was detected using high-performance liquid chromatography (HPLC). Figure 5 As shown in (a) and (b), the ESQ-3 type amino resin in the fixation bl LTA and sf It exhibits high fixation efficiency and activity during TDC.

[0060] Example 6 ory PNMT acl HMT immobilized enzyme efficiency and activity Unlike Example 5, 1 mg of [a specific ingredient] was added. ory PNMT enzyme and acl HMT enzyme was used to measure the A280 of the supernatant. After rotational binding at 25°C for 20 hours, the A280 of the supernatant was measured again. The supernatant was discarded, leaving solid particles, which were washed three times with pH 8.0 phosphate buffer. The reaction solution was added, and the product formation was detected by high-performance liquid chromatography. Figure 6 As shown in (a) and (b), the fixation was performed using LXTE-700 amino resin. ory PNMT exhibits the highest activity, with ESQ-3 type amino resin immobilization. acl HMT exhibits high fixation efficiency and activity.

[0061] Example 7 h PNMT, acl HMT-P8L / W27F / V265W Immobilized Enzyme Efficiency and Activity Unlike Example 6, 1 mg of [a specific ingredient] was added. h PNMT enzyme and acl HMT-P8L / W27F / V265W enzyme was used to measure the A280 of the supernatant. After rotational binding at 25℃ for 20 h, the A280 of the supernatant was measured again. The supernatant was discarded, leaving solid particles, which were washed three times with pH 8.0 phosphate buffer. The reaction solution was added, and the product formation was detected by high-performance liquid chromatography. Figure 7 As shown in (a) and (b), the fixation was performed using LXTE-700 amino resin. h PNMT exhibits the highest activity, with ESQ-3 type amino resin immobilization. acl HMT-P8L / W27F / V265W exhibits high fixation efficiency and activity.

[0062] Example 8: Modular multi-enzyme-level synthesis of adrenaline The system for synthesizing adrenaline and its analogues using the multi-enzyme-level combination provided in Example 1 was used.

[0063] 1) Preparation of norepinephrine synthesis module: Weigh 2 g of ESQ-3 amino resin carrier, wash three times with pH 8.0 phosphate buffer, add 10 mL of 2.5% glutaraldehyde, and incubate at 25℃ for 2 h by rotational binding. Wash three more times with phosphate buffer. Then, add an equal weight of wet cell lysate containing threonine aldolase and tyrosine decarboxylase, and incubate for 20 h on a rotary apparatus. Wash the unbound enzyme protein with pH 8.0 phosphate buffer, freeze-dry, and pack the immobilized enzyme into a 4.6 × 200 mm tubular reactor to prepare the first flow reactor for norepinephrine preparation; wherein, the cell lysate containing threonine aldolase is engineered bacteria BL21(DE3) / pET28(a)- bl The cell lysate for LTA and the cell lysate for tyrosine decarboxylase were engineered bacteria BL21(DE3) / pET28(a)- sf TDC cell lysis fluid.

[0064] 2) Preparation of methylation synthesis module: Weigh 0.7 g LXTE-700 and 0.3 g ESQ-3 amino resin carrier, wash three times with pH 8.0 phosphate buffer, add 10 mL 2.5% glutaraldehyde, and spin-bind for 2 h at 25℃. Wash three times with phosphate buffer, then add 12 mg phenylethanolamine N-methyltransferase and 3 mg methyltransferase, and bind for 20 h on a spin apparatus. Wash the unbound enzyme protein with pH 8.0 phosphate buffer, freeze-dry, and pack the immobilized enzyme into a 4.6 × 100 mm tubular reactor to prepare a flow reactor for methylation preparation.

[0065] 3) Continuous Flow Reaction: Prepare a 100 mL reaction solution system containing 50 mM protocatechuic aldehyde, 1 M glycine, and 0.5 mM pyridoxal phosphate, in a pH 6.5 phosphate buffer. The reaction solution is pumped into the norepinephrine synthesis reactor using a horizontal flow pump. Before entering the next stage reactor, a four-way valve is used to deliver 0.1 mM S-adenosyl-L-homocysteine ​​and 50 mM methyl p-toluenesulfonate. The concentration of norepinephrine exiting the first stage reactor is monitored in real time. If the concentration is low, the reaction can be circulated. After the reaction solution exits the methylation reactor, the final product, epinephrine, is obtained, and the yield is detected by liquid chromatography. Figure 8 As shown, the yield of the product adrenaline and the space-time conversion of the reactor are displayed at different flow rates. At a flow rate of 80 μL / min, the continuous flow system has a high yield and space-time conversion, reaching 3.31 g / L and 9.56 g / L / h, respectively.

[0066] Example 9 The difference between this embodiment and Embodiment 8 is that in this embodiment, the wet cell weight ratio (cell lysate weight ratio) of threonine aldolase and tyrosine decarboxylase in the norepinephrine module is adjusted to 0.5:1, 1:1, 2:1, 4:1, and 8:1. Following the method described in Embodiment 5, the content of synthesized adrenaline is detected at a flow rate of 80 μL / min. Figure 9 As shown, the continuous flow system exhibits the highest space-time yield of 14.3 g / L / h when the wet cell weight ratio of threonine aldolase to tyrosine decarboxylase is 4:1.

[0067] Example 10 The difference between this embodiment and Embodiment 8 is that in this embodiment, the flow rate ratio of the substrate supply pump (PUMP1) and the methyl cofactor supply pump (PUMP2) is adjusted to 1:1, 1:2, 1:3, 1:4, and 1:5, respectively. Following the method described in Embodiment 8, the content of synthesized adrenaline is detected. Figure 10 As shown, when the flow rate ratio is 1:4, the continuous flow system has the highest spacetime productivity, reaching 10.8 g / L / h.

[0068] Example 11 Modular multi-enzyme-level synthesis of ethyl epinephrine Ethyl epinephrine was synthesized using the multi-enzyme-level synthesis system for epinephrine and its analogues provided in Example 1.

[0069] 1) Preparation of norepinephrine synthesis module This embodiment is basically the same as that of embodiment 8, the main difference being: in this embodiment, the wet cell weight ratio of threonine aldolase and tyrosine decarboxylase is 4:1, and the other methods are the same; 2) Preparation of the ethylation synthesis module: This example is basically the same as Example 8, the main difference being that 20 mg of hPNMT and 5 mg of [unclear text] are added in this example. acl For HMT-P8L / W27F / V265W, the other methods are the same; 3) Continuous flow reaction: This example is basically the same as Example 8, the main difference being that the alkyl donor is replaced with iodoethane in this example, while the other methods are the same; such as Figure 11 As shown, the yield of the product adrenaline and the space-time conversion of the reactor are displayed at different flow rates. At a flow rate of 80 μL / min, the continuous flow system has a high yield and space-time conversion, reaching 0.53 g / L and 0.86 g / L / h, respectively.

[0070] Example 12 Modular continuous flow synthesis of adrenaline and ethyl adrenaline The system for synthesizing adrenaline and its analogues using the multi-enzyme-level combination provided in Example 1 was used to synthesize adrenaline. The flow reactor used for synthesizing norepinephrine was the same as in Example 10, the flow reactor used for synthesizing adrenaline was the same as in Example 8, and the flow reactor used for synthesizing ethyl adrenaline was the same as in Example 10.

[0071] A 100 mL reaction mixture containing 50 mM protocatechuic aldehyde, 1 M glycine, and 0.5 mM pyridoxal phosphate, along with pH 6.5 phosphate buffer, was prepared and fed into the norepinephrine synthesis reactor at a flow rate of 20 μL / min. Additionally, methyl and ethyl donors were fed via a constant flow pump at a flow rate of 80 μL / min. Products were collected every 2 h, and their concentrations were determined by HPLC. The methylation module contained 0.1 mM S-adenosyl-L-homocysteine ​​and 50 mM methyl p-toluenesulfonate, while the ethylation module contained 0.1 mM S-adenosyl-L-homocysteine ​​and 50 mM iodoethane. Figure 12 As shown, after 48 hours of continuous operation, the space-time yield of adrenaline remained at 8.83 g / L / h, and the space-time yield of ethyl adrenaline remained at 0.8 g / L / h. This figure demonstrates that the multi-enzyme-level synthesis system for adrenaline and its analogues provided in this embodiment exhibits good stability and can continuously and stably synthesize adrenaline and ethyl adrenaline.

[0072] Example 13: Application of the SAM cycle module in the synthesis of linderine and N-methyllinderine Prepare a 100 mL reaction solution system containing 10 mM norcopurulent, 0.1 mM SAM, 10 mM iodomethane, and pH 8 phosphate buffer. The continuous flow module consists of an oxygen-methylated copurulentine module and an nitrogen-methylated N-methylcopurulentine module, assembled in series. The copurulentine synthesis module contains... rn COMT and acl The mass ratio of HMT is 5:1, in the N-methylcodone synthesis module cj CNMT and acl The HMT mass ratio was 10:1, and the reaction solution was introduced into the reactor module at a flow rate of 20-150 μL / min. High-performance liquid chromatography (HPLC) was used to detect the formation of the effluent product. Figure 13 As shown, a flow rate of 50 μL / min exhibits a high space-time productivity.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A system for the synthesis of adrenaline and its analogues by a multi-enzyme-level conjugation, characterized in that, include: The norepinephrine synthesis module includes several first flow reactors, norepinephrine synthase, and a protocatechuic aldehyde-glycine storage tank that provides substrate to the first flow reactors. The norepinephrine synthase is encapsulated in several first flow reactors and is composed of immobilized threonine aldolase and tyrosine decarboxylase. A plurality of alkylation modules are provided, each alkylation module comprising an alkylation cofactor reservoir, a plurality of alkylation flow reactors, and an alkylating enzyme, wherein the alkylating enzyme is encapsulated within the plurality of the alkylation flow reactors; the alkylation cofactor reservoir and the first flow reactor are simultaneously connected to the alkylation flow reactors and respectively provide the alkylation cofactor substrate and norepinephrine to the alkylation flow reactors; the alkylating enzyme is composed of immobilized phenylethanolamine-N-methyltransferase and halide methyltransferase.

2. The system according to claim 1, characterized in that, The threonine aldolase mentioned is derived from the intestines of black bears. bl LTA, wherein the tyrosine decarboxylase is derived from Enterococcus faecalis. sf TDC, wherein the phenylethanolamine-N-methyltransferases are of human origin. h PNMT and European hare are the sources ory PNMT; the halide methyltransferase is derived from *Paederia cuspidatum*. acl HMT or its mutants acl HMT-P8L / W27F / V265W.

3. The system according to claim 1 or 2, characterized in that, The immobilized threonine aldolase and tyrosine decarboxylase are simultaneously encapsulated in the same first flow reactor or separately encapsulated in two first flow reactors; the immobilized phenylethanolamine-N-methyltransferase and halide methyltransferase are simultaneously encapsulated in the same alkylation flow reactor or separately encapsulated in two alkylation flow reactors.

4. The system according to claim 3, characterized in that, In the first flow reactor and the alkylation flow reactor, the carriers used to immobilize the corresponding enzymes are long-chain amino resins, such as amino resins of type LXTE-700, ESQ-3, ESR-3 or HA109.

5. The system according to claim 1, characterized in that, The norepinephrine synthesis module also includes a norepinephrine storage tank connected to the first flow reactor. The protocatechuic aldehyde-glycine storage tank, the first flow reactor, and the norepinephrine storage tank are connected by pipelines to form a circulation system. A substrate supply pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the first flow reactor. An intermediate material four-way valve is installed on the pipeline between the first flow reactor and the norepinephrine storage tank. A norepinephrine circulation pump is installed on the pipeline between the protocatechuic aldehyde-glycine storage tank and the norepinephrine storage tank.

6. The system according to claim 1, 2, or 5, characterized in that, The alkylation module further includes a target product storage tank connected to the alkylation flow reactor. The alkylation flow reactor is connected to the first flow reactor, the alkylation cofactor storage tank, and the target product storage tank via pipelines. An alkylation cofactor supply pump and an alkylation material four-way valve are installed on the pipeline between the alkylation flow reactor and the alkylation cofactor storage tank. The alkylation material four-way valve is connected to an intermediate material four-way valve. The target product is adrenaline or an analogue thereof. The alkylation cofactor storage tank is used to hold S-adenosine-L-homocysteine ​​and an alkyl donor, wherein the alkyl donor is methyl p-toluenesulfonate or a haloalkane.

7. The system according to claim 6, characterized in that, The device includes a methylation module and an ethylation module. The methylation module comprises a methyl flow reactor for synthesizing adrenaline, a methyl cofactor reservoir, and an adrenaline reservoir. These three components are connected by a pipeline. A second four-way valve and a methyl cofactor supply pump are installed on the pipeline between the methylation flow reactor and the methyl cofactor reservoir. The methylation flow reactor contains immobilized phenylethanolamine-N-methyltransferase and a halide methyltransferase. The methyl cofactor reservoir contains S-adenosine-L-homocysteine ​​and a methyl donor, where the methyl donor is methyl p-toluenesulfonate or a halomethane. The second four-way valve is connected to the intermediate material four-way valve. The ethylation module includes an ethyl flow reactor for synthesizing ethyl adrenaline, an ethyl cofactor reservoir, and an ethyl adrenaline reservoir. The ethyl flow reactor, ethyl cofactor reservoir, and ethyl adrenaline reservoir are connected by a pipeline. A third four-way valve and an ethyl cofactor supply pump are installed on the pipeline between the ethyl flow reactor and the ethyl cofactor reservoir. The ethyl flow reactor contains immobilized phenylethanolamine-N-methyltransferase and a halide methyltransferase mutant. The ethyl cofactor reservoir contains S-adenosyl-L-homocysteine ​​and haloethane. The third four-way valve is connected to the second four-way valve.

8. A method for synthesizing adrenaline and its analogues using the system according to any one of claims 1-7, comprising the steps of: Synthesizing norepinephrine: Protocatechuic aldehyde and glycine are added to the protocatechuic aldehyde-glycine storage tank and transported to the first flow reactor, where norepinephrine is synthesized. The synthesized norepinephrine is then transported to the alkylation flow reactor. Synthesis of the target product: The alkylation cofactor substrate in the alkylation cofactor reservoir is transported to the alkylation flow reactor, and norepinephrine and the alkylation cofactor substrate are used to synthesize adrenaline or its analogues in the alkylation flow reactor.

9. The method according to claim 8, characterized in that, When the concentration of norepinephrine synthesized in the first flow reactor does not reach the predetermined requirement, the step of synthesizing norepinephrine further includes: first collecting the norepinephrine synthesized in the first flow reactor into a norepinephrine storage tank by controlling the intermediate material four-way valve, and then transporting it to the protocatechuic aldehyde-glycine storage tank by the norepinephrine circulation pump, and continuing to repeat the cycle to synthesize norepinephrine until the concentration of synthesized norepinephrine reaches the predetermined requirement, and then switching the intermediate material four-way valve to transport it to the alkylation flow reactor.

10. The method according to claim 8 or 9, characterized in that, In the step of synthesizing norepinephrine, the first flow reactor is encapsulated with bl LTA cell lysis buffer and sf TDC cell lysis fluid, the bl LTA cell lysis buffer and sf The weight ratio of TDC cell lysis buffer is 0.5:1-8:1, the substrate supply pump flow rate is 10-200 μL / min, and the synthesis temperature is 20-40℃.

11. The method according to claim 10, characterized in that, The bl The weight ratio of LTA cell lysis buffer to sfTDC cell lysis buffer is 1:1-5:1, and the substrate supply pump has a flow rate of 20-100 μL / min.

12. The method according to claim 8, characterized in that, The steps for synthesizing the target product include: placing S-adenosine-L-homocysteine ​​and an alkyl donor in an alkylation cofactor reservoir; controlling a four-way valve for alkylation materials; and feeding S-adenosine-L-homocysteine, the alkyl donor, and norepinephrine together into the alkylation flow reactor via an alkylation cofactor supply pump and a substrate supply pump, respectively; synthesizing adrenaline or an analogue in the alkylation flow reactor; and collecting the product in the target product reservoir; wherein the alkyl donor is methyl p-toluenesulfonate or a haloalkane.

13. The method according to claim 12, characterized in that, The alkylation flow reactor contains pure enzyme solutions of phenylethanolamine-N-methyltransferase and halide methyltransferase, with a concentration ratio of phenylethanolamine-N-methyltransferase to halide methyltransferase of 1:1 to 10:

1. The synthesis temperature is 20-40℃. The flow rate of the cofactor supply pump is 10-200 μL / min, and the flow rate of the substrate supply pump is 10-200 μL / min.

14. The method according to claim 13, characterized in that, The steps for synthesizing the target product include synthesizing adrenaline: S-adenosine-L-homocysteine ​​and a methyl donor are placed in a methyl cofactor reservoir; a second four-way valve is controlled to deliver S-adenosine-L-homocysteine, the methyl donor, and norepinephrine together to a methyl flow reactor via a methyl cofactor supply pump and a substrate supply pump, respectively; adrenaline is synthesized in the methyl flow reactor and collected in the adrenaline reservoir; wherein the methyl donor is methyl p-toluenesulfonate or a halomethane; the methyl flow reactor is encapsulated with… ory PNMT pure enzyme solution and acl HMT pure enzyme solution, and in it ory PNMT enzyme and acl The concentration ratio of HMT enzyme is 1:1-5:1, and the flow rate of the methyl cofactor supply pump is 50-100 μL / min.

15. The method according to claim 13, characterized in that, The steps for synthesizing the target product include the synthesis of ethyl adrenaline: S-adenosine-L-homocysteine ​​and ethane halide are placed in the ethyl cofactor reservoir; the third four-way valve and the second four-way valve are controlled to deliver S-adenosine-L-homocysteine, ethane halide, and norepinephrine together to the ethyl flow reactor; ethyl adrenaline is synthesized in the ethyl flow reactor and collected in the ethyl adrenaline reservoir; the ethyl flow reactor contains encapsulated hPNMT pure enzyme solution and... acl HMT-P8L / W27F / V265W pure enzyme solution, and the hPNMT enzyme and acl The concentration ratio of HMT-P8L / W27F / V265W enzyme is 1:1-5:1, and the flow rate of the methyl cofactor supply pump is 50-100 μL / min.

16. The method according to any one of claims 12-15, characterized in that, The flow rate ratio of the substrate supply pump to each alkylation cofactor supply pump is 1:1 to 1:5.

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