A tyrosine phenol-lyase mutant, a recombinant vector, a recombinant engineering bacteria, a construction method and application thereof

Abstract

The application discloses a tyrosine phenol-lyase mutant, a recombinant vector, a recombinant engineering bacterium, a construction method and application, and belongs to the field of synthetic biology and biological catalysis technology. The application provides a tyrosine phenol-lyase mutant with high temperature tolerance, and further discloses a recombinant vector and an engineering bacterium for co-expressing the mutant and dopa decarboxylase, and a method for catalytically producing dopamine by using the whole cell of the recombinant engineering bacterium. Experiments prove that the tyrosine phenol-lyase mutant has significantly improved thermal stability, and the catalytic efficiency is better than that of the wild type. By using the recombinant engineering bacterium constructed by the application, dopamine can be efficiently produced in one step by using cheap substrates such as catechol, pyruvic acid and ammonium salt through an optimized two-stage variable-temperature catalytic process. The application solves the problems of high substrate cost and poor enzyme stability in the prior art, and has the advantages of simple operation, short cycle, environmental friendliness and suitability for industrial production.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and biocatalysis, and more specifically to a tyrosine phenol lyase mutant, a recombinant vector, a recombinant engineered bacterium, a construction method, and its applications. Background Technology

[0002] Dopamine is the simplest catecholamine neurotransmitter and is widely used clinically to treat acute myocardial infarction, congestive heart failure, and various shock syndromes. At the same time, dopamine is also the most abundant catecholamine neurotransmitter in the brain; its secretion and regulation disorders can lead to various neurological diseases such as Parkinson's disease and schizophrenia. Furthermore, using dopamine as a substrate, through β-hydroxylation and N-methylation modification, widely used catecholamine drugs such as norepinephrine and epinephrine can be further synthesized.

[0003] Currently, the industrial production of dopamine mainly employs chemical synthesis. Existing technologies disclose various chemical synthesis routes. For example, using veratral as a starting material, it undergoes a condensation reaction with nitromethane to generate 3,4-dimethoxy-β-nitrostyrene, followed by catalytic reduction, demethoxylation, and salt formation to obtain dopamine hydrochloride. Another example is the synthesis of dopamine from vanillin through multiple steps including condensation, reduction, and demethylation. However, chemical synthesis methods generally suffer from problems such as long process routes, cumbersome steps, harsh reaction conditions (e.g., high temperature and high pressure), large amounts of organic solvents used, and the generation of large quantities of wastewater, failing to meet the requirements of green and environmentally friendly industrial production.

[0004] Biosynthesis has attracted widespread attention in recent years due to its advantages such as mild reaction conditions and environmental friendliness. Existing technologies have reported the whole-cell catalytic production of dopamine using levodopa as a substrate and genetically engineered bacteria expressing dopa decarboxylase (DDC). This method achieves highly efficient whole-cell catalytic conversion of levodopa to dopamine, featuring high synthesis efficiency, no need for cofactors, and simple operation. However, levodopa itself is a relatively expensive pharmaceutical raw material, resulting in high production costs for dopamine and hindering large-scale industrial application.

[0005] On the other hand, some studies have attempted to synthesize dopamine through de novo fermentation using glucose as a substrate. For example, dopamine-producing strains have been constructed by knocking out genes such as tynA and tyrR in E. coli, overexpressing genes such as aroGfbr and tyrAfbr, and heterologously expressing the dopamine decarboxylase gene. However, this process has a long fermentation cycle (usually exceeding 60 hours), requires precise control of glucose feeding, is complex, and suffers from problems such as metabolic bypass competition, thus its production efficiency needs to be improved.

[0006] Tyrosine phenol-lyase (TPL) catalyzes the synthesis of levodopa from catechol, pyruvate, and ammonium salts. This reaction utilizes inexpensive substrates, offering a significant cost advantage. Existing techniques have reported on the enzymatic synthesis of levodopa using TPL, for example, by co-expressing tyrosine phenol-lyase and the cofactor pyridoxal-5-phosphate synthase (PdxST) in *E. coli*, which can increase levodopa yield. Theoretically, coupling TPL enzymes with dopamine decarboxylases could achieve a one-step synthesis of dopamine from inexpensive substrates. However, existing TPL enzymes generally exhibit poor thermostability, easily becoming inactive and exhibiting low catalytic efficiency at higher temperatures (e.g., 37-40°C), limiting their application in industrial production. Furthermore, optimizing the reaction conditions of the dual-enzyme coupling system to achieve efficient cascade catalysis remains a pressing technical challenge.

[0007] Therefore, how to provide a thermostable tyrosine phenol lyase mutant and construct a recombinant engineered bacterium that simultaneously expresses the mutant and dopa decarboxylase to achieve efficient one-step production of dopamine using inexpensive substrates such as catechol, pyruvate, and ammonium salts, and to achieve efficient cascade catalysis, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a tyrosine phenol lyase mutant, a recombinant vector, a recombinant engineered bacterium, a construction method, and applications. A tyrosine phenol lyase mutant with improved thermostability was successfully prepared, and a recombinant engineered bacterium expressing both the tyrosine phenol lyase mutant and dopa decarboxylase was constructed. This enabled the efficient one-step production of dopamine using inexpensive raw materials such as catechol, pyruvate, and ammonium salts as catalytic substrates. Furthermore, by optimizing the assembly sequence of the dual-enzyme recombinant vector, efficient cascade catalysis was achieved, increasing the yield of dopamine.

[0009] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this application is to provide a tyrosine phenol lyase mutant TPL-M1, the amino acid sequence of which is shown in SEQ ID NO.20.

[0010] Another object of this application is to provide a nucleic acid molecule encoding the tyrosine phenol lyase mutant TPL-M1, the nucleotide sequence of which is shown in SEQ ID NO.21.

[0011] Another object of this application is to provide: a recombinant vector comprising a first expression cassette and a second expression cassette; the first expression cassette comprising a sequence encoding a dopa decarboxylase; the second expression cassette comprising a sequence encoding a tyrosine phenol lyase mutant TPL-M1; the recombinant vector being pCSA-DDC-TPL, the nucleotide sequence of which is shown in SEQ ID NO.30.

[0012] Another objective of this application is to provide a method for constructing the recombinant vector, wherein a first expression cassette containing a dopa decarboxylase coding sequence and a second expression cassette containing a tyrosine phenol lyase mutant TPL-M1 coding sequence are sequentially assembled into a pCSA vector via a three-level assembly system of L0, L1, and L2 to obtain the recombinant vector pCSA-DDC-TPL. The PCSA vector is a high-copy-number vector. In the whole-cell catalytic synthesis of dopamine, strains containing the recombinant vector can produce a large amount of enzyme in a short time. High-copy-number vectors can better improve the expression level of the target protein than traditional medium / low-copy-number vectors like PET28. Simultaneously, the complex metabolic network within cells may lead to competition between the target product and the cell's own growth needs for substrates. The extremely high enzyme quantity provided by the high-copy-number vector can act like a pump, forcibly "siphoning" carbon sources or precursors into the target synthetic pathway, thereby achieving a large accumulation of the target product in a short time.

[0013] Another object of this application is to provide: a recombinant engineered bacterium comprising the said recombinant vector.

[0014] Another object of this application is to provide a method for constructing the recombinant engineered bacteria, wherein the recombinant vector pCSA-DDC-TPL is introduced into a host strain to construct the recombinant engineered bacteria.

[0015] Another object of this application is to provide the use of the recombinant engineered bacteria in the preparation of dopamine.

[0016] Another object of this application is to provide: a method for producing dopamine, comprising the following steps: (1) The recombinant engineered bacteria were cultured and the bacterial cells were collected after induction of expression; (2) Using the bacterial cells collected in step (1) as a catalyst, a catalytic reaction is carried out in a reaction system containing catechol, sodium pyruvate and ammonium acetate; (3) Collect dopamine from the reaction solution.

[0017] As a preferred technical solution, the catalytic reaction in step (2) is carried out in two stages: the first stage is carried out at 10-20℃ for 3-5 hours, and the second stage is carried out at 25-35℃ for 5-12 hours.

[0018] As a preferred technical solution, the reaction system in step (2) comprises the following components at final concentrations: catechol 6-18 g / L, sodium pyruvate 10-22 g / L, ammonium acetate 30 g / L, sodium sulfite 4 g / L, EDTA-2Na 2 g / L, pyridoxal phosphate 0.1 mM, Triton X-100 volume fraction of 5‰, ascorbic acid 0.1 mM, and pH 7.0-8.5. By adding 0.1%-0.5% Triton X-100 (a nonionic surfactant), the release of intracellular proteins is increased, which can significantly increase the substrate catalytic efficiency and increase the yield.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Technical effects at the enzyme molecule level Significantly improved thermal stability: By rationally designing and introducing mutations at three sites (S17K, A110M, and R142I), the resulting tyrosine phenol lyase mutant TPL-M1 exhibits significantly enhanced tolerance to high temperatures. Specifically, after placing the mutant-containing bacterial cells at 37°C for 4 hours, their enzyme activity was higher than that of the wild-type TPL under the same conditions.

[0020] Improved catalytic efficiency: While exhibiting enhanced thermal stability, the mutant TPL-M1 also demonstrated superior catalytic efficiency compared to the wild type. Experimental data showed that the mutant strain exhibited a higher catalytic rate for catechol substrates than the wild-type strain.

[0021] (2) Technical effects at the process level A two-stage temperature-controlled catalytic process (low temperature followed by optimal temperature) ranging from 15°C to 25°C effectively matched the optimal reaction temperatures of TPL and DDC enzymes, resulting in a significantly higher dopamine yield compared to a single isothermal (15°C or 25°C) catalytic process. This demonstrates that this temperature strategy can synergistically optimize the efficiency of the two-enzyme cascade reaction.

[0022] Synergistic effect of surfactants: Adding 5‰ Triton X-100 to the reaction system significantly increased dopamine production by increasing cell membrane permeability, promoting substrate entry and product release.

[0023] (3) Technical effects at the overall technical solution level The recombinant vector pCSA-DDC-TPL was assembled using the Golden Gatede method. This system boasts high layer-by-layer assembly accuracy and a short cycle time, significantly reducing working time and allowing for substantial flexibility in subsequent modifications to the recombinant vector. Furthermore, a one-step synthesis of dopamine from inexpensive substrates was achieved: a recombinant engineered bacterium (strain 001) co-expressing TPL-M1 and DDC was constructed, successfully enabling the direct one-step synthesis of dopamine using inexpensive industrial raw materials such as catechol, pyruvate, and ammonium acetate via whole-cell catalysis. This completely circumvents the cost bottleneck associated with using expensive substrates (L-DOPA).

[0024] Simple to operate and short cycle: Compared with the cumbersome steps of chemical synthesis and the long cycle of de novo fermentation (>60 hours), the whole-cell catalysis method of the present invention only requires simple cell culture, induction and catalytic reaction (reaction time 6-9 hours), the process is significantly simplified and suitable for industrial production.

[0025] Environmentally friendly: The entire reaction is carried out in an aqueous buffer system, eliminating the need for organic solvents, reducing wastewater generation, and meeting the requirements of green chemistry.

[0026] In summary, this application achieves the overall technical effect of efficiently producing the high-value chemical dopamine using inexpensive substrates by organically combining enzyme molecule modification (improved thermal stability) and process optimization (temperature regulation, surfactants, genome assembly), thus solving the two core problems of high substrate cost and poor enzyme stability in existing technologies. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 For: L1 and L2 recombinant vectors used in this patent.

[0029] Figure 2 The results of whole-cell catalytic experiments were performed to determine the enzyme activity of TPL enzymes with site mutations and those without modification after being placed under high-temperature conditions.

[0030] Figure 3 To: The effect of different temperatures on dopamine production during whole-cell catalysis. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0032] Construction of recombinant plasmids (L2-level plasmids constructed through L0→L1→L2 three-level assembly) and engineered bacteria ( Figure 1 ), as detailed below: S1. Construct L0 level components: (pUAP1 empty carrier + general-purpose component / special-purpose component) The L0-level element is a homologous recombination vector composed of a vector backbone and elements. The vector backbone contains BbsI and BsaI restriction endonuclease recognition sites. The elements include universal elements composed of promoter, RBS, and terminator fragments, and special elements composed of target gene CDS fragments. The ends of the universal element fragments are pre-programmed with BbsI recognition sequences; and all BbsI, BsaI, and SapI recognition sites within each element have been removed by synonymous mutations, as follows: (1) Vector backbone: pUAP1 (source: https: / / doi.org / 10.1111 / nph.13532, the properties and construction method of the plasmid are described in this link) is used as an empty vector. This empty vector has a BbsI recognition site (GAAGAC) added to the red fluorescent protein fragment. On the pUAP1 backbone, there is a BsaI recognition site (GGTCTC) in the direction of the red fluorescent protein fragment.

[0033] (2) General-purpose components: selected from promoters, RBSs, or terminators; The aforementioned promoter, RBS, or promoter each has a preset sequence at both ends, and the composition of the preset sequence is as follows: Two protective bases (gc) - BbsI recognition site (GAAGAC) - two spacer bases (tc) - BbsI cleavage site (CTCA) - BsaI pre-cleavage site.

[0034] When the general-purpose element is selected from the promoter, its BsaI pre-cleavage site is GGAG / TACT; When the general-purpose element is selected from RBS, its BsaI pre-cleavage site is TACT / AATG; When the general-purpose element is selected from the terminator, its BsaI precut site is GCTT / CGCT; Taking a general-purpose component as an example of a promoter: the preset sequence at both ends can be gcGAAGACtcCTCAGGAG, SEQ ID NO.1; or gcGAAGACtcCTCATACT, SEQ ID NO.2.

[0035] The following is a general L0-level component construction method and specific sequence: pUAP1-T7lac: 1) Preparation of dsDNA: The sequence for constructing the T7 promoter + lac operon was referenced from BBa K3633015 on the iGEM Registry website, with the sequence: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCC, SEQ ID NO.3. Primers were designed based on this sequence, and the following bidirectional primers were synthesized by Genewiz: 5'-gcGAAGACtcCTCAggagTAATACGACTCACTATAGGGGAATTGTG AGCGGATAACAATTCCtactCGAGagGTCTTCga-3', SEQ ID NO.4; 5'-tcGAAGACctCTCGagtaGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTActccTGAGgaGTCTTCgc-3', SEQ ID NO.5; The two artificially synthesized, complementary single-stranded DNA oligonucleotides (Oligos) were annealed to form double-stranded DNA (dsDNA) fragments, as follows: According to the DNA annealing buffer system in Table 1, the components were added sequentially, gently tapped to mix, centrifuged, and annealed in a PCR instrument. The annealing reaction program was: 95℃ for 5 min, cooling to 25℃ at 0.1℃ / s for 5 min, and 4℃ ∞ to obtain dsDNA.

[0036] Table 1 DNA annealing buffer system 2) Assembly of L0-level elements with pUAP1 vector: Following the L0-level element reaction system in Table 2 and the GoldenGate assembly procedure in Table 3, add the components sequentially, gently tap the bottom of the tube to mix, centrifuge, and perform Golden Gate assembly in a PCR instrument to prepare pUAP1-T7lac.

[0037] Table 2 L0-level component reaction system Table 3 GoldenGate Assembly Procedure pUAP1-MRBS (Medium ribosome binding site): 1) Preparation of dsDNA: The sequence for constructing MRBS was referenced from the iGEM Registry website: BBa B0034, specifically the sequence aaagaggagaaa, SEQ ID NO. 6. Primers were designed using this sequence as a reference, and the following bidirectional primers were synthesized by Genewiz: 5'-gcGAAGACtcCTCAtactaaagaggagaaaaatgCGAGagGTCTTCga-3', SEQ ID NO.7; 5'-tcGAAGACctCTCGcatttttctcctctttagtaTGAGgaGTCTTCgc-3', SEQ ID NO. 8.

[0038] The two artificially synthesized, complementary single-stranded DNA oligonucleotides (Oligos) were annealed to form double-stranded DNA (dsDNA) fragments, as follows: According to the DNA annealing buffer system in Table 1, the components were added sequentially, gently tapped to mix, centrifuged, and annealed in a PCR instrument. The annealing reaction program was: 95℃ for 5 min, cooling to 25℃ at 0.1℃ / s for 5 min, and 4℃ ∞ to obtain dsDNA.

[0039] 2) Assembly of L0-level elements with pUAP1 vector: Following the L0-level element reaction system in Table 2 and the GoldenGate assembly procedure in Table 3, add the components sequentially, gently tap the bottom of the tube to mix, centrifuge, and perform Golden Gate assembly in a PCR instrument to prepare pUAP1-MRBS.

[0040] pUAP1-T7Terminator: 1) Preparation of dsDNA: The T7 Terminator sequence was referenced from NCBI Reference Sequence: NC_001604.1, derived from T7 bacteriophage, and its nucleotide sequence is as follows: CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG, SEQ ID NO.9; primers were designed based on this sequence: the following bidirectional primers were synthesized by Genewiz. 5'-gcGAAGACtcCTCAgcttCTAGCATAACCCCTTGGGGCCTCTAAACG GGTCTTGAGGGGTTTTTTGcgctCGAGagGTCTTCga-3', SEQ ID NO.10; 5'-tcGAAGACctCTCGagcgCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGaagcTGAGgaGTCTTCgc-3', SEQ ID NO.11; The two artificially synthesized, complementary single-stranded DNA oligonucleotides (Oligos) were annealed to form double-stranded DNA (dsDNA) fragments, as follows: According to the DNA annealing buffer system in Table 1, the components were added sequentially, gently tapped to mix, centrifuged, and annealed in a PCR instrument. The annealing reaction program was: 95℃ for 5 min, cooling to 25℃ at 0.1℃ / s for 5 min, and 4℃ ∞ to obtain dsDNA.

[0041] 2) Assembly of L0-level elements with pUAP1 vector: Following the L0-level element reaction system in Table 2 and the GoldenGate assembly procedure in Table 3, add the components sequentially, gently tap the bottom of the tube to mix, centrifuge, and perform Golden Gate assembly in a PCR instrument to prepare pUAP1-T7Terminator.

[0042] (3) Special elements: selected from tyrosine phenol lyase (TPL) mutant TPL-M1, dopa decarboxylase (DDC), spacer sequence and repressor protein (LacI).

[0043] The following are the construction methods and specific sequences for special type L0 elements: pUAP1-cfTPL(M1): 1) Obtaining the mutant TPL-M1 gene: Tyrosine phenol lyase (TPL) is derived from *Citrobacter freundii*. Using the amino acid sequence (UniProtKB / Swiss-Prot: P31013.1) of the wild-type cfTPL gene (the TPL gene derived from *Citrobacter freundii*) as the starting point for designing the mutation, after codon optimization by GenScript, it was synthesized by Zhonghe Gene. The mutation site was determined through three rounds of PCR amplification, resulting in the tyrosine phenol lyase mutant strain cf, TPL-M1 (containing three mutations: S17K / A110M / R142I). After storage at high temperatures, the enzyme activity of the TPL-M1 strain with the designed mutation site was higher than that of the wild-type cfTPL strain. By modifying TPL with a mutant, the mutant enzyme does not require low-temperature storage during industrial use, reducing storage costs and making it suitable for industrial production. This sequence was used in subsequent experiments.

[0044] The primers for the above three rounds of PCR amplification are as follows: S17K: 5'-CCGTTAAGATGATCCCGCGCG-3', SEQ ID NO.12; 5'-GATCATCTTAACGGTTTCGACGCTTTTGAT-3', SEQ ID NO.13; A110M: 5'-CAGCTGATGATTAAACCGGGTCAGTAC-3', SEQ ID NO.14; 5'-GTTTAATCATCAGCTGACTCAGCAGATTTTCTG-3', SEQ ID NO.15; R142I: 5'-GATATTGTTATCGACGAAGCACACGACG-3', SEQ ID NO. 16; 5'-GCTTCGTCGATAACAATATCGACGAAGACC-3', SEQ ID NO. 17; The amino acid sequence of tyrosine phenol lyase TPL (wild type): MNYPAEPFRIKSVETVSMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLAIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVRDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTEAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYEYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*, SEQ ID NO.18。

[0045] Nucleotide sequence of tyrosine phenol lyase TPL (wild type):

[0046] The amino acid sequence of the tyrosine lyase mutant TPL-M1: MNYPAEPFRIKSVETVKMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLMIKPGQ YVAGNMYFTTTRYHQEKNGAVFVDIVIDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTEAHGIKVFYDATRCVENAYFIKEQEQ GFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYEYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAV FLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHRPKLETVRLLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*, SEQ ID NO.20.

[0047] The nucleotide sequence of the tyrosine lyase mutant TPL-M1 is as follows:

[0048] 2) Assembly of L0-level components with pUAP1 support: Assembly was carried out according to the GoldenGate assembly reaction system in Table 4 and the assembly reaction procedure in Table 3 to obtain pUAP1-cfTPL (M1).

[0049] Table 4 shows the GoldenGate assembly reaction system. pUAP1-ppDDC: Obtaining the PpDDC gene: Dopa decarboxylase (DDC) was derived from Pseudomonas putida, and its amino acid sequence is shown in NCBI:WP_404601127.1. At the same time, the nucleotide sequence of dopa decarboxylase was synthesized by Zhonghe Gene.

[0050] Dopa decarboxylase (DDC) amino acid sequence: MTPEQFRQYGHQLIDLIADYRQSVGDRPVMAQVEPGYLKAALPAGAPQQGEPFEAILDDINTLVMPGLSHWQHPDFYGYFPSNGTLSSVLGDFLSTGLGVLGLSWQSSPALSELEETT LDWVRQLLGLSGQWSGVIQDTASTSTLVALICARERATDYALVRGGLQAEAKPLIVYVSAHAHSSVDKAALLAGFGRTNIRLIATDEQFAMRPDALLAAIEQDLAEGNQPCAVVATTGT TTTTALDPLRQIGEIAQAQGLWLHVDSAMAGSAMILPECRWMWDGIELADSVVVNAHKWLGVAFDCSIYYVRDPQHLIRVMSTNPSYLQSAVDGEVKNLRDWGIPLGRRFRALKLWFML RSEGVEALQQRLRRDLDNARWLAEQVEAAGGWALLAPVQLQTLCIVHRPGGLEGEALDAHTKAWAERLNASGEAYVTPATLNGRWMVRVSVGALPTEREHVERLWQRLQAVVNG*, SEQ ID NO.22.

[0051] Dopa decarboxylase (DDC) nucleotide sequence:

[0052] L0-level component assembly with pUAP1 support: Assembly was carried out according to the GoldenGate assembly reaction system in Table 4 and the assembly reaction procedure in Table 3 to obtain pUAP1-ppDDC.

[0053] pUAP1-spacer: 1) Obtaining the spacer sequence: The spacer nucleotide sequence is: GTAGACACTTTATTAGTTAGATCCGCTGTTATTCTGCACGATTGCGCTTGCCGGTGGAAATTTAAGGACGAGTGTAGTGCACTCACCCTTAGCCTTCAGCGTGACAAGATTAGGGTAGCACCGGAAACAGCGATTTTACGGACAGATAGTACAGTCTGAGACTCAGTCACTGGTCCCTAAACGGGAGCCGGTACGTCGGC, SEQ ID NO.24, synthesized by the neutral gene.

[0054] 2) Assembly of L0-level components with pUAP1 support: Assembly was carried out according to the GoldenGate assembly reaction system in Table 4 and the assembly reaction procedure in Table 3 to obtain pUAP1-spacer.

[0055] pUAP1-LacI:

[0056] 2) Assembly of L0-level components with pUAP1 support: Assembly was carried out according to the GoldenGate assembly reaction system in Table 4 and the assembly reaction procedure in Table 3 to obtain pUAP1-LacI.

[0057] (4) Verification of L0 level components 1) Conversion and screening The L0-level elements (pUAP1-T7lac, pUAP1-MRBS, pUAP1-T7Terminator, pUAP1-cfTPL(M1), pUAP1-ppDDC, pUAP1-spacer, and pUAP1-LacI) obtained above were introduced into E. coli DH5α competent cells, and single clones were obtained through resistance selection. The specific process is as follows: Transformation: Take 5 μL of ligation product, add it to 100 μL of competent cells in an ice bath, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 45 s (to create pores in the cell membrane and allow the plasmid to enter the cell), and immediately incubate on ice for 2 min; add 900 μL of LB liquid medium (antibiotic-free), and culture at 37℃ and 200 rpm for 1 h (to allow the bacteria to recover and express the plasmid resistance gene).

[0058] Screening for single clones: Take 100 μL of culture and spread it evenly on an LB solid plate containing the corresponding antibiotic (pUAP1 contains the chloramphenicol resistance gene); incubate upside down at 37°C for 12-16 h, and single clones will form on the plate.

[0059] 2) Validation: The L0-level components were validated using PCR. The reaction system and procedure for validation are shown in Tables 5 and 6 below. The L0-level components that were validated correctly were then used for subsequent operations.

[0060] Table 5 PCR reaction system Note: The primers in Table 5 are common primers on the pUAP1 backbone, and all L0 level elements can be verified using these primers.

[0061] Table 6 PCR reaction procedure S2. Construct L1 level components: (pCK series empty carrier + L0 level components) The correctly verified L0-level components (pUAP1-T7lac, pUAP1-MRBS, pUAP1-T7Terminator, pUAP1-cfTPL(M1), pUAP1-ppDDC, pUAP1-spacer, and pUAP1-LacI) from step S1 are assembled into pCK series carriers to obtain the corresponding L1-level components. The specific process is as follows: (1) Vector backbone: The empty plasmids pCK1, pCK2, pCK3 or pCK4 are used as the vector backbone (source: https: / / doi.org / 10.1021 / acssynbio.9b00511, which describes the properties and construction methods of the plasmids). The empty pCK1, pCK2, pCK3 and pCK4 vectors all have a BsaI recognition site (GGTCTC) facing outward from the lacZ (blue-white screening) fragment, and the sticky ends generated after cutting are GGAG / CGCT; the pCK backbone has a SapI recognition site (GCTCTTC) facing the lacZ fragment, and the sticky ends generated after cutting are 3bp (pCK1: ATG / GCA, pCK2: GCA / TAC, pCK3: GCA / CAG, pCK4: CAG / GGT).

[0062] (2) Assembly of L1-level components: According to the GoldenGate assembly reaction system in Tables 7, 8 and 9 and the assembly procedure in Table 3, the corresponding L0-level components were assembled with the pCK empty carrier to obtain the following L1-level components: PCK1-T7lac-ppDDC-TT7: Contains the T7lac promoter, MRBS, ppDDC gene, and T7 terminator; PCK2-T7lac-cfTPL-TT7: contains the T7lac promoter, MRBS, cfTPL-M1 gene and T7 terminator; PCK3-spacer: Contains a spacer sequence; PCK4-LacI: Contains the LacI repressor protein gene; PCK1-T7lac-ppDDC-TT7 and PCK2-T7lac-cfTPL-TT7 are the target gene elements. PCK3-spacer represents an unintentional base sequence used only to support the connection between the front and back ends of the system. PCK4-LacI is intended to induce expression of the target gene by using IPTG to disable the repressor protein.

[0063] Table 7 GoldenGate Assembly Reaction System Table 8. GoldenGate assembly reaction system of PCK3-spacer Table 9. GoldenGate assembly reaction system of PCK4-LacI (3) Verification of L1 level components: 1) Conversion and screening: The L1-level elements obtained above were introduced into E. coli DH5α competent cells, and single clones were obtained through resistance selection. The specific process is as follows: Transformation: Take 5 μL of ligation product, add 100 μL of competent cells in an ice bath, mix gently, and incubate on ice for 30 min; Heat shock at 42℃ for 45 seconds (to create pores in the cell membrane and allow plasmids to enter the cell), then immediately place on ice for 2 minutes; add 900 μL LLB liquid culture medium (antibiotic-free), and culture at 37℃ with shaking at 200 rpm for 1 hour (to allow the bacteria to recover and express the plasmid resistance gene).

[0064] Screening for single clones: Take 100 μL of culture and spread it evenly on an LB solid plate containing the corresponding antibiotic (pCK1 contains the kanamycin gene); incubate upside down at 37°C for 12-16 h, and single clones will form on the plate.

[0065] 2) Verification: This verification method can be used (either by PCR or by sequencing companies). The primer sequences used are the common sequences on the pCK1-4 backbone. The PCR reaction system is shown in Table 10, and the PCR reaction procedure is shown in Table 11. Table 10 PCR Reaction System Table 11 PCR reaction procedure S3. Construct L2 level components (pCSA empty carrier + L1 level components) The L1-level elements verified in step S2 were assembled into the pCSA empty vector to obtain the final multi-gene co-expression L2-level plasmid. The specific process is as follows: (1) Vector backbone: pCSA empty plasmid was used as the vector backbone (source: https: / / doi.org / 10.1021 / acssynbio.9b00511, the properties and construction method of the plasmid are described in this link). pCSA empty plasmid has a SapI recognition site (gaagagc) and sticky ends generated by cutting are ATG / GGT. This vector has spectinomycin resistance gene and is used to receive the assembly of L1 level elements.

[0066] (2) Assembly of L2-level elements: Following the GoldenGate assembly reaction system in Table 12 and the assembly reaction procedure in Table 3, the L1-level elements were assembled with the pCSA empty vector to obtain the L2-level plasmid, named pCSA-T7lac-ppDDC-TT7-T7lac-cfTPLM1-TT7-LacI (abbreviated as pCSA-DDC-TPL). Nucleotide sequence:

[0067] Table 12 GoldenGate Assembly Reaction System (3) Verification of L2 level components: 1) Conversion and screening: Transformation: Take 5 μL of ligation product, add 100 μL of competent cells in an ice bath, mix gently, and incubate on ice for 30 min.

[0068] Heat shock at 42℃ for 45 seconds (to create pores in the cell membrane and allow plasmids to enter the cell), then immediately place on ice for 2 minutes; add 900 μL LLB liquid culture medium (antibiotic-free), and culture at 37℃ with shaking at 200 rpm for 1 hour (to allow the bacteria to recover and express the plasmid resistance gene).

[0069] Screening for single clones: Take 100 μL of culture and spread it evenly on an LB solid plate containing the corresponding antibiotic (pCSA contains the spectinomycin resistance gene); incubate upside down at 37°C for 12-16 h, and single clones will form on the plate.

[0070] 2) Validation: L2 level elements can be validated using PCR, with primer sequences from the pCSA backbone. The PCR reaction system is shown in Table 13, and the PCR reaction procedure is shown in Table 14. Table 13 PCR Reaction System Table 14 PCR Reaction Procedure S4. Construction of engineered bacteria The validated L2-level recombinant plasmid pCSA-DDC-TPL was transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing spectinomycin, and incubated overnight at 37°C. Single clones were picked, and after PCR validation, positive clones were activated in LB liquid medium (containing spectinomycin), and sterile glycerol was added to a final concentration of 20%, stored at -80°C, and named the engineered strain EC-BL21 / pCSA-DDC-TPL (or simply strain 001). Example 2

[0071] Thermal stability verification of tyrosine lyase mutant TPL-M1 Obtaining mutants: This embodiment is used to verify the improved thermal stability of the mutant TPL-M1 (containing mutations at three sites: S17K, A110M, and R142I) obtained through rational design compared to the wild-type cfTPL. The specific mutation process is as follows: Using the optimized cfTPL gene (synthesized by Zhonghe Gene) as a template, three rounds of PCR amplification were performed to determine the mutation sites, resulting in mutants TPL-S17K, TPL-A110M, and TPL-R142I, and mutations at sites 17, 11, 0, and 142, respectively, to obtain TPL-M1 containing the three mutation sites. The specific primer sequences for amplification are as follows: S17K: 5'-CCGTTAAGATGATCCCGCGCG-3', SEQ ID NO.12; 5'-GATCATCTTAACGGTTTCGACGCTTTTGAT-3', SEQ ID NO.13; A110M: 5'-CAGCTGATGATTAAACCGGGTCAGTAC-3', SEQ ID NO.14; 5'-GTTTAATCATCAGCTGACTCAGCAGATTTTCTG-3', SEQ ID NO.15; R142I: 5'-GATATTGTTATCGACGAAGCACACGACG-3', SEQ ID NO. 16; 5'-GCTTCGTCGATAACAATATCGACGAAGACC-3', SEQ ID NO. 17; The amino acids and nucleotides of TPL-S17K, TPL-A110M, TPL-R142I, and TPL-M1 obtained by the above amplification are as follows: The amino acid sequence of TPL-S17K is as follows: MNYPAEPFRIKSVETVKMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLAIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVRDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTAAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYWYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*, SEQ ID NO.33。

[0072] The nucleotide sequence of TPL-S17K is as follows:

[0073] The amino acid sequence of TPL-A110M is as follows: MNYPAEPFRIKSVETVSMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLMIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVRDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTAAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYWYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*, SEQ ID NO. 35.

[0074] The nucleotide sequence of TPL-A110M is as follows:

[0075] The amino acid sequence of TPL-R142I is as follows: MNYPAEPFRIKSVETVSMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLAIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVIDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTAAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYWYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*, SEQ ID NO.37.

[0076] The nucleotide sequence of TPL-R142I is as follows:

[0077] The amino acid sequence of TPL-M1 is shown in SEQ ID NO.20.

[0078] The TPL-M1 nucleotide sequence is shown in SEQ ID NO.21.

[0079] (2) Construction of the recombinant vector: The mutants obtained in step (1) were recombined with pUAP1 to construct TPL expression plasmids containing only a single site mutation, namely pUAP1-cfTPL-S17K, pUAP1-cfTPL-A110M, pUAP1-cfTPL-R142I, pUAP1-cfTPL-M1, and wild-type pUAP1-cfTPL-WT. The construction method was the same as that for pUAP1-cfTPL (M1) in Example 1, but only the corresponding single mutation was introduced.

[0080] (3) Validation of the recombinant vector: The recombinant vector obtained above was introduced into E. coli DH5α competent cells, and single clones were obtained through resistance selection. The specific process is as follows: Transformation: Take 5 μL of ligation product, add it to 100 μL of competent cells in an ice bath, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 45 s (to create pores in the cell membrane and allow the plasmid to enter the cell), and immediately incubate on ice for 2 min; add 900 μL of LB liquid medium (antibiotic-free), and culture at 37℃ and 200 rpm for 1 h (to allow the bacteria to recover and express the plasmid resistance gene).

[0081] Screening for single clones: Take 100 μL of culture and spread it evenly on an LB solid plate containing the corresponding antibiotic (pUAP1 contains the chloramphenicol resistance gene); incubate upside down at 37°C for 12-16 h, and single clones will form on the plate.

[0082] Validation: L0-level components were validated using PCR. The reaction system and procedure for validation are shown in Tables 5 and 6 below. Subsequent operations were performed on L0-level components that were validated correctly.

[0083] (4) Construction of recombinant strains: Following the method for constructing L1 and L2 in Example 1, four recombinant expression vectors were finally constructed: pCSA-T7lac-cfTPLs17k-TT7, pCSA-T7lac-cfTPLA110M-TT7, pCSA-T7lac-cfTPLR142I-TT7, and pCSA-T7lac-cfTPLM1-TT7. A wild-type control group, pCSA-T7lac-cfTPL-WT-TT7, was also set up. The above recombinant vectors were then transformed into BL21(DE3) to obtain recombinant strains, which were named 002, 003, 004, 005, and 006, respectively. Among them, strains 002, 003, 004, and 005 differed only in the amino acid sequence at the mutation site.

[0084] (5) Protein expression stage of the strain 1) Remove the strain from the -80℃ freezer and streak it on an LB agar plate (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, containing spectinomycin resistance) and incubate at 37℃ and 220 rpm for 16 h.

[0085] 2) Pick a single colony from the plate and inoculate it into liquid LB (containing spectinomycin resistance), 37℃, 220rpm, for 12h activation.

[0086] 3) The activated bacterial culture was inoculated at a 1% inoculum into TB medium (tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.54 g / L), and cultured at 37℃ and 220 rpm. When the OD value was between 0.6 and 0.8, 0.2 mM-0.5 mM IPTG was added, and the culture was incubated overnight at 20℃-25℃ with a shaker at 220 rpm. Under these induction conditions, the expression level of soluble protein was high and the number of inclusion bodies was low.

[0087] 4) Centrifuge the bacterial culture every other day (4℃, 4000rpm, 15min) to separate the bacterial cells from the culture medium and obtain wet bacterial cells containing the target enzyme (the target enzyme remains inside the cell) for subsequent catalytic reactions.

[0088] (6) Catalytic reaction stage (thermal stability verification) 1) High-temperature treatment of bacterial cells The wet bacterial cells collected in step (5) expressing tyrosine phenol lyase mutants S17K, A110M, R142I, and TPLM1 (corresponding to strains 002, 003, 004, and 005), as well as the wet bacterial cells of the wild-type strain (006) as a positive control and the wet bacterial cells of BL21(DE3) as a negative control (007), were placed in a 37°C incubator for 4 hours. The purpose of this step was to investigate the effect of mutations at different sites on the thermostability of tyrosine phenol lyase (TPL) by subjecting the bacterial cells to high-temperature stress treatment, i.e., to compare the residual enzyme activity of each mutant enzyme after high-temperature treatment.

[0089] 2) Catalytic reaction (detection of residual enzyme activity) Preparation of reaction solution: According to the formula in Table 15, weigh out catechol (10 g / L), sodium pyruvate (18 g / L), ammonium acetate (30 g / L), EDTA-2Na (2 g / L) and sodium sulfite (4 g / L), and dissolve them thoroughly in KPi buffer at pH 7.5 to prepare the reaction solution.

[0090] Resuspending the bacterial cells: Using the reaction solution prepared above, resuspend the bacterial cell precipitates of each strain (002, 003, 004, 005, 006, 007) after being treated at 37°C for 4 hours, and adjust the concentration of the resuspension solution to OD600 = 20.

[0091] Setting up the reaction system: 20 mL of the bacterial suspension with OD600=20 was placed in a 250 mL Erlenmeyer flask, and then the following components were added: Triton X-100 to a final volume concentration of 5‰; Pyridoxal phosphate (PLP) to a final concentration of 0.1 mM; The final concentration of ascorbic acid is 0.1 mM.

[0092] Reaction and Detection: The Erlenmeyer flask was placed in a shaker at 15°C and 220 rpm for 6 hours. After the reaction was complete, the reaction solution was collected, filtered through a microporous membrane, and the amount of levodopa produced was determined by high-performance liquid chromatography (HPLC). The experimental results are as follows: Figure 2 As shown (BL21 represents the negative control of the bacterial cell group (without plasmids)).

[0093] Table 15 Reaction Liquid System Results analysis: such as Figure 2 As shown, the engineered strain with the mutated site exhibited a higher catalytic efficiency for catechol substrates than the wild-type TPL strain, and the amino acid sequence after the mutated site was also used in subsequent experiments. Example 3

[0094] Effects of different temperature control strategies on dopamine production (1) Activation and induction of bacterial expression: The recombinant strain 001 (expressing TPL-M1 and DDC) constructed and preserved in Example 1 was taken out of the -80℃ freezer, streaked and revived, and expressed the protein according to the method in step (5) of Example 2. The bacterial solution was mixed and divided into 3 portions and centrifuged. The portions were placed in centrifuge tubes and centrifuged at 4℃ and 4000rpm for 15min. The bacterial precipitate was collected.

[0095] (2) Preparation of the reaction solution: According to the formula in Table 16, weigh out catechol (10 g / L), sodium pyruvate (18 g / L), ammonium acetate (30 g / L), EDTA-2Na (2 g / L) and sodium sulfite (4 g / L), and dissolve them thoroughly in KPi buffer at pH 7.5 to prepare the reaction solution.

[0096] (3) Bacterial resuspension and reaction system establishment: Using the prepared reaction solution, resuspend three portions of bacterial precipitate separately, and adjust the concentration of the resuspending solution to OD600 = 20. Add 20 mL of this bacterial suspension to each of three 250 mL Erlenmeyer flasks, and add Triton X-100 (final concentration 5‰), pyridoxal phosphate (final concentration 0.1 mM), and ascorbic acid (final concentration 0.1 mM), respectively.

[0097] (4) Catalytic reactions under different temperature conditions: Three samples were placed in a shaker and subjected to a catalytic reaction at the temperature conditions set in Table 16 (220 rpm). After the reaction was completed, the reaction solutions were collected, filtered through a microporous membrane, and the amount of dopamine produced was detected by HPLC. The effects of different temperature control strategies on the final yield were compared. The experimental results are as follows: Figure 3 As shown.

[0098] Table 16 Catalytic Reaction Conditions Results analysis: such as Figure 3 As shown, different temperature control strategies during the reaction have a significant impact on the yield of dopamine. Among them, 15℃→25℃ (reacting at 15℃ for 3 hours, then raising the temperature to 25℃ and continuing the reaction for 6 hours) can better match the activities of the two enzymes and increase the yield.

[0099] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0100] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tyrosine phenol lyase mutant TPL-M1, characterized in that, The amino acid sequence of the mutant TPL-M1 is shown in SEQ ID NO.

20.

2. A nucleic acid molecule encoding the tyrosine phenol lyase mutant TPL-M1 of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

21.

3. A recombinant vector, characterized in that, The recombinant vector includes a first expression cassette and a second expression cassette; the first expression cassette contains a sequence encoding dopa decarboxylase; the second expression cassette contains a sequence encoding a tyrosine phenol lyase mutant TPL-M1; the recombinant vector is pCSA-DDC-TPL, and its nucleotide sequence is shown in SEQ ID NO.

30.

4. The method for constructing the recombinant vector according to claim 3, characterized in that, The first expression cassette containing the coding sequence for dopa decarboxylase and the second expression cassette containing the coding sequence for the tyrosine phenol lyase mutant TPL-M1 were sequentially assembled into the pCSA vector using the L0, L1, and L2 three-level assembly system to obtain the recombinant vector pCSA-DDC-TPL.

5. A recombinant engineered bacterium comprising the recombinant vector of claim 3.

6. The method for constructing recombinant engineered bacteria according to claim 5, characterized in that, The recombinant vector pCSA-DDC-TPL described in claim 3 was introduced into a host strain to construct a recombinant engineered bacterium.

7. The use of the recombinant engineered bacteria constructed by the method of claim 5 or claim 6 in the preparation of dopamine.

8. A method for producing dopamine, characterized in that, Includes the following steps: (1) Cultivate the recombinant engineered bacteria as described in claim 5, and collect the bacterial cells after induction of expression; (2) Using the bacterial cells collected in step (1) as a catalyst, a catalytic reaction is carried out in a reaction system containing catechol, sodium pyruvate and ammonium acetate; (3) Collect dopamine from the reaction solution.

9. The method according to claim 8, characterized in that, The catalytic reaction in step (2) is carried out in two stages: the first stage is carried out at 10-20℃ for 3-5 hours, and the second stage is carried out at 25-35℃ for 5-12 hours.

10. The method according to claim 8, characterized in that, The reaction system in step (2) includes the following components at the following final concentrations: catechol 6-18 g / L, sodium pyruvate 10-22 g / L, ammonium acetate 30 g / L, sodium sulfite 4 g / L, EDTA-2Na 2 g / L, pyridoxal phosphate 0.1 mM, Triton X-100 volume fraction of 5‰, ascorbic acid 0.1 mM, and pH 7.0-8.5.

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