A dehydrogenase mutant for synthesizing duloxetine intermediate and its application

Abstract

The present invention relates to an alcohol dehydrogenase mutant, and the amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO.3. The alcohol dehydrogenase mutant of the present invention can catalyze the synthesis of MKTP into S-MHTP, and the conversion rate of the above catalytic reaction is 99.9%. The alcohol dehydrogenase mutant of the present invention has an activity increased by about 30 times compared with the wild type, and the proportion of the S-MHTP selective product is also higher than that of the wild type, having high potential for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a dehydrogenase mutant for synthesizing duloxetine intermediates. Background Art

[0002] Duloxetine is a selective serotonin reuptake inhibitor (SSRI) and serotonin and norepinephrine reuptake inhibitor (SNRI), and its pharmacodynamic performance is the S-isomer, which is used to treat depression, anxiety and chronic pain. Since its launch in the United States in 2004, it has been widely used because of its few side effects, quick onset, and long-term safety and effectiveness. Therefore, the efficient synthesis of (S)-duloxetine is of great significance for the treatment of related patients.

[0003] The industrial synthesis route of duloxetine mainly involves chemical synthesis with multiple steps, including the construction of an imidazole ring, the formation of a benzothiazole ring, and the introduction of a hexane ring. These steps require strict control of reaction conditions to ensure high-yield and high-purity products. An important chiral intermediate in the above reactions is S-N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (S-MHTP), which can be generated by the asymmetric reduction of N-methyl-3-keto-3-(2-thienyl)-1-propanamine (MKTP). Currently, the chemical synthesis process route of this precursor is long, with many side reactions and pollutants, and it cannot meet the requirements of green chemistry. In recent years, some enzyme catalysts have shown good effects in the synthesis process. These enzyme catalysts can react under mild conditions, reducing the generation of by-products. Therefore, the use of biocatalytic reduction of MKTP to asymmetrically synthesize S-MHTP has become an important means for the efficient preparation of duloxetine intermediates.

[0004] NAD(P)H-dependent alcohol dehydrogenases (ADHs), as an effective oxidoreductase, have been verified and applied in related catalytic processes. It is a reliable biocatalyst. However, enzymes in nature often cannot achieve large-scale industrial production due to their own low catalytic performance limitations. In order to increase the application potential of ADHs in industry, it is necessary to carry out molecular modification on related ADHs to obtain better catalytic activity, so as to lay a foundation for the large-scale industrial application of enzymatic preparation of S-MHTP in the future. Summary of the Invention

[0005] To solve the above technical problems, the present invention includes the following aspects:

[0006] The first aspect of the present invention provides an alcohol dehydrogenase mutant, and the amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO.3.

[0007] Preferably, the mutation information of the alcohol dehydrogenase mutant relative to the wild type is T2A, R40H, K46R, A94E, S96V, E145F, F147M, L153V, Y190P, M206W, I226V, D233G, V245I, and Y249W.

[0008] Preferably, the alcohol dehydrogenase mutant is derived from the wild-type Lactobacillus kefir strain.

[0009] The second aspect of the present invention provides a gene encoding the above-mentioned alcohol dehydrogenase mutant, and the nucleotide sequence of the gene is as shown in SEQ ID NO.4.

[0010] The third aspect of the present invention provides a recombinant expression vector, which contains the coding gene of the above-mentioned alcohol dehydrogenase mutant.

[0011] Preferably, the recombinant expression vector is the pET-24a plasmid.

[0012] Preferably, the coding gene is loaded between the NdeⅠ and Xhol I restriction enzyme sites of the pET-24a plasmid.

[0013] The fourth aspect of the present invention provides a recombinant engineering bacterium, which contains the above-mentioned recombinant expression vector.

[0014] Preferably, the recombinant engineering bacterium is Escherichia coli. More preferably, the recombinant engineering bacterium is Escherichia coli BL21(DE3).

[0015] The fifth aspect of the present invention provides an application of the above-mentioned recombinant genetic engineering bacterium in the catalytic synthesis reaction of duloxetine intermediate, and the synthesis reaction is the catalytic synthesis of S-N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (S-MHTP) from N-methyl-3-keto-3-(2-thienyl)-1-propanamine (MKTP).

[0016] Preferably, the reaction time of the synthesis reaction is 12 - 36 hours. More preferably, the reaction time of the synthesis reaction is 16 - 32 hours. Further preferably, the reaction time of the synthesis reaction is 20 - 28 hours. Further preferably, the reaction time of the synthesis reaction is 24 hours.

[0017] Preferably, the pH value of the synthesis reaction is 8.0 - 10.0. More preferably, the pH value of the synthesis reaction is 8.5 - 9.5. Further preferably, the pH value of the synthesis reaction is 8.8 - 9.2. Further preferably, the pH value of the synthesis reaction is 9.0.

[0018] Preferably, the temperature of the synthesis reaction is 25-45 °C. More preferably, the temperature of the synthesis reaction is 30-40 °C. Further preferably, the temperature of the synthesis reaction is 32-38 °C. Further preferably, the temperature of the synthesis reaction is 34-36 °C. Further preferably, the temperature of the synthesis reaction is 34 °C.

[0019] Technical effects produced by the present invention:

[0020] The present invention has developed an alcohol dehydrogenase mutant, which can catalyze the synthesis of MKTP into S-MHTP, and the conversion rate of the above catalytic reaction is 99.9%. The alcohol dehydrogenase mutant of the present invention has about 30 times higher activity than the wild type, and the proportion of the S-MHTP selective product is also higher than that of the wild type, having high industrial application potential. Description of the drawings

[0021] Figure 1 It is a detection protein gel diagram of LkADH;

[0022] Figure 2 It is a schematic diagram of the catalytic synthesis of duloxetine precursor;

[0023] Figure 3 It is a visualization diagram of the molecular docking of LkADH and the substrate;

[0024] Figure 4 It is the virtual alanine scanning result for the substrate MKTP;

[0025] Figure 5 It is the relevant activity change information during the mutant construction process. Detailed implementation manners

[0026] Gene source: The alcohol dehydrogenase LkADH involved in the present invention is derived from the wild-type Lactobacillus kefir strain, and has high stereoselectivity for catalyzing the formation of S-MHTP. Its gene sequence is optimized according to the amino acid sequence information (Genbank: AAP94029.1) in the NCBI database according to the codons of the Escherichia coli expression system, and obtained by gene synthesis, and loaded between the NdeⅠ and Xhol I restriction enzyme sites of the pET-24a expression vector. The amino acid sequence and gene sequence of this enzyme are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0027] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ(SEQ ID NO.1)

[0028] ATGACCGATCGTCTGAAGGGCAAAGTAGCCATCGTAACCGGCGGGACACTGGGTATCGGTTTGGCAATCGCCGATAAATTTGTAGAGGAGGGTGCGAAAGTAGTTATTACTGGCCGTcatGCGGATGTAGGTGAAAAAGCCGCCAAATCAATCGGCGGCACTGATGTTATTCGCTTTGTCCAGCACGATGCGTCCGATGAAGCAGGCTGGACGAAACTGTTCGACACCACCGAGGAGGCATTCGGCCCGGTTACGACCGTCGTGAACAATGCAGGGATTGCTGTTTCGAAAAGCGTTGAAGACACTACCACGGAGGAATGGCGTAAACTGCTGTCCGTTAATCTGGATGGTGTTTTTTTCGGCACCCGTCTGGGCATTCAGCGCATGAAAAATAAAGGCTTGGGCGCTAGCATCATCAATATGAGCAGTATTGAGGGGTTCGTAGGCGATCCGACGCTTGGGGCATACAACGCTTCCAAGGGGGCGGTACGTATCATGTCGAAAAGCGCAGCGCTGGATTGCGCACTGAAGGACTACGATGTGCGTGTCAACACAGTACATCCGGGCTACATCAAGACCCCGATGCTCGATGATGTGGAAGGTGCTGAGGAAATGATGTCACAGCGTACGAAAACCCCTATGGGCCACATTGGCGAACCGAATGACATCGCATGGATCTGTGTGTACCTGGCATCTGAAGAATCGAAATTTGCGACGGGTGCAGAATTTGTGGTTGACGGCGGGTACACCGCACAG(SEQ ID NO.2)

[0029] Experimental Example 1, Preparation of Alcohol Dehydrogenase LkADH

[0030] The synthesized pET24a-LkADH was transformed into competent E. coli BL21(DE3) cells and spread on an LB solid plate containing kanamycin at a final concentration of 50 mg / L. After culturing at 37 °C for 12 h, it was transferred to a fresh liquid LB shake flask medium and continued to be cultured in a shaker at 220 rpm and 37 °C until OD 600When reaching between 0.6 - 1.0, add 0.2 mM IPTG (isopropyl-β-D-thiogalactoside), induce expression at 25 °C and 220 rpm for 16 h, and collect the bacteria by centrifugation at 8000 r / min. Use an ultrasonic disruptor to break the cells, and the supernatant after centrifugation at 10000 r / min is the crude enzyme solution containing LkADH. This sample is detected by SDS-PAGE, and the results are as Figure 1 shown to match (the expected calculated protein molecular weight is 26.8 KD).

[0031] Experimental Example 2. Enzyme Activity Detection Method

[0032] Enzyme activity definition: At 35 °C, the amount of enzyme required to consume 1 micromole of NAD(P)H within 1 minute is defined as 1 U.

[0033] The detection principle of enzyme activity is carried out according to the amount of NAD(P)H consumed during the catalytic synthesis of S-MHTP from MKTP ( Figure 2 ), that is, by detecting the linear change of the absorbance value of the reaction system at 340 nm for calculation.

[0034] Preparation of reaction mother liquor:

[0035] Solution A: Weigh 0.01 g of NADH and dissolve it in 2 mL of 100 mM PBS buffer with pH = 7.4; Solution B: Weigh 0.25 g of the substrate MKTP and dissolve it in 1 mL of PBS buffer; Solution C: Pipette 0.1 mL of Solution A, 0.6 mL of Solution B, and 29 mL of 100 mM PBS with pH = 7.4 into a beaker, mix well, and place it in a water bath at 35 °C for preheating and standby.

[0036] Enzyme activity detection steps:

[0037] First, pipette 2900 μL of the preheated Solution C above into a cuvette, then add 100 μL of the diluted crude enzyme solution to make the total reaction volume 3 mL. Cover the cuvette lid, quickly invert and mix up and down, and use an ultraviolet spectrophotometer to measure the change rate of absorbance value ΔA per minute within 60 s at 340 nm.

[0038] Unified calculation formula for enzyme activity:

[0039] Enzyme activity (U / mL) = (ΔA × 10 6 × dilution factor × V 总 ) / (ε × b × V 酶 )

[0040] In the formula: V 总 is the total reaction volume = 3 mL; V 酶 is the amount of enzyme added in the reaction = 0.1 mL; ε is the molar extinction coefficient of NADH = 6220 L / (mol·cm); b is the optical path = 1 cm.

[0041] Experimental Example 3, Rational Design of Alcohol Dehydrogenase LkADH

[0042] The three-dimensional structure of the enzyme protein was modeled using the Aphalafold3 tool (https: / / golgi.sandbox.google.com / ). The SEQ ID NO:1 information was input, and the coenzyme ligand molecule was set as NADH.

[0043] Based on sequence conservation analysis (https: / / consurf.tau.ac.il / consurf_index.php), combined with the modeling results and previous studies, its catalytic active center was determined to be the Ser143-Tyr156-Lys160 triad, thereby locating the approximate position of the substrate binding pocket.

[0044] Subsequently, the substrate MKTP was docked to the catalytic center using the AutodockTools tool, and the docking results were visualized using the Pymol software. As Figure 3 shown, the carbonyl group of the substrate was correctly docked to the catalytic residues Ser143 and Tyr156. Among them, Ser143 mainly functions to stabilize the substrate and assist its electron transfer reaction with Tyr156, while Lys160 mediates the electron transfer between LkADH and the coenzyme (not marked in the figure). Taking the docked substrate as the center, amino acids within were selected as the key sites around the catalytic pocket. After excluding highly conserved sites, virtual alanine scanning was performed (for alanine residues in the selected sequence, direct virtual saturation mutagenesis was performed), and Figure 4 8 potential sites with significant binding energy contributions (>0.5 kcal / mol) were obtained. It is expected that the best active mutant M1 can be obtained by iterative saturation mutagenesis of the potential sites.

[0045] Based on M1, subsequently, the LigandMPNN model (https: / / github.com / dauparas / LigandMPNN) was used to design all non-conserved sites beyond from the substrate. The protein-ligand complex structure model after molecular docking and the target mutation point information were input. The calculation amino acid was set to be biased towards alanine, and finally, sequence-specific mutation analysis was generated, and the results were processed and output using a Python script. The top ten mutation sites with the highest average confidence of the redesigned residue sequences were selected (the results are shown in Table 1). It is expected that the optimal mutant M2 can be obtained by performing iterative mutation tests.

[0046] Table 1 Results of LigandMPNN Site Design

[0047] Overall confidence Mutations 0.736 R40H 0.721 R40Q 0.705 I226V 0.686 D233G 0.681 D233A 0.626 K46A 0.614 K46R 0.588 V245I 0.534 T2A 0.464 N131H

[0048] Test Example 4, Screening and Construction of Mutants

[0049] Using the pET-24a plasmid DNA containing the wild-type LkADH gene as a template, a pair of forward and reverse primers were designed to replace the original codons at key sites 94, 96, 145, 147, 153, 190, 206, and 249 with NNK codons, and saturation mutations were introduced by PCR using the high-fidelity DNA polymerase primeSTARmax (TaKaRa Bio). Taking the introduction of the A94E saturation mutation as an example: the forward primer was designed as pET24a-94-F: TGCAGGGATTNNKGTTGTTAAAAGC (SEQ ID NO.5), and the reverse primer was pET24a-94-R: GCTTTTAACAACMNNAATCCCTGCA (SEQ ID NO.6).

[0050] PCR reaction system: The total volume was 20 μL; 1.5 μL of each of the forward primer / reverse primer, 2 μL of the template, 5 μL of ddH2O, and 10 μL of 2X primeSTARmax.

[0051] The PCR reaction procedure was as follows: 95°C for 2 min; 95°C for 20 s, 56°C for 20 s, 72°C for 40 s, repeated for 25 cycles; continued extension at 72°C for 2 min.

[0052] After treating the PCR product with DpnI enzyme at 37°C for 1 h, it was transformed into E. coli BL21(DE3) competent cells, spread on an LB solid plate containing kanamycin resistance at a final concentration of 50 mg / L, and cultured at 37°C for 12 h to obtain 8 different saturation mutation libraries. Positive clones were selected and inoculated into a 96-well deep-well plate containing liquid LB medium with kanamycin resistance at a final concentration of 50 mg / L, and cultured overnight at 37°C and 220 rpm. Inoculate 10% of the above overnight bacterial solution into a fresh liquid LB medium containing kanamycin resistance at a final concentration of 50 mg / L, and shake culture at 37°C and 220 rpm until the OD is 0.6 - 0.8. Add IPTG at a final concentration of 0.2 mM and express at 25°C for 16 h. Take 100 μL of the whole-cell culture system as the crude enzyme, and perform activity detection on each colony according to the method in Test Example 2. Screen 90 single colonies for each library, and select the colonies with activity higher than that of the WT for sequencing.

[0053] Subsequently, based on the mutant M0 (M206W) with the highest activity improvement, each mutation site was selectively and preferably iterated, and new specific mutations were introduced by designing primers in the same method as above. For example, taking the simultaneous introduction of A94E and S96V as an example, when designing primers, a primer pair containing the A94E and S96V mutations was designed: pET24a-94E / 96V-F: TGCAGGGATTGAGGTTGTTAAAAGCGTTGA (SEQ ID NO.7); pET24a-94E / 96V-R: TCAACGCTTTTAACAACCTCAATCCCTGCA (SEQ ID NO.8). In the subsequent PCR procedure, the plasmid extracted from M0 was used as the template, and the other transformation and expression steps remained unchanged.

[0054] During the iteration process (as Figure 5 shown in 0-1 A), mutants M 0-2 (A94E, S96V, M206W), M 0-3 (A94E, S96V, E145F, F147M, L153V, M206W), and M

[0055] were mainly constructed. Finally, M1 was obtained (the mutation information for the wild type is A94E, S96V, E145F, F147M, L153V, Y190P, M206W, Y249W). Figure 5 Based on M1, according to the design results of the LigandMPNN site, mutants with mutations at relevant sites were constructed for activity testing. The expected mutation sites were gradually introduced according to the activity level. The activity test of the mutants in each iteration was repeated three times and the average value was calculated (the results are as shown in

[0056] B). Finally, the optimal mutant M2 was obtained. The amino acid and gene sequences of M2 are shown in SEQ ID NO.3 and SEQ ID NO.4, and the mutation information for the wild type is T2A, R40H, K46R, A94E, S96V, E145F, F147M, L153V, Y190P, M206W, I226V, D233G, V245I, and Y249W.MADRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRRADVGERAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIEVVKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIFGMVGDPTVGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPMLDDVEGAEEMWSQRTKTPMGHIGEPNDIAWVCVYLASGESKFATGAEFVIDGGWTAQ(SEQ ID NO.3)

[0057] ATGGCCGATCGTCTGAAGGGCAAAGTAGCCATCGTAACCGGCGGGACACTGGGTATCGGTTTGGCAATCGCCGATAAATTTGTAGAGGAGGGTGCGAAAGTAGTTATTACTGGCCGTCGTGCGGATGTAGGTGAACGTGCCGCCAAATCAATCGGCGGCACTGATGTTATTCGCTTTGTCCAGCACGATGCGTCCGATGAAGCAGGCTGGACGAAACTGTTCGACACCACCGAGGAGGCATTCGGCCCGGTTACGACCGTCGTGAACAATGCAGGGATTGAGGTTGTTAAAAGCGTTGAAGACACTACCACGGAGGAATGGCGTAAACTGCTGTCCGTTAATCTGGATGGTGTTTTTTTCGGCACCCGTCTGGGCATTCAGCGCATGAAAAATAAAGGCTTGGGCGCTAGCATCATCAATATGAGCAGTATTTTCGGGATGGTAGGCGATCCGACGGTTGGGGCATACAACGCTTCCAAGGGGGCGGTACGTATCATGTCGAAAAGCGCAGCGCTGGATTGCGCACTGAAGGACTACGATGTGCGTGTCAACACAGTACATCCGGGCCCCATCAAGACCCCGATGCTCGATGATGTGGAAGGTGCTGAGGAAATGTGGTCACAGCGTACGAAAACCCCTATGGGCCACATTGGCGAACCGAATGACATCGCATGGGTCTGTGTGTACCTGGCATCTGGTGAATCGAAATTTGCGACGGGTGCAGAATTTGTGATTGACGGCGGGTGGACCGCACAG(SEQ ID NO.4)

[0058] Experimental Example 5: Verification of the Catalytic Effect of the LkADH Mutant

[0059] Pick the original expression strain WT containing LkADH and its mutants M1 and M2, and inoculate them separately into liquid LB containing kanamycin resistance at a final concentration of 50 mg / L. After overnight culture at 37 °C with 220 rpm, transfer them to 1 L of TB medium (tryptone 10 g / L, yeast extract 6.67 g / L, glycerol 6.67 g / L, K2HPO4 3.34 g / L, ammonium sulfate 0.84 g / L, citric acid 1.75 g / L, sodium chloride 2.5 g / L) at an inoculation amount of 1%, and grow at 37 °C with 220 rpm for 4 h. Then add IPTG at a final concentration of 0.2 mM for induction expression, and let it express at 25 °C for 18 h. Centrifuge the above fermentation broth at 4 °C, discard the supernatant to obtain wet cells. Resuspend the cell pellet in 100 mM PBS buffer with pH = 7.4 at a ratio of 1:2, and then repeat the cell disruption of the mixture 3 times through a high-pressure homogenizer (ATS) to obtain cell debris. Subsequently, centrifuge the sample at 4 °C and 10,000 rpm for 30 min in a high-speed centrifuge and collect the supernatant to obtain the crude enzyme solution. Set two parallel experiments for each expression strain.

[0060] First, perform activity tests on each sample according to the method in Test Example 2. The corresponding amount of NADPH used is 0.013 g. Take a part of the above crude enzyme solution, dilute it 100 times with PBS buffer, add 100 μL to the reaction system, and measure the change rate of the absorbance per minute within 60 s at 340 nm and calculate the average catalytic activity respectively (the molar extinction coefficient of NADPH is 6859 L / (mol·cm)). Subsequently, verify the product through the reaction process of the large system.

[0061] (1) Substrate activation:

[0062] Take 40 g of MKTP substrate, add 100 g of 25% sodium hydroxide, add 32 g of isopropanol, control the temperature below 20 °C, stir for half an hour, let it stand for half an hour, and then discard the lower aqueous layer to obtain the upper activated substrate for standby.

[0063] (2) Enzyme conversion

[0064] Take a portion of the above substrate (the concentration should not exceed 10%), and sequentially add 56 g of pure water and 47 g of isopropanol. After stirring well and mixing evenly, adjust the pH to 9.0 with concentrated sulfuric acid, and continue to stir and mix evenly for 10 minutes. Set the temperature to 34°C. Add 0.8 g of triethanolamine, 20 - 40 g of the main enzyme solution, and a certain amount of coenzyme (not exceeding 0.1 g), and confirm that the pH is 9.0. If there is a deviation, fine-tune it to 9.0, and carry out the enzymatic reaction with sufficient stirring at 34°C. While catalyzing, carry out vacuum distillation on the reaction system to reduce the acetone concentration in the reaction system, and continuously add "isopropanol:water = 9:1" to keep the liquid level of the reaction system unchanged. During the reaction process, take samples and use HPLC to measure the products. Stop the reaction for all samples until the concentration of a certain group of substrates drops below 1%. The reaction time is about 24 h. Finally, calculate the conversion rate of the substrate and the proportion of the S-MHTP selective product. The test results are shown in Table 2.

[0065] Table 2 Catalytic effects of LkADH mutants

[0066]

[0067] As can be seen from the above table, the activities of mutants T2A, R40H, K46R, A94E, S96V, E145F, F147M, L153V, Y190P, M206W, I226V, D233G, V245I, and Y249W are about 30 times higher than that of the wild type, and their overall catalytic effects are significantly improved.

[0068] Although the specific implementation manners of the present invention have been described, those skilled in the art should recognize that various changes and modifications can be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the alcohol dehydrogenase mutant according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the gene encoding the alcohol dehydrogenase mutant as claimed in claim 2.

4. The recombinant expression vector according to claim 3, wherein The recombinant expression vector is the pET-24a plasmid.

5. A recombinant engineering bacterium, characterized in that, The recombinant engineered bacterium contains the recombinant expression vector as claimed in claim 3.

6. The recombinant engineering bacterium according to claim 5, characterized in that, The recombinant engineered bacterium is Escherichia coli.

7. The recombinant engineering bacterium according to claim 6, wherein The recombinant engineered bacterium is Escherichia coli BL21(DE3).

8. Use of the recombinant engineering bacterium according to any one of claims 5-7 in the catalytic synthesis reaction of duloxetine intermediate, characterized in that, The synthesis reaction is the catalytic synthesis of S-N-methyl-3-hydroxy-3-(2-thienyl)-1-propanamine (S-MHTP) from N-methyl-3-oxo-3-(2-thienyl)-1-propanamine (MKTP).

9. The application according to claim 8, wherein The temperature of the synthesis reaction is 25 - 45 °C.

10. The application according to claim 8, characterized in that The pH value of the synthesis reaction is 8.0 - 10.0.

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