Dehydrogenase mutant for synthesizing duloxetine intermediate and application of dehydrogenase mutant

Abstract

The invention relates to an alcohol dehydrogenase mutant. The amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO. 3. The alcohol dehydrogenase mutant provided by the invention can catalytically synthesize S-MHTP from MKTP, and the conversion rate of the catalytic reaction is 99.9%. Compared with a wild type, the alcohol dehydrogenase mutant disclosed by the invention has the advantages that the activity is improved by about 30 times, the proportion of an S-MHTP selective product is also higher than that of the wild type, and the alcohol dehydrogenase mutant has relatively high industrial application potential.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a dehydrogenase mutant for synthesizing a duloxetine intermediate. Background Art

[0002] Duloxetine is a selective serotonin reuptake inhibitor (SSRI) and serotonin and norepinephrine reuptake inhibitor (SNRI). Its pharmacodynamics are expressed as 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 due to its few side effects, rapid onset of action, and long-term safety and effectiveness. Therefore, achieving efficient synthesis of (S)-duloxetine is of great significance for the treatment of related patients.

[0003] The industrial synthesis route of duloxetine is mainly through chemical synthesis involving multi-step reactions, including the construction of the imidazole ring, the formation of the benzothiazole ring, and the introduction of the 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 reaction is SN-methyl-3-hydroxy-3-(2-thienyl)-1-propylamine (S-MHTP), which can be generated by asymmetric reduction of N-methyl-3-keto-3-(2-thienyl)-1-propylamine (MKTP). At present, the chemical synthesis process of this precursor is long, with many side reactions and pollutants, which cannot meet the needs 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 and reduce the generation of by-products. Therefore, the asymmetric synthesis of S-MHTP by biocatalytic reduction of MKTP has become an important means for the efficient preparation of duloxetine intermediates.

[0004] NAD(P)H-dependent alcohol dehydrogenases (ADHs) are effective oxidoreductases that have been verified and applied in related catalytic processes. They are reliable biocatalysts. However, natural enzymes are often limited by their own low catalytic performance and cannot be produced on a large scale in industry. In order to increase the application potential of ADHs in industry, it is necessary to molecularly modify the related ADHs to obtain better catalytic activity, so as to lay the foundation for the subsequent large-scale industrial application of enzymatic preparation of S-MHTP. 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, the amino acid sequence of the alcohol dehydrogenase mutant is 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 a wild-type Lactobacillus kefir strain.

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

[0010] The third aspect of the present invention provides a recombinant expression vector, which comprises a gene encoding the alcohol dehydrogenase mutant.

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

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

[0013] The fourth aspect of the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the above-mentioned recombinant expression vector.

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

[0015] The fifth aspect of the present invention provides an application of the above-mentioned recombinant genetically engineered bacteria in catalyzing the synthesis reaction of duloxetine intermediates, wherein the synthesis reaction is the synthesis of SN-methyl-3-hydroxy-3-(2-thienyl)-1-propylamine (S-MHTP) catalyzed by N-methyl-3-keto-3-(2-thienyl)-1-propylamine (MKTP).

[0016] Preferably, the synthesis reaction time is 12-36 hours. More preferably, the synthesis reaction time is 16-32 hours. Further preferably, the synthesis reaction time is 20-28 hours. Further preferably, the synthesis reaction time 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] The technical effects produced by the present invention are:

[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 an activity increased by about 30 times compared with the wild type, and the proportion of S-MHTP selective products is also higher than that of the wild type, and has a high potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the protein gel image of LkADH detection;

[0022] Figure 2 The schematic diagram of the catalytic synthesis of duloxetine precursor;

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

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

[0025] Figure 5 It is the relevant activity change information during the mutant construction process. DETAILED DESCRIPTION

[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 production of S-MHTP. Its gene sequence is obtained by total gene synthesis after codon optimization according to the amino acid sequence information in the NCBI database (Genbank: AAP94029.1) according to the Escherichia coli expression system, and loaded between the NdeⅠ and Xhol I restriction sites of the pET-24a expression vector. The amino acid sequence and gene sequence of the enzyme are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0027] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ(SEQ ID NO.1)

[0028] (SEQ ID NO.2)

[0029] Test Example 1. Preparation of alcohol dehydrogenase LkADH

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

[0031] Test Example 2: Enzyme Activity Detection Method

[0032] Enzyme activity definition: The amount of enzyme required to consume 1 micromole of NAD(P)H in 1 min at 35°C is defined as 1 U.

[0033] The detection principle of enzyme activity is based on the process of catalyzing MKTP to synthesize S-MHTP ( Figure 2 ) is calculated by measuring the linear change of the absorbance of the reaction system at 340 nm.

[0034] Preparation of reaction mother solution:

[0035] Solution A: weigh 0.01 g of NADH and dissolve it in 2 mL of 100 mM pH = 7.4 PBS buffer; Solution B: weigh 0.25 g of 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 pH = 7.4 PBS into a beaker, mix well, and preheat in a 35°C water bath for later use.

[0036] Enzyme activity detection steps:

[0037] First, pipette 2900 μL of the preheated C solution 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 upside down to mix, and use a UV spectrophotometer at 340 nm to test the rate of change ΔA of the absorbance value per min within 60 s.

[0038] Unified calculation formula for enzyme activity:

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

[0040] Where: V 总 The total reaction volume = 3 mL; V 酶 is the amount of enzyme added to the reaction = 0.1 mL; ε is the molar absorption coefficient of NADH = 6220 L / (mol·cm); b is the light path = 1 cm.

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

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

[0043] Based on the results of sequence conservation analysis (https: / / consurf.tau.ac.il / consurf_index.php) combined with modeling 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] The AutodockTools tool was then used to dock the substrate MKTP to the catalytic center, and the docking results were visualized using Pymol software. Figure 3 As shown in the figure, the carbonyl group of the substrate is correctly docked to the catalytic residues Ser143 and Tyr156, of which Ser143 mainly stabilizes the substrate and assists it in the electron transfer reaction with Tyr156, while Lys160 mediates the electron transfer between LkADH and the coenzyme (not marked in the figure). The amino acids within the range of 1 and 2 were used as key sites around the catalytic pocket. After excluding the highly conserved sites, virtual alanine scanning was performed (virtual saturation mutation was performed directly for the alanine residues in the selected sequence). Figure 4 Eight potential sites with significant binding energy contribution (>0.5 kcal / mol) were identified. It is expected that the best active mutant M1 will be obtained by iterative saturation mutagenesis of potential sites.

[0045] Based on M1, the LigandMPNN model (https: / / github.com / dauparas / LigandMPNN) was then used to quantify the distance to the substrate. All non-conserved sites except for were designed. The protein-ligand complex structure model after molecular docking and the target mutation point information were input, and the calculation of amino acids was set to be biased towards alanine. Finally, sequence-specific mutation analysis was generated, and the results were processed and output using Python scripts. The top ten mutation sites with the highest average confidence in the redesigned residue sequence were selected (the results are shown in Table 1), and it is expected that the optimal mutant M2 will be obtained through iterative mutation testing.

[0046] Table 1 LigandMPNN site design results

[0047] Overallconfidence 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] Experimental 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 using NNK codons to replace the original codons at key sites 94, 96, 145, 147, 153, 190, 206 and 249, and saturation mutations were introduced by PCR using high-fidelity DNA polymerase primeSTARmax (TaKaRa Bio). Take the introduction of A94E saturation mutation as an example: design forward primer: pET24a-94-F: TGCAGGGATTNNKGTTGTTAAAAGC (SEQ ID NO.5), reverse primer: pET24a-94-R: GCTTTTAACAACMNNAATCCCTGCA (SEQ ID NO.6).

[0050] PCR reaction system: total volume 20 μL; forward primer / reverse primer 1.5 μL each, template 2 μL, ddH2O 5 μL, 2XprimeSTARmax 10 μL.

[0051] The PCR reaction program 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; and further extension at 72°C for 2 min.

[0052] The PCR product was treated with DpnI enzyme at 37°C for 1 hour, then transformed into E.coli BL21 (DE3) competent medium, coated on LB solid plate containing kanamycin resistance at a final concentration of 50 mg / L, and cultured at 37°C for 12 hours to obtain 8 different saturated mutation libraries. The positive clones were selected and inoculated into 96 deep-well plates containing liquid LB medium containing kanamycin resistance at a final concentration of 50 mg / L, and cultured overnight at 37°C and 220 rpm. 10% of the inoculum from the above overnight bacterial solution was inoculated into liquid LB medium containing fresh kanamycin resistance at a final concentration of 50 mg / L, and cultured at 37°C and 220 rpm with shaking until the OD was 0.6-0.8, and IPTG was added at a final concentration of 0.2 mM, and expressed at 25°C for 16 hours. 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 Experimental Example 2. Screen 90 single colonies for each library, and select colonies with activity higher than WT for sequencing.

[0053] Then, based on the mutant M0 (M206W) with the highest activity improvement, each mutation point was selected for iteration, and new specific mutations were introduced by designing primers in the same way as above. For example, when introducing A94E and S96V at the same time, a primer pair containing A94E and S96V mutations was designed: pET24a-94E / 96V-F: TGCAGGGATTGAGGTTGTTAAAAGCGTTGA (SEQ ID NO.7); pET24a-94E / 96V-R: TCAACGCTTTTTAACAACCTCAATCCCTGCA (SEQ ID NO.8). In the subsequent PCR program, the plasmid extracted from M0 was used as a template, and the other transformation and expression steps remained unchanged.

[0054] In the iteration process (such as Figure 5 A) mainly constructed mutant M 0-1 (A94E, S96V, M206W), M 0-2 (A94E, S96V, E145F, F147M, L153V, M206W), M 0-3 (A94E, S96V, E145F, F147M, L153V, Y190P, M206W), and finally obtained M1 (the mutation information for the wild type is A94E, S96V, E145F, F147M, L153V, Y190P, M206W, Y249W).

[0055] Based on M1, according to the results of LigandMPNN site design, mutants of relevant sites were constructed for activity testing. The expected mutation points were gradually introduced according to the activity level. The activity test of each iterative mutant was repeated three times to calculate the average value (the results are shown in Figure 5 B), and 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 relative to the wild type is T2A, R40H, K46R, A94E, S96V, E145F, F147M, L153V, Y190P, M206W, I226V, D233G, V245I and Y249W.

[0056] 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] The original expression strain WT containing LkADH and its mutants M1 and M2 were selected and inoculated into liquid LB containing a final concentration of 50 mg / L kanamycin resistance, 220 rpm, 37°C overnight culture, and then transferred to 1L 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 a 1% inoculation amount, and after growing at 220 rpm, 37°C for 4 hours, IPTG with a final concentration of 0.2 mM was added to induce expression, and it was expressed at 25°C for 18 hours. After the above fermentation broth was centrifuged in a 4°C centrifuge, the supernatant was discarded to obtain wet bacteria. The bacterial sludge was resuspended in 100 mM pH = 7.4 PBS buffer at a ratio of 1:2, and the mixture was then repeatedly disrupted by a high-pressure homogenizer (ATS) for 3 times to obtain cell fragments. The sample was then centrifuged at 4°C, 10000 rpm for 30 min in a high-speed centrifuge to collect the supernatant to obtain the crude enzyme solution. Two parallel experiments were set up for each expression strain.

[0060] First, the activity of each sample was tested according to the method in Test Example 2, wherein the corresponding NADPH dosage was 0.013 g, and part of the above crude enzyme solution was diluted 100 times with PBS buffer and then 100 μL was added into the reaction system, and the change rate of the absorbance value per min within 60 s was tested at 340 nm and the average catalytic activity was calculated respectively (the relative molar absorption coefficient of NADPH was 6859 L / (mol·cm)). Then the reaction process of the large system was carried out to verify the product.

[0061] (1) Substrate activation:

[0062] Take 40g MKTP substrate, add 100g 25% sodium hydroxide, add 32g isopropanol, control the temperature below 20 degrees, stir for half an hour, let it stand for half an hour, discard the lower water layer, and obtain the upper activated substrate for use.

[0063] (2) Enzyme conversion

[0064] Take part of the above substrate (concentration cannot exceed 10%), add 56g pure water and 47g isopropanol in turn, stir and mix thoroughly, adjust pH to 9.0 with concentrated sulfuric acid, continue to stir and mix for 10 minutes, and set the temperature to 34°C. Add 0.8g triethanolamine, 20-40g main enzyme solution, a certain amount of coenzyme (not more than 0.1g), and confirm that the pH is 9.0. If there is a deviation, fine-tune to 9.0, and stir well at 34°C for enzymatic reaction. While catalyzing, the reaction system is subjected to reduced pressure distillation to reduce the acetone concentration in the reaction system, and "isopropanol: water = 9:1" is added to maintain the liquid level of the reaction system unchanged. During the reaction, samples are taken and the product is determined by HPLC until the concentration of a certain group of substrates drops below 1%, and all samples are stopped from reacting. The reaction time is about 24h, and the conversion rate of the substrate and the proportion of S-MHTP selective products are finally calculated. The test results are shown in Table 2.

[0065] Table 2 Catalytic effect of LkADH mutants

[0066]

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

[0068] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may 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 comprises the gene encoding the alcohol dehydrogenase mutant according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pET-24a plasmid.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the recombinant expression vector according to claim 3 or 4.

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

7. The recombinant engineered bacterium according to claim 6, characterized in that The recombinant engineering bacteria is Escherichia coli BL21 (DE3).

8. Use of the recombinant genetically engineered bacteria according to any one of claims 5 to 7 in catalyzing the synthesis reaction of duloxetine intermediates, characterized in that: The synthesis reaction is to synthesize SN-methyl-3-hydroxy-3-(2-thienyl)-1-propylamine (S-MHTP) by catalysis of N-methyl-3-keto-3-(2-thienyl)-1-propylamine (MKTP).

9. The use according to claim 8, characterized in that: The temperature of the synthesis reaction is 25-45°C.

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

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