Imine reductase mutants and their use in the synthesis of chiral 1,3-disubstituted tetrahydroisoquinolines

Abstract

The application discloses an imine reductase mutant and application thereof in synthesis of chiral 1,3-disubstituted tetrahydroisoquinoline, and belongs to the technical field of bioengineering. The imine reductase of different sources is screened, and the imine reductase mutant SnIR-L175A / M245A is constructed, which can convert the substrate racemic imine into (1S,3R)-4a, and the conversion rate is increased to 50% compared with the starting sequence. The imine reductase mutant constructed by the application is used for synthesizing 1,3-disubstituted THIQ from imine, and the de value and ee value of the 1,3-disubstituted THIQ can reach 99%, so the application has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to imine reductase mutants and their use in the synthesis of chiral 1,3-disubstituted tetrahydroisoquinolines, belonging to the field of bioengineering technology. BACKGROUND

[0002] Tetrahydroisoquinoline (THIQ) alkaloids are one of the largest classes of natural products, and play an indispensable role in drug discovery. In recent decades, THIQ natural products have been intensively studied, and they exhibit a wide range of structural diversity and biological activities.

[0003] Among these diverse THIQs, 1,3-disubstituted THIQs, which contain at least two chiral carbon centers, are a special class of THIQ alkaloids, distinct from the THIQs that are generally believed to be formed via Pictet-Spengler (P-S) reactions between aldehydes and aryl ethylamines derived from aromatic amino acids. Instead, they are formed from polyketides generated via the acetate-malonate pathway, which involves a polyketide synthase and other enzymes that have not been identified. However, the unusual substitution pattern of 1,3-disubstituted THIQs, especially the presence of a methyl group at both C1 and C3, poses a significant challenge for their construction via traditional P-S pathways, as it is difficult to preinstall an a-methyl group at C3 via biocatalysis and to install a C1 methyl group from acetaldehyde via NCS substrates.

[0004] Despite the challenges in their biosynthesis, many pioneering chemical synthetic methods have been developed to construct these valuable chiral structures, including transition-metal-catalyzed asymmetric reductions of 1,3-disubstituted isoquinolines, asymmetric nucleophilic additions to chiral 3,4-dihydroisoquinolines, alkylations of chiral tetrahydroisoquinolines, P-S reactions between chiral acylphenethylamine derivatives and aldehydes, Bischler-Napieralski (B-N) / reduction cascades of chiral acylphenethylamines, and palladium-catalyzed three-component Catellani / cyclization / reduction cascades of aryl iodides. Although these strategies are effective, they often rely on complex substrates with preinstalled chiral centers, heavy metal catalysts, flammable and hazardous hydrogen gas, and harsh conditions of high temperature and high pressure. Therefore, it is highly desirable to develop a novel, efficient, and green method to construct chiral 1,3-disubstituted THIQs from simple achiral substrates. SUMMARY

[0005] The present application provides imine reductase mutants, which have one or more mutations in (a)-(f) based on the imine reductase with the amino acid sequence shown in SEQ ID NO. 3 as the parent:

[0006] (a) the threonine at position 123 of the parent is mutated to alanine;

[0007] (b) mutating the parent proline at position 124 to alanine;

[0008] (c) mutating the parent leucine at position 175 to alanine;

[0009] (d) mutating the parent methionine at position 216 to alanine;

[0010] (e) mutating the parent threonine at position 223 to alanine;

[0011] (f) mutating the parent methionine at position 245 to alanine.

[0012] The present application also provides a gene encoding the imine reductase mutant.

[0013] The present application also provides a recombinant plasmid carrying the gene.

[0014] The present application also provides a recombinant microorganism expressing the imine reductase mutant.

[0015] In one embodiment, the microorganism includes, but is not limited to, Escherichia coli.

[0016] In one embodiment, the imine reductase mutant is expressed in Escherichia coli BL21 (DE3) with pET-21b (+) as the vector.

[0017] The present application also provides a method for preparing the imine reductase mutant.

[0018] In one embodiment, the method is inoculating the recombinant Escherichia coli expressing the transaminase into a culture medium, culturing the strain to the logarithmic growth phase, inducing, collecting the bacterial cells, resuspending, breaking, centrifuging to collect the lysate, and purifying the enzyme protein.

[0019] The present application also provides an enzyme preparation containing the imine reductase mutant.

[0020] The present application also provides the use of the imine reductase mutant in the synthesis of 1,3-disubstituted THIQ.

[0021] In one embodiment, the use includes the following steps:

[0022] (1) mixing the transaminase protein with a substrate in the presence of a lactate dehydrogenase (LDH) cycle system to prepare amine 2; the substrate is ketone 1, D-alanine or L-alanine, pyridoxal phosphate (PLP); the LDH cycle system includes lactate dehydrogenase (LDH), glucose dehydrogenase (GDH), NADH, and glucose;

[0023] (2) reacting the amine 2 obtained in step (1) with an acyl chloride, collecting the reaction product, and then reacting the obtained product with phosphorus oxychloride to obtain an imine 3;

[0024] (3) catalyzing the reaction of the imine 3 obtained in step (2) as a substrate with an imine reductase protein and a GDH cycle system to obtain a 1,3-disubstituted THIQ; the GDH cycle system comprises GDH, NADP + , glucose.

[0025] In an embodiment, the transaminase is derived from Arthrobacter sp., and the amino acid sequence thereof is shown in SEQ ID NO. 1.

[0026] In an embodiment, the glucose dehydrogenase is derived from Bacillus subtilis, and the amino acid sequence thereof is shown in SEQ ID NO. 5.

[0027] In an embodiment, the lactate dehydrogenase is derived from Lactobacillus fermentum JN248, and the amino acid sequence thereof is shown in SEQ ID NO. 7.

[0028] In an embodiment, the amount of the imine 3 added in step (3) is 1 mM, the amount of the imine reductase mutant added is 1.6-3 mol%, the amount of the GDH added is 1 mol%, the amount of the NADP + added is 10 mol%, and the amount of the glucose added is 100 mM; wherein all the molar percentages are calculated based on the substrate imine 3.

[0029] In an embodiment, the reaction system of step (2) contains 0.5 M amine 2 dissolved in dichloromethane, 1.5 equivalents of triethylamine, at least 1 equivalent of acyl chloride, 0.5 M amide dissolved in acetonitrile, and 5 equivalents of phosphorus oxychloride; the acetonitrile is preferably dry acetonitrile; and the phosphorus oxychloride is preferably freshly distilled phosphorus oxychloride.

[0030] In an embodiment, the reaction temperature in step (2) is 90°C.

[0031] In an embodiment, the substrate comprises ketone 1, D-alanine or L-alanine, and pyridoxal phosphate (PLP).

[0032] In an embodiment, the LDH cycle system comprises LDH, glucose dehydrogenase (GDH), NADH, and glucose.

[0033] In an embodiment, the temperature of the reaction is 20-40°C, and the pH of the reaction is 7.0-8.0.

[0034] In one embodiment, the reaction time is 12-24h, or the reaction time is no less than 12h.

[0035] In one embodiment, the ketone 1 is added in an amount of 5-10mM; the D-alanine or L-alanine is added in an amount of 100mM, the PLP is added in an amount of 1mM, the glucose is added in an amount of 100mM, and the NADH is added in an amount of 1mM.

[0036] In one embodiment, the transaminase is added in an amount of 0.2-0.4mol%, the GDH is added in an amount of 0.15-0.3mol%, and the LDH is added in an amount of 0.03-0.06mol%, wherein all the mol% are calculated based on the substrate ketone 1.

[0037] In one embodiment, the ketone 1 is added in an amount of 5-10mM; the D-alanine or L-alanine is added in an amount of 100mM, the PLP is added in an amount of 1mM, the glucose is added in an amount of 100mM, and the NADH is added in an amount of 1mM.

[0038] In one embodiment, the transaminase is added in an amount of 0.2-0.4mol%, the GDH is added in an amount of 0.15-0.3mol%, and the LDH is added in an amount of 0.03-0.06mol%.

[0039] In one embodiment, the ketone 1 comprises at least one of the following 1a-1d, 1f:

[0040]

[0041] In one embodiment, the acyl chloride comprises at least one of the following A-G:

[0042]

[0043] In one embodiment, the imine 3 comprises at least one of the following 3a-3l:

[0044]

[0045] In one embodiment, the 1,3-disubstituted THIQ has the following structure:

[0046]

[0047] The present application also provides the use of the imine reductase mutant or the gene encoding the imine reductase mutant, or the recombinant cell expressing the mutant in the synthesis of 1,3-disubstituted THIQ.

[0048] The application also provides the mutant, or the recombinant cell, or the method in the preparation of 1,3-disubstituted THIQ.

[0049] Beneficial effects:

[0050] (1) The application screens imine reductase from different sources, wherein the imine reductase SnIR can convert the substrate racemic imine into (1S,3R)-4a, the conversion rate is 11%, the de value is 44, and the ee value is 59%;

[0051] (2) The application is based on the imine reductase SnIR obtained by screening, and the imine reductase mutant SnIR-L175A / M245A is constructed, which can convert the substrate racemic imine into (1S,3R)-4a, the conversion rate is 50%, the de value is > 99%, and the ee value is > 99%;

[0052] (3) The application also provides a method for synthesizing 1,3-disubstituted THIQ from imine, and the HPLC yield of (1S,3R)-4a-(1S,3R)-4l is 85%, 83%, 92%, 36%, 98%, 68%, 92%, 95%, 84%, 95%, 99%, and 99%, respectively; the de value is > 99%, > 99%, > 99%, > 99%, > 99%, 99%, > 99%, 99%, > 99%, > 99%, > 99%, and > 99%, respectively; and the ee value is > 99%, > 99%, 97%, > 99%, > 99%, > 99%, 97%, > 99%, > 99%, > 99%, > 99%, and > 99%, respectively.

[0053] (4) The method of the application synthesizes 179.8 mg of (1S,3R)-4a from the ketone 1a as a raw material by a chemical enzyme modularization level, and the yield is 81%, > 99de, and > 99ee. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is a synthesis route of 1,3-disubstituted THIQ.

[0055] Figure 2 It is an imine reductase module liquid phase analysis diagram.

[0056] Figure 3 It is a chiral identification of enzyme-catalyzed synthesis of (1S,3R)-4a.

[0057] Figure 4 It is a nuclear magnetic resonance spectrum analysis of enzyme-catalyzed synthesis of (1S,3R)-4a 1 H NMR and 13 C NMR.

[0058] Figure 5 NMR spectroscopy of the enzyme-catalyzed synthesis of (1S,3R)-4a was analyzed by NOE spectrum.

[0059] Figure 6 For the use of imine reductase mutants to expand the substrate spectrum. DETAILED DESCRIPTION

[0060] Table 1 amino acid sequences of related proteins

[0061]

[0062]

[0063] Table 2 gene sequences encoding related proteins

[0064]

[0065]

[0066] DETAILED DESCRIPTION

[0068] The present application will be further described below in conjunction with specific examples, so that those skilled in the art can better understand the present application and implement it. The examples are not intended to limit the present application.

[0069] Reagent materials purchase source: The antibiotics such as ampicillin sodium, kanamycin sulfate, streptomycin sulfate used in the present application are from Shanghai Generay Biotech Co., Ltd.; NADP + , NADH are from Shanghai Biotech Co., Ltd.; The PCR enzyme involved in the molecular experiment in the present application is purchased from Baosheng Bioengineering (Dalian) Co., Ltd.; The chemical reagents used in the present application such as methanol, dichloromethane, ethyl acetate (EtOAc), petroleum ether (PE), acetonitrile (MeCN), sodium sulfate, sodium carbonate, sodium bicarbonate, diethylamine (DEA), ammonia, sodium hydroxide, hydrochloric acid and the like are purchased from China Pharmaceutical Group Co., Ltd. and Shanghai Titan Science and Technology Co., Ltd.

[0070] Transaminase activity assay: Substrate 1a (10 mM), transaminase (0.2 mol%), GDH (0.15 mol%), LDH (0.03 mol%), D,L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM), and DMSO (5% v / v) were incubated in a 200 μL reaction system (pH = 8) containing 100 mM at 30 °C for 24 h. After the reaction, 24 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20000 g for 5 min. 20 μL of the sample was loaded onto a chromatographic column for conversion analysis. The remaining reaction solution was then adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers were combined and concentrated under vacuum to remove the organic solvent. The ee value of the product was analyzed by forward HPLC.

[0071] Chemical module detection: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acetyl chloride was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered, and concentrated to obtain crude amide, which could be used directly for the next step without purification. A two-necked round-bottom flask (50 mL) equipped with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added to the solution, and the mixture was refluxed at 90 °C for 2 hours. A 1 μL sample was diluted 1000 times with chromatographic grade methanol for liquid chromatography analysis.

[0072] Imine reductase activity assay: imine rac-3a (1 mM), imine reductase (1.6 mol%), GDH (1 mol%), NADP + 10 mol%, glucose (100 mM), and DMSO (5% v / v) were incubated at 30°C for 24 hours in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction was complete, 30 μL of the reaction mixture was diluted to 300 μL with chromatographic grade methanol, and the conversion and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers were combined and concentrated under vacuum to remove the organic solvent, and the ee value of the product was analyzed by forward HPLC.

[0073] High performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature 25 °C, detection wavelength 280 nm. The detection method uses a three-phase solvent system of methanol (containing 0.1% (v / v) trifluoroacetic acid), acetonitrile (containing 0.1% (v / v) trifluoroacetic acid) and double distilled water (containing 0.1% (v / v) trifluoroacetic acid) for gradient separation, and the flow rate of the mobile phase is 1 mL min -1 . The linear gradient is as follows:

[0074] Table 3 High performance liquid chromatography (HPLC) detection conditions

[0075]

[0076] Protein purification method: The collected bacteria were resuspended with lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH = 8), and then broken by a high-pressure homogenizer. The resuspended solution was subjected to low-temperature high-speed centrifugation at 4 °C, 10000 rpm for 30 min to obtain a crude enzyme solution. The resuspended solution was subjected to nickel column affinity chromatography and desalting column (Histrap™ 5 mL Desalting) desalting, and finally the purified protein was obtained.

[0077] Example 1: SnIR protein expression and purification

[0078] 1. Obtaining of SnIR plasmid

[0079] The target gene SnIR from Stackebrandtia nassauensis was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 4. The gene was synthesized and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-SnIR.

[0080] 2. Protein expression and purification

[0081] A single colony of E. coli BL21 containing the target gene was picked and inoculated in 5 mL LB medium containing antibiotics (ampicillin) and incubated at 37 °C, 220 rpm for 8 h. Then, 1% (v / v) of the inoculum was transferred to 500 mL 2xYT medium, and 500 ul of 100 mg / mL ampicillin was added to the medium. The culture was incubated at 37 °C until the optical density at 600 nm (OD600) of the bacteria reached 0.5-0.7, and then the culture was cooled to 18 °C. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM for protein induction expression.

[0082] The cells were collected by centrifugation (8000 rpm, 5 min, 4°C) and the supernatant was discarded. The cell pellet was then resuspended in 35 mL of pre-chilled (4°C) Tris-HCl buffer (25 mM, pH 8.0) containing 0.1 mM pyridoxal-5-phosphate (PLP), 0.3 M sodium chloride, 20 mM imidazole and 10% (v / v) glycerol. The resuspended cells were broken by a high pressure homogenizer (800 bar) and the cell debris was removed by centrifugation (18300 rpm, 30 min, 4°C). The protein was purified by immobilized metal affinity chromatography (IMAC) and the nickel column was equilibrated with 10 column volumes of lysis buffer. Non-specifically bound proteins were removed by applying ten column volumes of 20 mM imidazole. The specifically bound proteins were eluted from the nickel column with two column volumes of 250 mM imidazole. The purified transaminase fractions were pooled and then concentrated by a 30 KDa ultrafiltration tube and the storage buffer (25 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, 0.1 mM PLP, pH 8.0) was exchanged. The purified enzyme solution was aliquoted and stored at -80°C at a final concentration of 10 mg / mL until use.

[0083] According to the above similar method, imine reductase GF3546 (amino acid sequence as shown in SEQ ID NO. 9) derived from Streptomyces sp., imine reductase CfIR (amino acid sequence as shown in SEQ ID NO. 10) derived from Cystobacter ferrugineus were expressed in E. coli to prepare imine reductase proteins.

[0084] 1. Construction of plasmid for expressing transaminase

[0085] Taking ArR-TA as an example, the target gene ArR-TA derived from Arthrobacter sp. was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 2. The gene was synthesized and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-ArR-TA.

[0086] 2. Protein expression and purification

[0087] The recombinant plasmids constructed in step 1 were transformed into E. coli BL21 competent cells, respectively, and the correct strains were verified as recombinant E. coli expressing corresponding transaminase. According to the method of Example 1, protein expression and purification were performed to obtain ArR-TA enzyme protein, which was aliquoted and adjusted to a final concentration of 10 mg / mL and stored at -80°C for use.

[0088] Example 3: Preparation of LDH and GDH

[0089] 1. Construction of plasmid expressing LDH, GDH

[0090] The target gene GDH from Bacillus subtilis was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 6, and the gene was synthesized and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-GDH.

[0091] The target gene LDH from Lactobacillus fermentum JN248 was codon-optimized to obtain the gene sequence shown in SEQ ID NO. 8, and the gene was synthesized and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-LDH.

[0092] 2. Protein expression and purification

[0093] The protein expression and purification were performed according to the method of Example 1, and LDH with a specific enzyme activity of 903 U / mg and GDH with a specific enzyme activity of 885 U / mg were obtained, respectively, which were aliquoted and adjusted to a final concentration of 10 mg / mL and stored at -80°C for use.

[0094] Example 4: Imine reductase screening

[0095] The rac-3a was converted to the corresponding 4a by imine reductase, glucose, GDH, and co-factor NADH.

[0096] The specific steps are as follows: imine rac-3a (1 mM), imine reductase (1.6 mol%), GDH (1 mol%), NADP + (10 mol%), glucose (100 mM), and DMSO (5% v / v), wherein the molar percentage of each reactant is based on the substrate imine rac-3a. Incubation was performed at 30°C for 24 hours in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction was completed, 30 μL of the reaction mixture was diluted to 300 μL with chromatographic grade methanol, and the conversion rate and de value were determined by HPLC and standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution, and extracted with ethyl acetate for 3 times. The organic layer was combined and concentrated under vacuum to remove the organic solvent, and the ee value of the product was analyzed by normal phase HPLC.

[0097] Table 4 Screening results of imine reductase with 3a as substrate

[0098]

[0099]

[0100] Example 5: Construction of imine reductase mutants

[0101] The rate-limiting enzyme SnIR in the chemical enzyme modular cascade reaction pathway was engineered. First, the substrate (R)-3a was docked into the substrate pocket of SnIR protein by molecular docking software (Discovery Studio), and then 15 amino acids in the range of 1.5 A from the docked substrate (R)-3a were selected for alanine mutation.

[0102] The primers used for SnIR mutant design are shown in Table 2.

[0103] Table 5 Primer sequences of SnIR mutants

[0104]

[0105] The reaction was carried out according to the procedure of Example 4, and the conversion rate was determined by the standard curve of substrate rac-3a to represent the catalytic efficiency of different mutants.

[0106] As shown in Table 6, five single mutants SnIR-T123A, SnIR-P124A, SnIR-L175A, SnIR-M216A and SnIR-T223A with higher conversion rates were obtained, among which SnIR-L175A had higher de value and ee value. Secondly, the optimal mutant SnIR-L175A / M245A was obtained by semi-rational design and combinatorial mutation strategy. Among them, SnIR-L175A / T223A, SnIR-L175A / L225A, SnIR-L175A / M246A, SnIR-L175A / W287A also had better results. Compared with wild-type SnIR, the activity and stereoselectivity of mutant SnIR-L175A / M245A were both improved.

[0107] Table 6 Relative enzyme activity of SnIR mutants

[0108]

[0109]

[0110] As Figure 1 ​The first step (validation of transaminase module) is shown: with (3,5-dimethoxyphenyl) ethanone (1a) as a model substrate, conversion to the corresponding amine (R-(2a) or S-(2a)) by transaminase, co-factor PLP, D,L-alanine, glucose, LDH, GDH constructed in Example 3, co-factor NADH. The first step (validation of chemical module): racemic (3,5-dimethoxyphenyl) ethylamine 2a reacts with acyl chloride to form amide, followed by the formation of racemic 6,8-dimethoxy-1,3-dimethyl-3,4-dihydroisoquinoline (rac-3a) by the action of phosphorus oxychloride. The third step (validation of imine reductase module): with rac-3a as substrate, conversion to the corresponding 4a by imine reductase, glucose, GDH, co-factor NADH.

[0111] Example 6: Application of imine reductase mutants in the preparation of 1,3-disubstituted THIQs

[0112] (1) The first step reaction: substrate 1a (10 mM), ArR-TA (0.2 mol%), GDH (0.15 mol%, specific enzyme activity 885 U / mg), LDH (0.03 mol%, specific enzyme activity 903 U / mg), D,L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) in a 200 μL reaction system containing 100 mM (pH = 8) at 30°C for 24 hours, after the reaction is completed, 24 times the volume of methanol is added to quench the reaction, shake, low temperature high speed centrifugation, 20000g centrifugation for 5 min, take 200 μL sample for analysis of conversion rate on the chromatographic column. Then the remaining reaction mixture is alkalized to pH 10 with 5M sodium hydroxide solution, extracted with dichloromethane 3 times. The solvent in the combined organic layer is removed by vacuum concentration, and the ee value is determined by normal phase HPLC. The results show that xx

[0113] (2) The second step reaction: amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalents) are dissolved in dichloromethane (0.5 M), and acetyl chloride is added dropwise at 0°C. After the reaction is completed, the solution is diluted with dichloromethane, washed with brine twice, dried with sodium sulfate, filtered and concentrated to obtain the crude amide, which is directly used in the next step without purification. A double-necked round-bottom flask (50 mL) is equipped with a magnetic stirring rod, and flushed thoroughly with argon for 3 cycles. The crude amide (1 equivalent) is dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalents) is added. Add to the mixture, reflux at 90°C for 2 hours. Take 1 μL sample, dilute 1000 times with chromatographic grade methanol, and analyze the reaction by liquid chromatography.

[0114] (3) Third step reaction: imine rac-3a (1 mM), imine reductase mutant SnIR-L175A / M245A (1.6 mol%), GDH (1 mol%), NADP + (10 mol%), glucose (100 mM) and DMSO (5% v / v), where the molar percentage of each reactant is based on the substrate imine rac-3a. The 200 μΐ, reaction system containing 100 mM Tris-HCl was incubated at 30 °C for 24 hours. After the reaction was completed, the reaction mixture was diluted to 300 μΐ, with chromatographic grade methanol, and the conversion and de values were determined by HPLC and standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution, and extracted with ethyl acetate three times. The organic layers were combined and concentrated under vacuum to remove the organic solvent, and the ee value of the product was analyzed by normal phase HPLC.

[0115] Example 7: Substrate spectrum expansion

[0116] According to the experimental results, the main rate-limiting step is the imine reductase module, and the yield calculation in the substrate spectrum expansion is mainly focused on the imine reduction module. The de value and ee value can represent the results of the chemical enzyme modular cascade reaction to prepare the final product from the substrate ketone. The structural formula of the corresponding product is shown in Figure 6 .

[0117] Preparation of enantiomerically enriched imine 3:

[0118] Substrate 1 (5 mM), ArR-TA (0.4-1 mol%), GDH (0.15-0.3 mol%), LDH (0.03-0.06 mol%), D, L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were incubated in a 200 μΐ, reaction system containing 100 mM (pH = 8) at 30 °C for 24 hours. After the reaction was completed, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution, and extracted with dichloromethane three times. The organic layers were combined, dried with sodium sulfate, and concentrated under vacuum to remove the organic solvent. The crude amine 2 was directly used in the next step reaction.

[0119] Amine 2 (1 equivalent) and triethylamine (TEA) (1.5 equivalent) were dissolved in dichloromethane (0.5 M), and acyl chloride (≥1 equivalent) was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered, and concentrated to obtain crude amide, which was used directly for the next step without purification. A two-necked round-bottom flask (50 mL) with a magnetic stir bar was thoroughly rinsed with argon for 3 cycles. Crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalent) was added. The mixture was added, and the mixture was refluxed at 90 °C for 2 hours. The solvent was evaporated under vacuum, the residue was quenched with ammonia, extracted three times with ethyl acetate, washed three times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated under vacuum, and the residue was purified by column chromatography to obtain imine 3.

[0120] Imine 3 (1 mM), imine reductase mutant SnIR-L175A / M245A (1.6 mol%), GDH (1 mol%), NADP + 10 mol%, glucose (100 mM), and DMSO (5% v / v) were incubated at 30°C for 24 hours in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction was complete, 30 μL of the reaction mixture was diluted to 300 μL with chromatographic grade methanol, and the conversion and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted three times with ethyl acetate. The organic layers were combined and concentrated under vacuum to remove the organic solvent, and the ee value of the product was analyzed by forward HPLC.

[0121] The results showed that all substrates ( Figure 6 All of them can be converted into the corresponding products (1S,3R)-4a to (1S,3R)-4l. The specific yields, de values ​​and ee values ​​are shown in the table below.

[0122] Table 7. Yields, de, and ee of 1,3-disubstituted THIQ

[0123]

[0124] Comparative Example 1: Comparison before and after imine reductase mutation

[0125] The specific implementation method is the same as in Example 5, except that a mutant Y171A was also constructed and reacted under the same conditions as in Example 4. The results showed that the single-point mutant SnIR-Y171A basically lost its activity, which may be related to the fact that it is a key catalytic site.

[0126] Table 8. Relative enzyme activities of imine reductase mutants

[0127]

[0128] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. An imine reductase mutant, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine.

2. The imine reductase mutant according to claim 1, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine, and threonine at position 223 was mutated to alanine.

3. The imine reductase mutant according to claim 1, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine, and methionine at position 245 was mutated to alanine.

4. The imine reductase mutant according to claim 1, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine, and leucine at position 225 was mutated to alanine.

5. The imine reductase mutant according to claim 1, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine, and methionine at position 246 was mutated to alanine.

6. The imine reductase mutant according to claim 1, characterized in that, Using imine reductase with the amino acid sequence shown in SEQ ID NO.3 as the parent, leucine at position 175 was mutated to alanine, and tryptophan at position 287 was mutated to alanine.

7. A gene encoding the imine reductase mutant of any one of claims 1 to 6.

8. A recombinant plasmid carrying the gene of claim 7.

9. A recombinant microorganism expressing any one of the imine reductase mutants according to claims 1 to 6.

10. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET-21b(+) as the vector, the imine reductase mutant described in claims 1 to 6 was expressed.

11. A method for preparing the imine reductase mutant according to any one of claims 1 to 6, characterized in that, The recombinant Escherichia coli of claim 10 is cultured in a culture medium to induce enzyme production, and the imine reductase mutant is collected.

12. An enzyme preparation containing any one of the imine reductases according to claims 1 to 6.

13. The application of the imine reductase mutant according to claim 1 in the synthesis of 1,3-disubstituted THIQ, characterized in that, The 1,3-disubstituted THIQ includes, but is not limited to, any of the following:

14. The application according to claim 13, characterized in that, The application includes the following steps: (1) Amine 2 is prepared by mixing and reacting transaminase protein with substrate in the presence of lactate dehydrogenase cycle system; the substrate is ketone 1, D-alanine or L-alanine, pyridoxal phosphate; the LDH cycle system includes lactate dehydrogenase, glucose dehydrogenase, NADH, and glucose. (2) The amine 2 obtained in step (1) is reacted with acyl chloride, the reaction product is collected, and the product is then reacted with phosphorus oxychloride to obtain imine 3. (3) Using the imine 3 obtained in step (2) as a substrate, a catalytic reaction is carried out with the imine reductase mutant described in claim 1 and the GDH cycle system to obtain 1,3-disubstituted THIQ; the GDH cycle system includes GDH and NADP. + ,glucose.

15. The application according to claim 14, characterized in that, The ketone 1 includes at least one of the following 1a-1f: The acyl chloride includes at least one of the following A and B: The imine includes at least one of the following:

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