An imine reductase mutant, a recombinant genetic engineering bacterium and their application in the synthesis of (R)-2-methylpyrrolidine

Abstract

The present invention discloses an imine reductase mutant, a recombinant genetic engineering bacterium and their application in the synthesis of (R)-2-methylpyrrolidine. The imine reductase mutant uses the wild-type imine reductase IR23 with the amino acid sequence shown in SEQ ID NO.1 as a template, and the mutant V249T obtained by mutating valine at position 249, and the mutant V249T / W224F obtained by mutating valine at position 249 and tryptophan at position 224. Through the molecular modification method of semi-rational design, the present invention significantly improves the ability of imine reductase to catalyze the formation of the corresponding cyclic amine from the cyclic imine 2-methylpyrroline. When the mutant V249T / W224F catalyzes 2-methylpyrroline, the substrate conversion rate is 93.3%, which is increased by 49.8% compared with the wild type. The present invention effectively improves the ability of imine reductase to asymmetrically synthesize chiral amines, which is of great significance for the green catalytic synthesis of chiral amine compounds.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and particularly relates to an imine reductase mutant, a coding gene, a recombinant plasmid containing these coding genes, a recombinant genetically engineered bacterium, and their application in catalyzing the synthesis of (R)-2-methylpyrrolidine from 2-methylpyrroline. Background Art

[0002] As an important structural building block of chemical substances, chiral amine structures are widely used in the pharmaceutical industry, fine chemicals, and agrochemicals. It is statistically shown that chiral amine structures are present in many drug molecules, and approximately 40% of drugs have at least one chiral amine structure in their structures. Among 200 small molecule drugs, drugs with chiral amine structures account for 40%, and chiral amine functional group compounds account for 20% in agrochemicals. The Pharmaceutical Roundtable of the American Chemical Society - Green Chemistry Institute has proposed that the synthesis of chiral amine compounds is one of the great challenges faced by the current pharmaceutical industry. Thus, it is of great significance to construct a green and efficient synthesis process for chiral amine compounds.

[0003] The synthetic methods of chiral amine compounds are mainly divided into chemical methods and biological methods. Traditional chemical methods use chiral transition metals or organic small molecules to catalyze reactions, and the catalysts include rhodium, cobalt, palladium, chiral phosphoric acid, etc. The use of metal catalysts requires effective steps to separate them, increasing the industrial production cost and difficulty. Chemical synthesis also faces problems such as low enantioselectivity, environmental pollution, and harsh reaction conditions. Therefore, the industry is committed to developing sustainable catalytic methods. Compared with chemical synthesis, biocatalysis has the advantages of high stereoselectivity, mild reaction conditions, less environmental pollution, and simple reaction routes, and is widely used in the synthesis of various chiral amine compounds. At present, several biocatalysts have been applied to the synthesis of chiral amines, including imine reductase, transaminase, amine dehydrogenase, etc. Among them, three types of enzymes have received extensive attention due to their ability to catalyze the synthesis of chiral amines. The first type of enzyme is transaminase. ω-Transaminase can catalyze the conversion of aldehydes and ketones into chiral amine compounds, realizing the asymmetric transfer of amino groups. The industrial application of transaminase faces problems such as thermodynamic equilibrium, narrow substrate spectrum, and low catalytic substrate concentration. In addition, its catalytic transfer of amino groups can only produce primary amine compounds, and chiral secondary amine and chiral tertiary amine compounds need to be obtained through other synthetic routes. The second type of enzyme is amine dehydrogenase, which can catalyze the coupling of prochiral ketones and ammonia to achieve asymmetric reductive amination of aldehyde and ketone substrates. Similar to transaminase, the products of amine dehydrogenase are also limited to primary amine compounds. In recent years, researchers have found that imine reductase can be applied to the synthesis of chiral amines, asymmetrically reducing prochiral imines to form chiral amines, and it is the most promising catalyst for the synthesis of optically pure cyclic amines. Imine reductase can also directly start from prochiral ketones and combine with suitable amine donors to form imine compounds, and then perform asymmetric reduction to generate chiral amines. Its ability to directly generate primary and secondary amines provides a new route for the synthesis of chiral amines.

[0004] Imine reductase is a class of NADPH-dependent oxidoreductases with a strict preference for NADPH, which can asymmetrically catalyze the reduction of prochiral ketones and imines to chiral amines. Therefore, according to the catalyzed reactions and substrate types, it can be divided into two categories: 1) enzymes that can only catalyze the reduction of prochiral imines and 2) enzymes that catalyze the reductive amination of ketones and the reduction of prochiral imines. The first category can catalyze the asymmetric reduction of imine substrates. Due to the low bond energy and electrophilicity of the C=N bond, the C=N bond is extremely vulnerable to attack by nucleophilic molecules, and acyclic imine substances are very unstable in aqueous solution, while cyclic imines are relatively stable. Therefore, imine reductase is widely used in the reduction reaction of cyclic imines. The other type of imine reductase can catalyze the synthesis of imine substrates from prochiral ketones and suitable amines, and then catalyze the asymmetric reduction of imine substrates to achieve the reductive amination of prochiral ketones. However, the existing imine reductases still have the defect of low chiral amine production ability and cannot meet the current requirements for the green industrial catalytic synthesis of chiral amines. Summary of the Invention

[0005] The object of the present invention is to provide an imine reductase mutant with improved chiral amine generating ability, a coding gene, a recombinant plasmid, a recombinant genetically engineered bacterium and their application in catalyzing the synthesis of corresponding chiral amines from 2-methylpyrroline, so as to solve the problem that the imine reductase in the prior art still has a relatively low chiral amine generating ability and cannot meet the requirements of the current green industrial catalytic synthesis of chiral amines.

[0006] To solve the above problems, the present invention adopts the following technical solutions:

[0007] According to the first aspect of the present invention, there is provided an imine reductase mutant with improved chiral amine generating ability, including an imine reductase mutant formed by mutating the wild-type imine reductase IR23 with the amino acid sequence shown in SEQ ID NO.1 as follows: the imine reductase mutant V249T in which valine at position 249 is mutated to threonine, and the amino acid sequence is as shown in SEQ ID NO.3; the imine reductase mutant V249T / W224F in which valine at position 249 is mutated to threonine and tryptophan at position 224 is mutated to phenylalanine, and the amino acid sequence is as shown in SEQ ID NO.4.

[0008] According to the second aspect of the present invention, there is provided a coding gene for the imine reductase mutant.

[0009] According to the third aspect of the present invention, there is provided a recombinant vector containing the coding gene for the imine reductase mutant.

[0010] According to the fourth aspect of the present invention, there is provided a recombinant genetically engineered bacterium prepared by transformation with the recombinant vector. The host cell used can be various conventional host cells in the art. Preferably, the host cell is Escherichia coli BL21(DE3).

[0011] According to the fifth aspect of the present invention, there is also provided an application of using the imine reductase mutant to catalyze the preparation of (R)-2-methylpyrrolidine from 2-methylpyrroline.

[0012] According to a preferred embodiment of the present invention, the application includes: S1: Culturing the recombinant genetically engineered bacterium, inducing expression to obtain a crude enzyme solution or wet cells or freeze-dried cell powder; S2: Adding a buffer solution to the crude enzyme solution or wet cells or freeze-dried cell powder, and adding a mother liquor containing 2-methylpyrroline, NADP + , glucose, and fully mixing all components, and then placing them in a shaker at 25-30 °C for reaction, and the efficient synthesis of (R)-2-methylpyrrolidine can be achieved.

[0013] The present invention first selected the imine reductase IR23 gene cloned from Streptomyces viridochromogenes Tue57 as a template. The amino acid sequence of the imine reductase IR23 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. Homology modeling was performed using the crystal structure of the imine reductase from Actinoalloteichus hymeniacidonis (PDB ID: 7wnw) as a template, and the substrate was docked into the imine reductase IR23 model. By analyzing the conformation of the substrate-enzyme docking complex, two amino acids near the catalytic active center of IR23 were identified, which may affect the synthesis efficiency of chiral amines. Therefore, saturation mutations were performed on these two sites (the 249th and 224th positions) respectively, and combinatorial mutations were performed on the forward single-point mutants, and the catalytic conversion of 2-methylpyrroline to the corresponding chiral amine by the mutant library was measured. Through these protein engineering strategies, multiple mutants with improved chiral amine production ability were obtained, and the acquisition of these mutants is of great significance for the catalytic synthesis of chiral amine compounds. Among them, when the mutant V249T catalyzed 2-methylpyrroline, the substrate conversion rate was 82.6%, which was 39.1% higher than that of the wild type. When the mutant V249T / W224F catalyzed 2-methylpyrroline, the substrate conversion rate was 93.3%, which was 49.8% higher than that of the wild type.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0015] 1) The present invention performs molecular modification on the imine reductase IR23 with low catalytic efficiency through semi-rational design, and obtains mutants with significantly improved chiral amine production ability. The substrate conversion rate of the best mutant is increased from 43.5% of the wild type to 93.3%, and ee > 99.9%. The catalytic efficiency of the mutant is significantly improved, realizing the efficient synthesis of (R)-2-methylpyrrolidine.

[0016] 2) The improvement of the catalytic efficiency of the imine reductase enables it to be applied to the green industrial catalytic synthesis of chiral amines, which has important application value.

[0017] In summary, the present invention performs molecular modification on the imine reductase derived from Streptomyces viridochromogenes Tue57, determines the amino acid sites that play a key role in the production of chiral amine products through analysis, performs saturation mutations and combinatorial mutations, and constructs an imine reductase mutant with improved catalytic ability for 2-methylpyrroline. The present invention has important reference significance for regulating the natural imine reduction activity of imine reductase, also provides a supplementary scheme for the synthesis of chiral amine compounds, and is of great significance for the green catalytic synthesis of chiral amine compounds. Brief Description of the Drawings

[0018] Figure 1 The docking structure of the imine reductase IR23 model with the substrate and the spatial position distribution of the key mutant residues V249 and W224 are shown. Detailed Description of the Invention

[0019] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art, or the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0020] LB liquid medium: Dissolve 10 g of NaCl, 10 g of peptone, and 5 g of yeast extract into 1 L of deionized water. Measure 50 mL with a measuring cylinder and dispense it into a 250 mL Erlenmeyer flask. Wrap it with a sealing film and sterilize it at 121 °C for 20 min.

[0021] LB solid medium: Measure 100 mL of LB liquid medium with a measuring cylinder and dispense it into a 250 mL Erlenmeyer flask. Add 1.5 g of agarose to each flask. Wrap it with a sealing film and sterilize it at 121 °C for 20 min.

[0022] Selection of mutation sites in Example 1

[0023] Since the corresponding crystal structure of imine reductase IR23 has not been resolved, the inventors performed homology modeling through the SWISS-MODEL online server (http: / / www.swissmodel.expasy.org / ). Select the imine reductase (PDB ID: 7wnw derived from Actinoalloteichus hymeniacidonis) as the template. After the modeling is completed, download and optimize the obtained structure. Then dock the prepared substrate molecules into the model. After the docking is completed, screen and save the ideal docking complex according to the binding energy of the complex, the dominant orientation of the substrate, and the distance between the catalytic key groups.

[0024] Through the analysis of the docking structure, the inventors screened out two key residues, V249 and W224, near the catalytic active center of imine reductase IR23 that may affect the catalytic efficiency. The docking structure of the imine reductase IR23 model with the substrate and the spatial position distribution of the key mutant residues V249 and W224 are as Figure 1 shown. Subsequently, saturation mutagenesis was performed on these two potential sites, and combined mutagenesis was performed on the forward mutants to construct a mutant library.

[0025] Construction of imine reductase mutants in Example 2

[0026] The Escherichia coli with the pET-22b(+)-IR23 recombinant plasmid was cultured in a test tube of LB liquid medium for 10 - 12 h, and the plasmid was extracted as a template for subsequent mutant construction. The primers used for mutagenesis (SEQ ID NO.5 - 12) are shown in Table 1.

[0027] Table 1 Primer information for mutants

[0028]

[0029]

[0030] The PCR reaction system (20 μL) is as follows:

[0031]

[0032] PCR reaction conditions:

[0033] (1) Pre-denaturation at 95°C for 5 min

[0034] (2) Denaturation at 95°C for 30 s

[0035] (3) Annealing at 60°C for 30 s

[0036] (4) Extension at 72°C for 1 min 10 s

[0037] (5) Final extension at 72°C for 10 min

[0038] (6) Incubation at 4°C

[0039] Steps (2) - (4) are set for 35 cycles.

[0040] After the PCR product is verified as positive by agarose gel electrophoresis, 0.5 μL of restriction endonuclease Dpn I is added, and the template is removed by water bath at 37°C for 2.5 h. Subsequently, transformation, colony picking, and sequencing can be carried out.

[0041] Induced expression of imine reductase mutants in Example 3

[0042] Take out a test tube containing 5 mL of liquid medium, add 0.1% volume of the resistance, and use a pipette to aspirate an appropriate amount of bacterial liquid and inoculate it into the resistance test tube. Incubate overnight at 37°C for 12 hours. Add 0.1% volume of the resistance to a conical flask containing 50 mL of medium. Take out the cultured test tube, use a pipette to aspirate 500 μL of bacterial liquid and add it to the medium, and place it in a shaker at 37°C for 2 hours until the OD600 value is between 0.6 and 0.8. Add 0.1% v / v of IPTG inducer in a laminar flow hood for induction, place it in a shaker at 20°C, and induce for about 18 - 20 hours.

[0043] After the induction culture is completed, perform the operation of collecting bacteria. Add deionized water to the centrifuge tube containing the bacterial liquid to balance it, and then place it in a pre-cooled centrifuge at 4°C for centrifugation (8,000 rpm, 10 min). After centrifugation, pour out the supernatant, add 20 mL of 0.5% NaCl solution to resuspend the bacterial cells, balance it and centrifuge again under the same conditions. After centrifugation, remove the supernatant as much as possible, and then place it in a -40°C refrigerator for freezing for more than 3 h to perform freeze-drying. After the sample is placed in a vacuum freeze-dryer for about 10 h, take it out to obtain the freeze-dried powder of imine reductase.

[0044] Screening of mutants in Example 4

[0045] Using the freeze-dried powder of imine reductase prepared in Example 3 as a catalyst, perform a conversion reaction with 2-methylpyrroline as a substrate to screen for mutants with improved ability to generate chiral amines.

[0046] Prepare the reaction system according to the composition shown in Table 2: First, weigh the freeze-dried powder and place it in a 2 mL centrifuge tube, add buffer solution to dissolve the freeze-dried powder, and finally add the mother liquor containing 2-methylpyrroline (100 mM), NADP + (1 mM), and glucose (125 mM). After adding all components, mix well. Do three parallel samples for each reaction. Place it in a shaker at 30°C for reaction for 2 hours. After the reaction is completed, add 50 μL of sodium hydroxide solution (10 M) to terminate the reaction, and centrifuge at 14,000 rpm for 10 min. The reaction solution needs to be further processed for gas / liquid detection.

[0047] Table 2

[0048]

[0049] The conversion rate of the reaction was detected by gas chromatography. 400 μL of the terminated reaction solution was taken into a new 2 mL EP tube, and 2 volumes of ethyl acetate (with internal standard) were added. After vortexing for 15 s for sufficient extraction and centrifuging at 14,000 rpm for 10 min, 400 μL of the supernatant organic phase was taken into a new centrifuge tube, an appropriate amount of anhydrous sodium sulfate was added, and then centrifuged at 14,000 rpm for 10 min. 200 μL of the supernatant was taken into a small tube for gas chromatography detection.

[0050] The gas chromatography detection conditions were as follows: chromatographic column DB-1701, internal standard o-xylene (10 mM), injection volume 1 μL, split ratio 2:1; temperature programming: 50 °C for 5 min, heating rate 25 °C / min to 190 °C, heating rate 100 °C / min to 240 °C and maintained for 5 min.

[0051] The ee value of the reaction sample needed to be detected by liquid chromatography after derivation. A 1.5 mL centrifuge tube was taken and the derivation system was added. The derivation system was as follows (200 μL): 100 μL of 0.8% (w / v) GITC (dissolved in acetonitrile), 50 μL of 0.2% (v / v) triethylamine (dissolved in acetonitrile), and 50 μL of the treated sample. The prepared derivation system was placed in a 40 °C water bath for 30 min of derivation. After the derivation was completed, it was centrifuged at 14,000 rpm for 10 min, and 50 μL of the supernatant was transferred to a liquid chromatography small tube for HPLC liquid chromatography detection.

[0052] Liquid chromatography detection conditions: Extend C18 chromatographic column (5 μm, 4.6 x 250 mm), mobile phase methanol: water = 45:55, column temperature 30 °C, flow rate 0.8 mL / min, injection volume 3 μL, ultraviolet absorption wavelength 254 nm.

[0053] Using 2-methylpyrroline as the substrate for the catalytic reaction, the constructed mutant library was screened and analyzed by the above method. The results are shown in Table 3. The chiral amine generation ability of the mutants obtained according to the present invention was higher than that of the wild type. It can be seen from the table that among the single-point mutants, the substrate conversion rate of V249T was the highest, reaching 82.6%, which was 39.1% higher than that of the wild type. Among the double-point mutants, the substrate conversion rate of V249T / W224F reached 93.3%, which was 49.8% higher than that of the wild type. The forward mutants V249S, V249T, V249Y, W224F, V249S / W224F, V249T / W224F, and V249Y / W224F all had improved catalytic efficiency for 2-methylpyrroline compared to the wild type.

[0054] Table 3 Catalytic chiral amine generation by imine reductase mutants (3 parallels were set for each experiment)

[0055]

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of protection of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. An imine reductase mutant with improved chiral amine generating ability, characterized in that, Comprising an imine reductase mutant formed by the following mutations using the wild-type imine reductase IR23 with the amino acid sequence shown in SEQ ID NO.1 as a template: The imine reductase mutant V249T with valine at position 249 mutated to threonine, and the amino acid sequence is as shown in SEQ ID NO.3; The imine reductase mutant V249T / W224F with valine at position 249 mutated to threonine and tryptophan at position 224 mutated to phenylalanine, and the amino acid sequence is as shown in SEQ ID NO.

4.

2. A coding gene encoding the imine reductase mutant according to claim 1.

3. A recombinant plasmid containing the coding gene according to claim 2.

4. A recombinant genetically engineered bacterium containing the recombinant plasmid according to claim 3.

5. The recombinant genetic engineering bacterium according to claim 4, characterized in that, The host cell is Escherichia coli BL21(DE3).

6. A method for preparing an imine reductase mutant, characterized in that, Including the following steps: 1) Culturing the recombinant genetically engineered bacterium according to claim 4 and inducing the expression of the imine reductase mutant; 2) Isolating the imine reductase mutant according to claim 1 from the culture obtained in 1).

7. Use of the imine reductase mutant according to claim 1 in the catalytic synthesis of (R)-2-methylpyrrolidine from 2-methylpyrroline.

8. The application according to claim 7, wherein The said use includes: S1: Culturing the recombinant genetically engineered bacterium according to claim 4, inducing expression, and obtaining a crude enzyme solution or wet bacterial cells or freeze-dried bacterial powder; S2: Add buffer to the crude enzyme solution or wet bacterial cells or freeze-dried bacterial powder, and add a mother liquor containing 2-methylpyrroline, NADP + , glucose. After adding all components, mix well and place it in a shaker at 25-30 °C for reaction, then the efficient synthesis of (R)-2-methylpyrrolidine can be achieved.

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