An imine reductase and its use in the preparation of chiral arylpyrrolidines

Abstract

The application belongs to the technical field of enzyme engineering and biological catalysis, and provides an imine reductase and application thereof in preparation of chiral arylpyrrolidine. The amino acid sequence of the imine reductase is shown as SEQ ID NO:1. The enzyme can convert aryl-substituted pyrroline into chiral arylpyrrolidine. The product of the imine reductase provided in the application has an enantiomeric excess value greater than 99%, and can tolerate a higher concentration of organic cosolvent in a reaction system, and exhibits excellent enantioselectivity, significantly enhanced organic solvent tolerance and good industrial application prospect. The imine reductase of the application is very suitable for scale-up, green enzymatic synthesis of chiral arylpyrrolidine intermediates, and has great industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to a mutant imine reductase and its application in the preparation of chiral arylpyrrolidines. Background Technology

[0002] Larotrectinib is a neurotrophic tyrosine receptor kinase (NTRK) inhibitor, developed as an anti-solid tumor drug targeting the ATP-binding site of the TRK receptor family. NTRK fusions are pan-tumor markers and drug targets for the treatment of various cancers. Larotrectinib has shown excellent efficacy against various solid tumors in adults and children, and it was launched in the United States in 2018. (R)-2-(2,5-difluorophenyl)pyrrolidine is a key intermediate in the synthesis of larotrectinib, which can be obtained by asymmetric reduction of 2-(2,5-difluorophenyl)pyrrolidine using a catalyst. Chemical synthesis methods using metal catalysts such as rhodium, iridium, and ruthenium are costly, have low yields, and involve complex processes, making them unsuitable for large-scale industrial production. Biochemical enzymatic catalysis offers advantages such as high chiral selectivity, low cost, and mild reaction conditions; therefore, green, efficient, and simple enzymatic synthesis is attracting increasing attention.

[0003] To address the enzymatic asymmetric reduction of 2-(2,5-difluorophenyl)pyrrololine to (R)-2-(2,5-difluorophenyl)pyrrolidine, in 2016, Turrer's group (Stereoselectivity and Structural Characterization of anImine Reductase (IRED) from Amycolatopsis orientalis, ACS Catalysis, 2016, 6, 3880-3889) identified a naturally derived S-selective imine reductase, AoIRED, which can catalyze the reduction of 2-substituted pyrrololines. It can achieve highly efficient catalysis for 2-methylpyrrololine compounds with smaller substituents, but its catalytic activity is lower for pyrrololines with larger substituents, such as 2-aryl-fluorophenyl-substituted pyrrololines. Recently, Professor Gaowei Zheng's research group (Identification of anImine Reductase for Asymmetric Reduction of Bulky Dihydroisoquinolines, Org. Lett., 2017, 19, 3151-3154) screened an imine reductase (IRED) from the genome of Streptomyces clavuligerus. After modification (Engineered Imine Reductase for Larotrectinib Intermediate Manufacture, Acs Catal., 2022, 12, 14795-14803), the enzyme activity reached a specific activity of 19.2 U / mg. A common challenge in industrial production is that the substrate 2-(2,5-difluorophenyl)pyrrolline is a water-insoluble oil; high concentrations of the substrate strongly inhibit the enzyme, resulting in slow reaction rates and low conversion rates. Meanwhile, in the existing asymmetric catalytic synthesis reaction system of imine reductase, the amount of organic cosolvent added is maintained at about 1% of the total system volume. The reaction system is very prone to emulsification, which makes post-processing difficult. Increasing the content of organic cosolvent can alleviate the problem of system emulsification, but high content of organic solvent will seriously affect the conformation of enzyme protein, thereby affecting enzyme activity and causing enzyme inactivation. The above factors together restrict its industrial application.

[0004] Therefore, developing an imine reductase that possesses high stereoselectivity, high substrate tolerance, and high organic solvent tolerance to the target substrate is of great significance for achieving green and efficient industrial production of larotrectinib chiral intermediates. Summary of the Invention

[0005] To address the problems of insufficient enantioselectivity, poor tolerance to high concentrations of hydrophobic substrates, and easy inactivation in the presence of organic solvents in existing biocatalysts for the industrial production of larotrectinib chiral intermediates, this invention aims to provide a novel imine reductase. This enzyme should be able to efficiently and selectively catalyze the asymmetric reduction of 2-(2,5-difluorophenyl)pyrrolline, and significantly improve its tolerance to substrates and organic solvents, thus making it suitable for industrial production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] An imine reductase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] To enable the purification, detection, and functional study of the aforementioned imine reductase, this invention also provides a fusion protein capable of realizing the catalytic function of the imine reductase. Preferably, the fusion protein comprises: the amino acid sequence of the aforementioned imine reductase and a tag sequence; or, the amino acid sequence of the aforementioned imine reductase and the amino acid sequence of a carrier protein. The tag sequence is used to label the fusion protein, and the carrier protein is used to display the imine reductase surface on the cell surface. To ensure the functional integrity or high activity of the imine reductase, the fusion protein may further include a linker sequence for connecting the imine reductase and the tag sequence or carrier protein. The tag sequence may be selected from His tags, GST tags, MBP tags, Strep-tag II, SUMO tags, Avi tags, etc., used for protein purification, and GFP, mCherry, YFP, CFP fluorescent proteins, etc., used for imaging and localization. The carrier protein is selected from outer membrane proteins, lipoproteins, autotransporters, ice nuclei proteins, cell wall binding proteins, cell membrane anchoring proteins, spore surface proteins, surface adhesion proteins, etc.

[0009] In order to enable the fermentation production or surface display of the above-mentioned imine reductase or fusion protein, the present invention also provides an expression cassette, vector or engineered bacteria capable of expressing the above-mentioned imine reductase.

[0010] Preferably, the expression cassette and vector described above are used to express the imine reductase or fusion protein in prokaryotic cells (engineered bacteria). The prokaryotic cells are preferably *Escherichia coli*, *Bacillus subtilis*, or *Streptomyces*. In some embodiments, the prokaryotic cells are *Escherichia coli*.

[0011] The present invention also provides a coding sequence for the above-mentioned imine reductase or fusion protein. To improve the expression of the expression cassette in engineered bacteria, the coding sequence is modified according to the codon preferences of the engineered bacteria. In some embodiments, the nucleotides of the coding sequence are as shown in SEQ ID NO: 2.

[0012] The engineered bacteria provided by this invention can be obtained using genetic engineering techniques commonly used in the field.

[0013] To enable prokaryotic cells to express the aforementioned imine reductase, engineered bacteria were obtained using the following method: the encoding gene of imine reductase or fusion protein was ligated to an expression vector to obtain a recombinant expression vector; the recombinant expression vector was transformed into competent cells of the starting strain, and engineered bacteria were obtained through screening.

[0014] To display the aforementioned imine reductase on the surface of prokaryotic cells, engineered bacteria were obtained using the following method: the encoding gene for the imine reductase or fusion protein was ligated to a surface display vector to obtain a recombinant display vector; the recombinant display vector was transformed into competent cells of the starting strain, and engineered bacteria were screened. The engineered bacteria were inoculated into a culture medium and cultured and induced to express expression sequentially. Preferably, the surface display vector can express the periplasmic secretion signal peptide and the β-barrel structure protein of Ag43.

[0015] The present invention also provides a fixed-bed bioreactor comprising an inert carrier immobilized with the aforementioned imine reductase, fusion protein, or engineered bacteria. The inert carrier provides stable, static attachment support and allows the reaction fluid to contact the imine reductase, fusion protein, or engineered bacteria.

[0016] An asymmetric synthesis method for chiral arylpyrrolidines includes the following steps: Aryl-substituted pyrroline and other necessary substrates are added to a feed solution containing the aforementioned imine reductase, fusion protein, and engineered bacteria, and reacted under appropriate conditions; or, The feed solution containing aryl-substituted pyrroline and other necessary substrates is contacted with a fixed-bed reactor and reacted under appropriate conditions. The reaction solution was separated to obtain (R)-aryl-substituted pyrrolidine.

[0017] The aryl-substituted pyrrolidine is selected from 2-phenylpyrrolidine, 2-(4-fluorophenyl)pyrrolidine, 2-(2,5-difluorophenyl)pyrrolidine, 2-(3,4-difluorophenyl)pyrrolidine, 5-(3-chlorophenyl)-3,4-dihydro-2H-pyrrole, 2-(3,4-dihydro-2H-pyrrole-5-yl)-1H-pyrrole, or 5-phenyl-3,4-dihydro-2H-pyrrole. The resulting chiral arylpyrrolidine compounds can be used as intermediates for chiral drugs or pesticides.

[0018] The present invention has the following advantages: The imine reductase provided by this invention exhibits excellent enantioselectivity, significantly enhanced tolerance to organic solvents, and promising prospects for industrial application. Specifically, when the enzyme catalyzes the reduction of 2-(2,5-difluorophenyl)pyrrolidine to (R)-2-(2,5-difluorophenyl)pyrrolidine, the enantiomeric excess (ee) of the product is greater than 99%, far exceeding that of the wild-type enzyme, thus meeting the stringent requirements for the synthesis of chiral pharmaceutical intermediates. It can tolerate high concentrations of organic co-solvents (such as DMSO and isopropanol) in the reaction system, which helps increase the solubility of hydrophobic substrates, further improving reaction efficiency and avoiding separation processes such as centrifugation, thus preventing severe emulsification. The imine reductase of this invention is highly suitable for the large-scale, green enzymatic synthesis of chiral arylpyrrolidine intermediates, possessing significant industrial application value. Attached Figure Description

[0019] Figure 1 The chiral HPLC chromatograms of a mixture of standards for 2-(2,5-difluorophenyl)pyrrolidine, R-configuration and S-configuration 2-(2,5-difluorophenyl)pyrrolidine are shown. Figure 2 This is the HPLC chromatogram of the wild-type imine reductase reaction product (ee=81%). Figure 3 This is an HPLC chromatogram of the reaction product of mutant imine reductase (ee>99%). Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Experimental conditions not specified in the embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the embodiments can be obtained commercially.

[0021] Example 1 Preparation of mutant imine reductase 1. Construction of IRED mutant engineered bacteria Based on the imine reductase shown in the amino acid sequence WP_119928229 (NCBI), multiple rounds of site-directed mutagenesis were performed. Analysis of the protein structure and protein-substrate complex model identified key amino acid residues affecting substrate binding, product release, and enzyme conformational stability. Site-directed mutagenesis was used to modify the substrate binding pocket inlet and surrounding residues, aiming to optimize the substrate binding mode, expand the substrate channel, and enhance the stability of the enzyme protein in non-aqueous environments. Specifically, the mutations include: replacing arginine at position 129 with serine (R129S), replacing glutamic acid at position 131 with glycine (E131G), replacing serine at position 155 with arginine (S155R), replacing serine at position 183 with glycine (S183G), replacing methionine at position 212 with leucine (M212L), replacing glutamine at position 216 with leucine (Q216L), and replacing valine at position 232 with serine (V232S), resulting in the amino acid sequence shown in SEQ ID NO: 1.

[0022] The above amino acid sequence was codon-optimized to match the codon preference of *E. coli*, resulting in the nucleotide sequence SEQ ID NO: 2. NcoⅠ and XhoⅠ restriction enzyme sites were added to both ends, and the target gene fragment was commercially synthesized. The target gene fragment and the vector pET28a(+) were digested with restriction endonucleases Nco1 and Xho1, followed by ligation, transformation, and screening to obtain positive plasmids. These positive plasmids were transformed into *E. coli* competent cells BL21(DE3), and high-expression strains were screened to obtain engineered bacteria expressing the mutant IRED.

[0023] The same method was used to obtain codon-optimized wild-type enzyme expression engineered bacteria.

[0024] 2. Expression and purification of recombinant imine reductase The above-mentioned engineered bacterial strains were inoculated at a ratio of 1:1000 into LB liquid medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C. The overnight culture was then inoculated into TB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 3 hours until the bacterial concentration reached OD500. 600 When the light absorbance reaches 0.6-0.8, add IPTG-induced protein at a final concentration of 0.5 mM and incubate overnight at 16°C. Collect the bacterial cells by centrifugation at 6000 rpm and 4°C for 15 min.

[0025] The bacterial cells were resuspended in 100 mM potassium phosphate buffer (KPB buffer) at pH 7.0, and then autoclaved at 16,000 rpm and centrifuged at 4°C for 60 min to collect the supernatant. The target protein was purified from the supernatant using nickel affinity chromatography, as detailed below: (1) Equilibrate the nickel medium with KPB buffer (100 mM, pH 7.0) containing 10 mM imidazole; (2) Add the equilibrated nickel medium to the supernatant, gently shake until the medium is suspended, and incubate for 10-30 min. Let the mixture stand for 10 min, and remove the supernatant after the medium has precipitated. (3) Rinse the medium with KPB buffer (100 mM, pH 7.0) containing 10-50 mM imidazole to remove any adsorbed proteins; (4) Elute the medium with 100 mM KPB buffer at pH 7.0 containing 250 mM imidazole, collect the eluent, and obtain wild-type and mutant imine reductases; the N-terminus of the mutant imine reductase is fused with 6×His, and its amino acid sequence is shown in SEQ ID NO: 3.

[0026] The results showed that, under the same conditions, the purified mutant protein yielded approximately 35 mg / L of fermentation broth, with a specific activity of 18.5 U / mg Pr, compared to 6.2 U / mg Pr for the wild type; the specific activity of the mutant enzyme was increased by about 3 times.

[0027] 3. Enantioselectivity of wild-type and mutant imine reductases Construct a 1 mL reaction system with the following concentrations: 100 mM potassium phosphate buffer (pH 7.0), 5 mM 2-(2,5-difluorophenyl)pyrrolidone (dissolved in 0.2 mL DMSO), 0.2 mM NADPH, 0.015 mg / mL purified imine reductase, 1 U / mL glucose dehydrogenase (GDH), and 20 mM glucose.

[0028] After adding imine reductase, the above system was rapidly mixed and reacted at 30°C and 200 rpm for 12 hours. Then, 1M NaOH solution was added to stop the reaction at pH 13, and the mixture was extracted three times with an equal volume of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and analyzed by chiral high-performance liquid chromatography (HPLC). The reaction of wild-type imine reductase under the same conditions served as a control.

[0029] Chromatographic conditions: Chiralpak AD-H chiral column (4.6 mm × 250 mm, 5 μm), mobile phase n-hexane / isopropanol = 90:10, flow rate 0.8 mL / min, detection wavelength 268 nm, column temperature 30℃, injection 20 μL, run time 25 min.

[0030] A mixed standard solution was prepared by mixing the substrate 2-(2,5-difluorophenyl)pyrrolidine and the R or S configuration product 2-(2,5-difluorophenyl)pyrrolidine at a mass ratio of 1:49.5:49.5. The mixture was then analyzed under the same chromatographic conditions. The substrate and product were located based on the peak times of the standards, and the enantiomeric excess (ee) was calculated using the following formula: ee (%) = (C R - C S ) / (C R + C S ) × 100%; Among them, C R For the peak area of ​​the R configuration product, C S The peak area is for the S-configuration product.

[0031] Chromatogram of mixed standards as shown Figure 1 As shown: the peak time of 2-(2,5-difluorophenyl)pyrrolidine was 7.861 min, the peak time of (R)2-(2,5-difluorophenyl)pyrrolidine was 9.556 min, and the peak time of (S)2-(2,5-difluorophenyl)pyrrolidine was 11.241 min.

[0032] The conversion results of 2-(2,5-difluorophenyl)pyrrololine by wild-type and mutant imine reductases are as follows: Figure 2 and 3 As shown in the figure, the ee value of the wild-type imine reductase catalyzed product was calculated to be 81%, while the ee value of the mutant imine reductase catalyzed product was greater than 99%. This indicates that the enantioselectivity of imine reductase was significantly improved by modification.

[0033] 4. Substrate tolerance First, establish the relationship between NADPH concentration and OD. 340 A standard curve was obtained, and then a 1 mL reaction system was constructed with the following concentrations: 100 mM potassium phosphate buffer (pH 7.0), 0.2 mM NADPH, 0.015 mg / mL purified imine reductase, and 10 mM-50 mM 2-(2,5-difluorophenyl)pyrrolidine dissolved in 0.2 mL DMSO. After adding the imine reductase, the mixture was rapidly mixed and reacted at 30 °C and 200 rpm for 5 min. The OD of the system was then measured. 340 The remaining NADPH in the system is calculated based on the standard curve. The substrate tolerance of the enzyme is assessed by the rate of decrease of substrate NADPH. The relative enzyme activity is calculated with the enzyme activity at the lowest substrate concentration of 10 mM as 100%: Relative enzyme activity = (initial rate at each concentration / initial rate at 10 mM concentration) × 100%.

[0034] Table 1. Relative enzyme activities at different substrate concentrations The results showed that when the substrate concentration reached 50 mM, the activity of the wild-type enzyme was almost completely inhibited, while the mutant enzyme could still maintain about 55% of the initial reaction rate, showing strong substrate tolerance.

[0035] 5. Organic solvent resistance In a standard reaction system (1 mL, containing 5 mM 2-(2,5-difluorophenyl)pyrrolidine), different volume fractions of isopropanol were added as organic co-solvents (final concentrations of 10%, 20%, and 30%, respectively). Specifically, the substrate was directly dissolved in the corresponding volume of isopropanol, and then mixed with buffer and other components to ensure complete substrate dissolution. The concentrations of other components were: 100 mM potassium phosphate buffer (pH 7.0), 0.2 mM NADPH, 0.015 mg / mL purified mutant or wild-type enzyme protein, 1 U / mL glucose dehydrogenase, and 20 mM glucose. After reacting at 30°C and 200 rpm for 12 hours, the reaction was completed, and the mixture was extracted with an equal volume of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and the total amount of 2-(2,5-difluorophenyl)pyrrolidine produced was determined by high-performance liquid chromatography (HPLC). The conversion rates of wild-type and mutant enzymes in different solvent systems were calculated. The conversion rate is calculated using the formula: Conversion rate (%) = (Product peak area) / (Substrate peak area + Product peak area) × 100%. The conversion rates of wild-type and mutant enzymes in different solvent systems are calculated separately.

[0036] Table 2 Substrate conversion rates at different organic solvent concentrations The above results demonstrate that the organic solvent tolerance of the mutant enzyme is significantly enhanced.

[0037] Application Example 1: Catalytic preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine from 2-(2,5-difluorophenyl)pyrrolidine The engineered bacteria expressing mutant IRED prepared in Example 1 were fermented: inoculated into TB medium containing 50 μg / mL kanamycin at a ratio of 1:1000 and cultured at 37°C until OD. 600 =0.6-0.8, add 0.5 mM IPTG, and induce at 16℃ for 20 h. Centrifuge to collect wet cells, resuspend in 100 mM KPB (pH 7.0) to 0.2 g cells / mL, and autoclave to obtain a wet cell lysate.

[0038] 1 g of 2-(2,5-difluorophenyl)pyrrolidine, 4 mL of isopropanol, 20 mM glucose, 0.2 mM NADPH, 20 U glucose dehydrogenase powder, and 5 mL of lysate (containing 1 g of bacterial cells) of wet cells expressing mutant imine reductase were added to a round-bottom flask. The volume was brought up to 20 mL with 100 mM KPB 7.0 phosphate buffer. The mixture was magnetically stirred at 30 °C and 240 rpm for 12 hours. After the reaction was completed by TLC, 6 M KOH aqueous solution was added to adjust the pH to 14. The mixture was extracted three times with an equal volume of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain 0.85 g of (R)-2-(2,5-difluorophenyl)pyrrolidine. High-performance liquid chromatography analysis showed that the product purity was 99.5% and the ee value was 99.1%.

[0039] Application Example 2: Catalytic preparation of (R)-2-(2,5-difluorophenyl)pyrrolidine from 2-(2,5-difluorophenyl)pyrrolidine The lysate of wet cells of the mutant IRED engineered bacteria (containing 50 g of cells, total volume 250 mL) was obtained according to the method in Application Example 1. Then, 550 mL of 100 mM KPB 7.0 phosphate buffer and 0.2 mM NADPH were added to a round-bottom flask. 50 g of 2-(2,5-difluorophenyl)pyrrolidone was dissolved in 200 mL of DMSO to prepare a substrate solution, which was then added to the reaction system. A total of 1000 U of glucose dehydrogenase (GDH) was added in two batches: 500 U at the start of the reaction and another 500 U at the 6th hour.

[0040] Glucose was added at a rate of 1.2 g every 4 hours, for a total of 3.6 g (final concentration 20 mM). The total reaction volume was brought to 1 L with 100 mM KPB 7.0 phosphate buffer. The reaction was carried out at 30 °C and 240 rpm with magnetic stirring for 12 hours, during which the pH was monitored and maintained at approximately 7. After TLC analysis showed that the reaction was complete, the pH was adjusted to 13 with 6 M KOH aqueous solution, and the mixture was extracted three times with 1 L ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain 45.2 g of (R)-2-(2,5-difluorophenyl)pyrrolidine. High performance liquid chromatography analysis showed that the product purity was 99.2% and the ee value was 99.0%.

[0041] Example 2: Preparation of a fixed-bed bioreactor containing mutant imine reductase Diatomaceous earth was used as a carrier. It was first boiled in dilute sulfuric acid for 2-3 hours, then washed with water until neutral, and then immersed in 100 mM KPB (pH 7.0) for equilibration for 2 hours to obtain activated diatomaceous earth. The purified mutant imine reductase from Example 1 was prepared into an enzyme solution of appropriate concentration using 100 mM KPB (pH 7.0). The activated diatomaceous earth was added at a carrier:enzyme ratio of (3-5):1 (m / m), and the mixture was shaken at 30℃ and 240 rpm for 2-3 hours for adsorption. After low-temperature drying, immobilized enzyme was obtained. The specific activity of the immobilized enzyme was measured to be between 8.3 and 10.2 U / mg. The immobilized enzyme was then packed into a reactor to obtain a fixed-bed bioreactor.

[0042] The fixed-bed bioreactor described above was used to prepare (R)-2-(2,5-difluorophenyl)pyrrolidine according to the method in Application Example 2, except that 20% isopropanol was replaced with 30% DMSO. The results showed that the bioreactor could tolerate the addition of up to 30% DMSO, and the substrate conversion rate was still above 90% after 5 cycles.

Claims

1. An imine reductase, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A fusion protein, characterized in that, The fusion protein has the catalytic function of the imine reductase as described in claim 1; and the fusion protein comprises: the amino acid sequence of the imine reductase and a tag sequence; the tag sequence is used to label the fusion protein; The tag sequence is selected from His tag, GST tag, MBP tag, Strep-tag II, SUMO tag, and Avi tag for protein purification; or GFP, mCherry, YFP, and CFP fluorescent proteins for imaging and localization.

3. The fusion protein according to claim 2, characterized in that, The fusion protein also includes a linker sequence for linking the imine reductase and the tag sequence.

4. An expression cassette, vector, or engineered bacteria for expressing the imine reductase as described in claim 1 or the fusion protein as described in claim 2 or 3.

5. The expression cassette, vector, or engineered bacteria according to claim 4, characterized in that, This is used to express the imine reductase or fusion protein in prokaryotic cells; the prokaryotic cells are selected from Escherichia coli, Bacillus subtilis, or Streptomyces.

6. A nucleic acid encoding an imine reductase as described in claim 1 or a fusion protein as described in claim 2 or 3.

7. A method for constructing an engineered bacterium expressing the imine reductase as described in claim 1 or the fusion protein as described in claim 2 or 3, comprising the following steps: The encoding gene of imine reductase or fusion protein is ligated to an expression vector to obtain a recombinant expression vector; the recombinant expression vector is transformed into competent cells of the starting strain, and engineered bacteria are obtained by screening. or, The encoding gene of imine reductase or fusion protein is linked to a surface display vector to obtain a recombinant display vector; the recombinant display vector is transformed into competent cells of the starting strain, and engineered bacteria are obtained by screening.

8. A fixed-bed bioreactor, characterized in that, It includes an inert carrier immobilized with the imine reductase as described in claim 1, the fusion protein as described in claim 2 or 3, or the engineered bacteria as described in claim 4 or 5.

9. An asymmetric synthesis method for chiral arylpyrrolidines, characterized in that, Includes the following steps: 2-(2,5-difluorophenyl)pyrrololine and other necessary substrates are added to a feed solution containing the imine reductase as described in claim 1, the fusion protein as described in claim 2 or 3, or the engineered bacteria as described in claim 4 or 5; or, The feed solution of 2-(2,5-difluorophenyl)pyrrolidone and other necessary substrates is contacted and reacted with the fixed-bed bioreactor as described in claim 8; Then, the reaction solution was separated to obtain (R) 2-(2,5-difluorophenyl)pyrrolidine; The other necessary substrates are NADPH, glucose, and glucose dehydrogenase.

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