Imine reductase mutant and application of imine reductase mutant in synthesis of vibeglone

Abstract

The invention discloses an imine reductase mutant and an application of the imine reductase mutant in synthesis of vibeglone. The invention discloses an imine reductase mutant, which is characterized in that on the basis of an imine reductase mutant IR-13-M5, the imine reductase mutant has the following mutations: M203P-F269W-I149G-S241Q-L200T-G268Q, M203P-F269W-I149G-L200T, M203P-F269W-I149G-S241Q-L200T, M203P-F269W-I149G-S241Q-L200T or M203P-F269W-I149G-L200T-G268Q, and the imine reductase mutant has the following mutations: M203P-F269W-I149G The mutant can be used for asymmetric catalytic synthesis of an S-selective chiral amine product vibeglone, the conversion rate reaches 78%, the stereoselectivity reaches up to 99%, and the mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to an imine reductase mutant with high stereoselectivity and high catalytic activity, and the application of this mutant in the asymmetric catalytic synthesis of key chiral amine intermediates of vebergerone. Background Technology

[0002] Vibegron is a β3-adrenergic receptor agonist used to treat overactive bladder (OAB). Its molecular structure contains a key chiral amine fragment, and the efficient and stereoselective synthesis of this fragment is a core challenge for industrial production. Currently, the chemical catalytic synthesis of this chiral amine mainly relies on noble metal chiral catalysts, high-pressure catalytic environments, or multi-step continuous reactions, which suffers from poor atom economy, severe environmental pollution, and insufficient optical purity.

[0003] Biocatalysis, especially the direct asymmetric reduction of imines to chiral amines catalyzed by imine reductases, offers advantages such as mild reaction conditions, high selectivity, and environmental friendliness. In previous research, the inventors cloned an imine reductase, IR-13, from *Actinoalloteichushymeniacidonis*. The mutant IR-13-M5, obtained through engineering modification of the active site, was used for the asymmetric catalytic reduction of imines to synthesize the Vibegron pharmaceutical intermediate (hereinafter referred to as IR-M5).

[0004] IR-M5 exhibits good catalytic activity towards the five-membered ring imine structure of the Vibegron drug intermediate, but its stereoselectivity is poor (ee value of 36% R), which cannot meet the requirements for industrial production. Therefore, further engineering modification of this mutant to obtain an enzyme capable of efficiently and selectively catalyzing the Vibegron drug intermediate has significant industrial value and practical implications. Summary of the Invention

[0005] The purpose of this invention is to provide engineered imine reductase mutants to overcome the technical bottlenecks of harsh reaction conditions and substandard stereoselectivity (S configuration) in the catalysis of key imine intermediates in the drug vebergren.

[0006] This invention provides a mutant obtained by further mutation of the imine reductase mutant IR-13-M5, wherein the further mutation is selected from: M203P-F269W-I149G-S241Q-L200T-G268Q, M203P-F269W-I149G-L200T, M203P-F269W-I149G-S241Q, M203P-F269W-I149G-S241Q-L200T, and M203P-F269W-I149G-L200T-G268Q. The amino acid sequence of the imine reductase mutant IR-13-M5 of this invention is shown in SEQ ID NO: 2.

[0007] The present invention also provides the encoding nucleic acid of the mutant.

[0008] The present invention further provides a recombinant vector containing the encoded nucleic acid.

[0009] The present invention also provides a recombinant host bacterium containing the encoded nucleic acid or the recombinant vector.

[0010] The present invention particularly provides the mutants that encode nucleic acids for use in the synthesis of vibergron, specifically, through the reduction of vibergron imine to obtain the key chiral amine intermediate of vibergron.

[0011] This invention provides a method for preparing vibergron, which involves asymmetric catalysis of the imine reduction reaction of (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol under NADPH cycle conditions, using the mutant described in claim 1 as a catalyst, to obtain the chiral amine precursor compound of vibergron.

[0012] Furthermore, the coenzyme NADPH cycle system includes the coenzyme NADP. + Salt, glucose, and glucose dehydrogenase;

[0013] Specifically, through the presence of the substrate vibergrenimide, the mutant, D-glucose, and NADP... + The reaction system of glucose dehydrogenase and phosphate was carried out to obtain the chiral amine compound Vibergron.

[0014] Furthermore, the reaction system uses DMSO as a solvent; the glucose dehydrogenase is a glucose dehydrogenase extract made from Escherichia coli with the nitroreductase gene knocked out; the mutant is added to the reaction system as a pure enzyme, a cell lysate of the recombinant host bacterium, or a wet cell of the recombinant host bacterium.

[0015] In a specific embodiment, the reaction system contains a substrate concentration of 20-80 mM, a mutant reaction mixture with a mass-to-volume ratio of 8-16 g / L, a D-glucose to substrate molar ratio of 1-2:1, and NADP... + The concentrations of the DMSO were 0.6-1.5 mmol / L, the glucose dehydrogenase extract was 0.2-0.6 g / L, the phosphate concentration was 60-150 mmol / L, and the volume of DMSO was 15-50% of the total volume of the reaction solution.

[0016] The solution used in the reaction has a pH of 6.5-7.5; the reaction temperature is 25-35 ℃; and the reaction time is 10-30 hours.

[0017] More specifically, the reaction steps include: adding DMSO and sodium phosphate buffer to the reactor in the reaction volume ratio; adding a mixture of vibergrenimide substrate, the mutant or the wet cells, D-glucose and nicotinamide adenine dinucleotide disodium phosphate to the reactor to react and obtain the product;

[0018] For gram-scale reactions, after 20-28 hours of reaction, the following steps are also included: quenching the reaction with sodium carbonate solution to obtain a solution with pH 8.0-9.0, extracting with ethyl acetate two to four times; filtering the remaining mixture after extraction on a diatomaceous earth layer and washing with ethyl acetate to obtain a filtrate; extracting again with ethyl acetate two to four times and combining the organic phase extracts; washing the organic phase with water and drying the organic phase with anhydrous sodium sulfate; concentrating under reduced pressure to obtain the product.

[0019] This invention utilizes semi-rational design of the enzyme's active site, engineering modifications based on previous research of IR-M5 to obtain mutants with high selectivity and high conversion rates. The best mutant, V1, is a combination mutant based on IR-M5, incorporating mutations at six sites: M203P, F269W, I149G, S241Q, L200T, and G268Q. It exhibits highly efficient catalysis of a single configuration for key intermediates of Vibegron, achieving a substrate loading of 12.4 g / L and an ee value greater than 99%S. Therefore, the enzymatic synthesis method of this invention provides a new enzymatic catalytic solution for the green, high-chiral purity industrial production of Vibegron. Attached Figure Description

[0020] Figure 1 Enzymatic synthesis route.

[0021] Figure 2 The effect of nitroreductase gene knockout on whole-cell catalytic side reactions.

[0022] Figure 3Chiral HPLC chromatograms of product 2 racemic standard, enzyme IR-13 mutant, IR-13-M5 mutant, and imine reduction products catalyzed by enzyme IR-M5 mutant V1.

[0023] Figure 4 Plasmid map of AzoR nitroreductase gene knockout.

[0024] Figure 5 Plasmid map of NfsA nitroreductase gene knockout.

[0025] Figure 6 Plasmid map of NfsB nitroreductase gene knockout.

[0026] Figure 7 . 1H NMR spectrum of V1-catalyzed reductive amination product 2.

[0027] Figure 8 Carbon 2 spectrum of the reductive amination product catalyzed by V1. Detailed Implementation

[0028] Example 1

[0029] 1. Semi-rational design of protein structure based on IR-13 mutant IR-M5

[0030] Based on the reductive amination enzyme IR-13 previously discovered by our research group from the actinomycete *Actinoalloteichus hymeniacidonis*, we obtained an engineered mutant IR-M5 through rational design and directed evolution. IR-M5 possesses the DNA sequence described in SEQ ID NO.1 and the amino acid sequence described in SEQ ID NO.2. Co-crystallization results with the cofactor NADP(H) show that its structure is a classic homodimer. The two monomers consist of an N-terminal Rossman domain and a C-terminal helical bundle, respectively. Two catalytic active sites are formed at the junctions of the C-terminal and N-terminal domains of the two monomers, and the cofactor NADP(H) binds to both active pockets.

[0031] This invention selects (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrolo-2-yl)(phenyl)methanol as the substrate for the IR-M5 catalytic imine reduction reaction. Figure 1To identify potential mutation sites, the imine intermediate was docked to the active site of IR-M5 using Autodock. The docking results showed that substrate 1 contains 16 amino acid residues within a 5 Å range of the IR-M5 active pocket, including I149, M203, Q265, Y204, W234, L200, and F269, forming a complex hydrophobic microenvironment. In this microenvironment, the nitrophenyl group faces the NADPH opening, while the benzene ring faces the lateral opening of the active pocket. Based on the large volume, rigid framework, and hydrophobic aromatic ring at both ends of the imine substrate 1 molecule, and combined with our previous research, we hypothesize that residues near the M5 active pocket channel and the NADPH binding inlet are potential key sites for regulating the stereoselectivity of the reaction.

[0032] Therefore, combining computer simulations (molecular docking and molecular dynamics simulations), and observing the position of these residues in the protein structure, subsequent studies systematically evaluated these residues from three dimensions: amino acid side chain steric hindrance effect, hydrophilic / hydrophobic properties, and rigid regulation of protein conformation (A / L / F / / P) (Table 1). Based on the results, residues near the IR-M5 active pocket channel and the NADPH binding inlet were identified as potential key sites for regulating the stereoselectivity of the reaction (I149, M203, and F269). Subsequently, single-point saturation mutagenesis was performed at each of the three sites, followed by whole-cell catalytic stereoselectivity and activity screening. The overall results, as shown in Tables 2-4, indicate that the mutants of M203 and F269 significantly enhanced the S-configuration product.

[0033] Table 1. Stereoselectivity of mutants produced at the selected M5 site by site-directed mutagenesis (A / L / F / P).

[0034]

[0035] Reaction conditions: imine substrate 1 (20 mM), D-glucose (30 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL), NADP + (1 mM) whole cells of *E. coli* containing imine reductase (IRED) with complete endogenous nitroreductase (based on 1, the amount used was 0.56 mol%, approximately 0.75 mg of wet cells), were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. nd: Enantiomer ratio was not determined because the mutant was almost inactivated. [a] The conversion rate was determined by high performance liquid chromatography (HPLC). [b] The enantiomeric ratio (S:R) was determined by chiral high-performance liquid chromatography.

[0036] Table 2. Results of ee value determination for whole-cell catalysis in the saturated mutant at the M203 site.

[0037]

[0038] Reaction conditions: imine substrate 1 (20 mM), D-glucose (30 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL), NADP + Whole cells of *E. coli* containing imine reductase (IRED) with intact endogenous nitroreductase (1 mM) (0.56 mol%, approximately 0.75 mg of wet cells, based on 1 mM) were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. nd: Enantiomer ratios were not determined due to near-inactivation in the mutant. Enantiomer ee values ​​were determined by chiral high-performance liquid chromatography.

[0039] Table 3. Results of whole-cell ee value determination for the saturated mutant at site I149

[0040]

[0041] Reaction conditions: imine substrate 1 (20 mM), D-glucose (30 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL), NADP + Whole cells of *E. coli* containing imine reductase (IRED) with complete endogenous nitroreductase (1 mM) (0.56 mol%, approximately 0.75 mg of wet cells, based on 1 mM) were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. Enantiomer ee values ​​were determined by chiral high-performance liquid chromatography.

[0042] Table 4. Results of whole-cell ee value determination for the saturated mutant at the F269 site.

[0043]

[0044] Reaction conditions: imine substrate 1 (20 mM), D-glucose (30 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL), NADP +Whole cells containing imine reductase (IRED) with intact endogenous nitroreductase (1 mM) (0.56 mol%, approximately 0.75 mg of wet cells, based on 1 mM) were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. nd: Enantiomer ratios were not determined due to near-inactivation in the mutant. Enantiomer ee values ​​were determined by chiral high-performance liquid chromatography.

[0045] To further improve the stereoselectivity of S, the dominant mutants were combined and mutated according to their spatial location. The mutants and their corresponding ee values ​​are shown in Table 5.

[0046] Next, the conversion rate of mutants with S selectivity reaching 99% was screened and improved. As shown in Table 6, under the conditions of substrate concentration of 50 mmol / L and enzyme amount of 0.03 mol%, the six-mutant M5-M203P-F269W-I149G-S241Q-L200T-G268Q had the highest whole-cell conversion rate, reaching 94%. Among them, the five-mutant M5-M203P-F269W-I149G-S241Q-L200T and M5-M203P-F269W-I149G-L200T-G268Q had a conversion rate of 88%. Therefore, M5-M203P-F269W-I149G-S241Q-L200T-G268Q was finally selected as the optimal mutant, abbreviated as V1, with the ee value still maintained at >99% S ( Figure 3 ).

[0047] Table 5. Results of ee value determination for combined mutations

[0048]

[0049] Reaction conditions: imine substrate 1 (30 mM and 60 mM), D-glucose (1.5 times that of imine substrate 1), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL, from a nitroreductase-deficient *E. coli* host), NADP + Whole cells of *E. coli* with 1 mM imine reductase (IRED) knocked out (based on 1, the amounts used were 0.28 mol% and 0.14 mol%, approximately 0.75 mg of wet cells), were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. [a] The conversion rate was determined by high performance liquid chromatography (HPLC). [b] The enantiomeric ee value was determined by chiral high-performance liquid chromatography.

[0050] Example 2

[0051] The presence of the nitroreductase gene in the genome of common E. coli resulted in the detection of two small byproduct peaks [M+H] in the IR-M5 catalytic reaction mass spectrum. + =283 and 281 (respectively) Figure 2 The byproducts (3a and 2a) are difficult to isolate and purify in subsequent steps. To address the generation and isolation of these byproducts at their source, the CRISPR-Cas9 gene editing system was used to target and knock out the major nitroreductase encoding genes (nfsA, nfsB, and azoR) in the host bacterium BL21(DE3), constructing a three-gene deletion mutant strain ΔnfsA-nfsB-azoR. Details are as follows:

[0052] Based on the design of the plasmid for constructing the knockout ( Figure 4 , Figure 5 and Figure 6 First, four pairs of primers were designed to amplify approximately 320 bp homologous arms upstream / downstream of the target gene, a vector fragment containing Cas9 and gRNA, and a vector fragment containing araC, respectively. After recovering the PCR products from agarose gel electrophoresis and determining their concentrations, the four fragments were ligated using a seamless cloning kit to construct a knockout plasmid. The ligation product was then transformed into Trelief. ® 5α competent cells were selected, and single clones were identified by colony PCR. Positive clones were cultured, plasmids were extracted, and sequencing was performed for confirmation.

[0053] The correctly sequenced knockout plasmid was transferred into the strain to be knocked out and cultured at 30 °C for approximately 12 h as a seed culture. Subsequently, it was inoculated 1:100 into fresh LB liquid medium containing ampicillin and grown to OD. 600 Add 10 mM L-arabinose to a pH of 0.1-0.2 to induce Cas9 protein expression, and culture overnight to achieve gene knockout. Dilute the overnight culture 30-50 times and plate it on plates containing Amp and 10 mM L-Ara. Pick single clones and perform preliminary verification of knockout by colony PCR.

[0054] To rule out false positives, genomic DNA was extracted from clones that were correctly identified by colony PCR, and PCR was performed again for verification. Sequencing was then conducted to finally confirm the knockout results.

[0055] Since the pSC101 promoter replication depends on the temperature-sensitive protein Rep101, plasmid elimination can be achieved by continuous subculturing at 40 °C for 3-4 generations. Finally, the success of the knockout strain in losing plasmid background was verified by parallel plating on plates containing Amp resistance and those without.

[0056] Example 3

[0057] The gene encoding the mutant V1 (M5-M203P-F269W-I149G-S241Q-L200T-G268Q) was ligated into the pET28a plasmid and transformed into *E. coli* BL21(DE3) competent cells with the nitroreductase gene knocked out for protein expression. Single colonies were picked and cultured overnight in 10 mL of LB medium containing 50 µg / mL kanamycin at 37 °C and 220 rpm. The bacterial culture was then inoculated at a 1:100 ratio into LB medium containing 50 µg / mL kanamycin and cultured at 37 °C and 220 rpm until OD500. 600 The value was 0.6-0.8. IPTG (0.2 mM / L) was added to the culture medium to induce gene expression, and the culture was continued at 20 ℃ and 200 rpm for 16 hours. The cells were then harvested by centrifugation at 6000 rpm for 20 minutes and resuspended in sodium phosphate buffer (100 mmol / L, pH 7.0), followed by centrifugation at 6000 rpm for 10 minutes. The cells were washed twice with sodium phosphate buffer, resuspended in sodium phosphate buffer, and then disrupted using an ultrasonic cell disruptor. The disruption solution was centrifuged at 12000 rpm, the supernatant was collected, and then purified using Nitrogen buffer. +2 Affinity chromatography was used to purify proteins.

[0058] Example 4

[0059] The gene encoding the mutant M5-M203P-F269W-I149G-L200T was ligated into the pET28a plasmid and transformed into *E. coli* BL21(DE3) competent cells with the nitroreductase gene knocked out for protein expression. Single colonies were picked and cultured overnight in 10 mL of LB medium containing 50 µg / mL kanamycin at 37 °C and 220 rpm. The bacterial culture was then inoculated at a 1:100 ratio into LB medium containing 50 µg / mL kanamycin and cultured at 37 °C and 220 rpm until OD500. 600 The value was 0.6-0.8. IPTG (0.2 mM / L) was added to the culture medium to induce gene expression, and the culture was continued at 20 ℃ and 200 rpm for 16 hours. The cells were then harvested by centrifugation at 6000 rpm for 20 minutes and resuspended in sodium phosphate buffer (100 mmol / L, pH 7.0), followed by centrifugation at 6000 rpm for 10 minutes. The cells were washed twice with sodium phosphate buffer, resuspended in sodium phosphate buffer, and then disrupted using an ultrasonic cell disruptor. The disruption solution was centrifuged at 12000 rpm, the supernatant was collected, and then purified using Nitrogen buffer. +2Affinity chromatography was used to purify proteins.

[0060] The purification process for other combined mutant proteins such as M5-M203P-F269W-I149G-L200T-G268Q, M5-M203P-F269W-I149G-S241Q, and M5-M203P-F269W-I149G-S241Q-L200T is the same as described above.

[0061] Example 5

[0062] The purified optimal mutant protein V1 (M5-M203P-F269W-I149G-S241Q-L200T-G268Q) was used to determine the asymmetric reduction reaction of the substrate (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol.

[0063] Add 1 mmol / L NADP to a 250 µL reaction mixture. + 0.5 mg / mL purified enzyme (0.03 mol% enzyme content) was added, along with substrate (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol (50 mM), D-glucose to substrate at a molar ratio of 1.5:1, glucose dehydrogenase powder (0.4 mg / mL GDH, from a nitroreductase-deficient *E. coli* host), and 20% DMSO dissolved in sodium phosphate buffer (100 mM, pH 7.0). All reactions were carried out at 30°C and 900 rpm for 20 hours, followed by quenching with 500 μL of acetonitrile containing 1 M acetic acid. After centrifugation at 12000 rpm for 15 minutes, the supernatant was analyzed by high-performance liquid chromatography to determine the conversion rate.

[0064] The procedure for other mutant pure enzyme reactions is the same as described above.

[0065] Example 6

[0066] The enzymatic activity and stereoselective reduction of the substrate (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrolo-2-yl)(phenyl)methanol 1 were evaluated using nitroreductase-deficient whole-cell culture. The gene encoding the target mutant was ligated into the pET28a plasmid and transformed into *E. coli* BL21(DE3) competent cells with the nitroreductase gene knocked out for protein expression. Single colonies were picked and cultured overnight in 5 mL LB medium containing 50 µg / mL kanamycin at 37 °C and 220 rpm. The bacterial culture was then inoculated at a 1:100 ratio into 10 mL LB medium containing 50 µg / mL kanamycin and cultured at 37 °C and 220 rpm until OD500. 600 The value was 0.6-0.8. IPTG (0.2 mM) was added to the culture medium to induce gene expression, and the culture was continued at 20 ℃ and 200 rpm for 16 hours. 1 mL of the above bacterial suspension was taken and harvested by centrifugation at 4000 rpm for 10 minutes. The cells were then resuspended in sodium phosphate buffer (100 mmol / L, pH 7.0) and collected by centrifugation at 4000 rpm for another 10 minutes.

[0067] The target coding mutants used in the experiment were: M203P-I149S and M203P-F269W. , M203P-F269V, M203P-F269L, M203P-F269C, M203P-F269W-I149S, M203P-F269W-I149N, M203P-F269W-I149A, M203P-F269W-I149G, M203P- F269W-I149P, M203P-F269W-Y204M, M203P-F269W-Y204L, M203P-F269W-I149G-T121S, M203P-F269W-I149G-T121M, M203P-F269W-I149G-G2 68Q, M203P-F269W-I149G-L272I, M203P-F269W-I149G-S241Q, M203P-F269W-I149G-S241M, M203P-F269W-I149G-W234L, M203P-F269W-I14 9G-W234I, M203P-F269W-I149G-L200T, M203P-F269W-I149G-S241Q-T 121S, M203P-F269W-I149G-S241Q-W234L, M203P-F269W-I149G-S241Q -W234I, M203P-F269W-I149G-S241Q-G268Q, M203P-F269W-I149G-S241Q-A238T, M203P-F269W-I149G-S241Q-L200T, M203P-F269W-I149G- L200T-T121S, M203P-F269W-I149G-L200T-W234L, M203P-F269W-I149G-L200T-W234I, M203P-F269W-I149G-L200T-G268Q, M203P-F269W-I1 49G-L200T-A238T, M203P-F269W-I149G-L200T-S241M, M203P-F269W- I149G-W234I-T121S, M203P-F269W-I149G-W234I-G268Q, M203P-F269 W-I149G-W234I-S241M, M203P-F269W-I149G-W234I-A238T, M203P-F269W-I149G-W234I-L200T, M203P-F269W-I149G-S241Q-L200T-G268Q.

[0068] The 250 µL reaction mixture contained 1 mM NADP + The reaction mixture consisted of 0.15 mg wet cell mutant (approximately 0.03 mol% enzyme), 50 mM (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol, D-glucose (75 mM), 0.4 mg / mL glucose dehydrogenase (a cell extract prepared from a nitroreductase-deficient *E. coli* host), and 20% dimethyl sulfoxide in sodium phosphate buffer (100 mM, pH 7.0). After incubation at 30°C with shaking at 900 rpm for 20 hours, the reaction was quenched by adding 500 µL of acetonitrile containing 1 mol / L acetic acid. The mixture was centrifuged at 12,000 rpm for 15 min, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the conversion. This analysis yielded a conversion of 72% at a substrate loading of 50 mM (Table 6).

[0069] Table 6. Results of Screening Tests for Activity of Combination Mutants

[0070]

[0071] Reaction conditions: imine substrate 1 (50 mM), D-glucose (75 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL, from a nitroreductase-deficient *E. coli* host), NADP + Whole cells of *E. coli* with 1 mM imine reductase (IRED) knocked out (based on 1 mM, the amount used was 0.03 mol%, approximately 0.15 mg of wet cells), were reacted in 250 μL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. [a] The conversion rate was determined by high performance liquid chromatography (HPLC). [b] The enantiomeric ee value was determined by chiral high-performance liquid chromatography.

[0072] Example 7

[0073] Whole-cell enzyme catalysis of 1 mL reaction by mutant M203P-F269W-I149G-L200T. NADP was added to 1 mL of the whole-cell catalytic reaction system. +Add 0.6 mg of *E. coli* wet cells (approximately 0.03% mol) of the M203P-F269W-I149G-L200T mutant (nitroreductase gene knockout) to a solution of 1 mmol / L. Add 15.5 mg of substrate (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol (50 mM), add D-glucose (75 mM), add glucose dehydrogenase powder (0.4 mg / mL GDH, from a nitroreductase-deficient *E. coli* host), and DMSO (20%) dissolved in sodium phosphate buffer (100 mM, pH 7.0). The reaction was carried out at 30°C and 800 rpm for 20 hours. After the reaction was complete, add sodium carbonate aqueous solution to adjust the pH to 8.0-9.0. After quenching the reaction with ethyl acetate, the mixture was filtered through diatomaceous earth. The filtrate was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with water (5 mL × 3) and dried over anhydrous sodium sulfate. The combined organic solvents after extraction were removed under reduced pressure to obtain the chiral amine product.

[0074] The operation steps for other mutants are the same as in Example 7. The different mutants, reaction systems, conversion rates and ee values ​​in the reaction are shown in Table 7.

[0075] Table 7. 1 mL reaction system of substrate 1 catalyzed by the combined mutant

[0076]

[0077] Reaction conditions: imine substrate 1 (50 mM), D-glucose (75 mM), glucose dehydrogenase (GDH) wet cell extract (0.4 mg / mL, from a nitroreductase-deficient *E. coli* host), NADP + Whole cells of *E. coli* with endogenous nitroreductase knocked out (IRED) (based on 1 mM, the amount used was 0.03 mol%, approximately 0.6 mg of wet cells), were reacted in 1 mL sodium phosphate buffer (100 mM, pH 7.0) at 30 °C and 900 rpm for 20 hours to test stereoselectivity. [a] The conversion rate was determined by high performance liquid chromatography (HPLC). [b] The enantiomeric ee value was determined by chiral high-performance liquid chromatography.

[0078] Example 8

[0079] Whole-cell enzymatic gram-scale synthesis of (R)-((2R,5S)-5-(4-nitrobenzyl)pyrrolidine-2-yl)(phenyl)methanol Figure 1 ).

[0080]

[0081] NADP was added to a 40 mL whole-cell catalytic reaction system. + Add 2.4 g of *E. coli* wet cells (approximately 0.11% mol) of mutant V1 (nitroreductase gene knocked out), 1.0 g of substrate (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol (80 mM), D-glucose to substrate at a molar ratio of 1.5:1, glucose dehydrogenase powder (0.4 mg / mL GDH, from a nitroreductase-deficient *E. coli* host), and 40% DMSO dissolved in sodium phosphate buffer (100 mM, pH 7.0). The reaction was carried out at 30°C and 200 rpm for 24 hours, achieving a conversion rate of 96%. After the reaction, sodium carbonate aqueous solution was added to adjust the pH to 8.0-9.0. After quenching the reaction with ethyl acetate, the mixture was filtered through diatomaceous earth, and the filtrate was extracted with ethyl acetate (300 mL × 3). The organic phase was washed with water (100 mL × 3) and dried over anhydrous sodium sulfate. After vacuum distillation, the organic phase was purified by silica gel column chromatography (methanol:dichloromethane = 1:10) to obtain 780 mg of a red oily product, yield 78%, ee value > 99% (S). (1H NMR spectroscopy, 400 MHz, deuterated chloroform) 8.18 (m, 2H), 7.45 - 7.23 (m, 7H), 4.45 (d, J = 5.8 Hz, 1H), 3.72 (s, 2H), 3.57 - 3.36 (m, 2H), 3.03 - 2.84 (m, 2H), 1.96 - 1.87 (m, 1H),1.85 - 1.76 (m, 2H), 1.67 - 1.55 (m, 1H). Note: The broad signal peak at 3.72 MHz is attributed to exchangeable protons (such as NH / OH) and does not affect the identification of the compound's structure. (C-carbon spectrum, 100 MHz, deuterated chloroform) 147.80, 146.67, 143.26, 129.93, 128.42, 127.52, 126.20, 123.70, 75.53, 64.17, 59.85, 43.27, 31.00, 27.99 Figure 7 and Figure 8 ).

Claims

1. An imine reductase mutant, characterized in that, It is based on the imine reductase mutant IR-13-M5 with the following mutations: M203P-F269W-I149G-S241Q-L200T-G268Q, M203P-F269W-I149G-L200T, M203P-F269W-I149G-S241Q, M203P-F269W-I149G-S241Q-L200T or M203P-F269W-I149G-L200T-G268Q, wherein the amino acid sequence of the imine reductase mutant IR-13-M5 is shown in SEQ ID NO:

2.

2. The encoded nucleic acid of the mutant as described in claim 1.

3. A recombinant vector containing the nucleic acid encoding as described in claim 2, such as the pET28a plasmid.

4. A recombinant host bacterium containing the encoded nucleic acid as described in claim 2, or the recombinant vector as described in claim 3, preferably Escherichia coli, such as Escherichia coli BL21(DE3).

5. The recombinant host bacterium as described in claim 4, characterized in that, The nitroreductase gene was knocked out or weakened in E. coli.

6. The application of the mutant or its encoded nucleic acid as described in claim 1, or the recombinant host bacteria as described in claim 4 or 5, in the synthesis of vilbergerone, specifically, the vilbergerone chiral amine precursor is obtained by vilbergerone imine reduction reaction.

7. A method for preparing a key chiral amine intermediate of vibergron, characterized in that, Under NADPH cycle conditions, using the mutant described in claim 1 as a catalyst, the substrate vebergerone imine, namely (R)-((R)-5-(4-nitrobenzyl)-3,4-dihydro-2H-pyrrole-2-yl)(phenyl)methanol, was asymmetrically catalyzed to obtain the vebergerone precursor compound. Furthermore, the coenzyme NADPH cycle system includes the coenzyme NADP. + Salt, glucose, and glucose dehydrogenase; Specifically, through the presence of the substrate vibergrenimide, the mutant as described in claim 1, D-glucose, and NADP. + The reaction system of glucose dehydrogenase and phosphate was carried out to obtain the chiral amine intermediate of Vibergron.

8. The method as described in claim 7, characterized in that, The reaction system uses phosphate buffer as solvent and DMSO as co-solvent; the glucose dehydrogenase is a glucose dehydrogenase extract made from Escherichia coli with the nitroreductase gene knocked out; the mutant as described in claim 1 is added to the reaction system as a pure enzyme or as a cell lysate of the recombinant host bacterium as described in claim 4 or as a wet cell of the recombinant host bacterium as described in claim 4.

9. The method as described in claim 8, characterized in that, In the reaction system, the substrate concentration is 20-80 mM, the mass-to-volume ratio of the mutant reaction mixture is 8-16 g / L, the molar ratio of D-glucose to substrate is 1-2:1, the amount of NADP+ is 0.6-1.5 mmol / L, the amount of glucose dehydrogenase extract is 0.2-0.6 g / L, the amount of phosphate is 60-150 mmol / L, and the volume of DMSO used is 15-50% of the total volume of the reaction solution. The solution pH for the reaction is 6.5-7.5; the reaction temperature is 25-35 ℃; and the reaction time is 10-30 hours.

10. The method as described in claim 8, characterized in that, The reaction steps include: adding a buffer solution of DMSO and sodium phosphate to the reactor according to the reaction volume ratio; adding a mixture of substrate vibergroimide, the mutant or the wet cells, D-glucose and nicotinamide adenine dinucleotide disodium phosphate to the reactor to react and obtain the product; For gram-scale reactions, after 20-28 hours of reaction, the following steps are also included: quenching the reaction with sodium carbonate solution to obtain a solution with pH 8.0-9.0, extracting with ethyl acetate two to four times; filtering the remaining mixture after extraction on a diatomaceous earth layer and washing with ethyl acetate to obtain a filtrate; extracting again with ethyl acetate two to four times and combining the organic phase extracts; washing the organic phase with water and drying the organic phase with anhydrous sodium sulfate; concentrating under reduced pressure to obtain the product.

Images