Styrene monooxygenase combined mutant and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly discloses a styrene monooxygenase combined mutant with high enantioselectivity. The mutant enzyme can catalyze 4-chlorostyrene, 4-fluorostyrene, 4-bromostyrene, 4-trifluoromethylstyrene and 4-cyano styrene to synthesize corresponding (R)-epoxide, the enantiomeric excess is 95-99%, and the mutant enzyme is an excellent catalyst for synthesizing various drug intermediates.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to a styrene monooxygenase combination mutant catalyzing a para-substituted styrene compound to generate optically pure (R)-epoxide. Background Art

[0002] Optically pure styrene epoxides are important building blocks for medicines, chiral chemicals and functional polymers. Asymmetric epoxidation of alkenes is one of the most economical and concise methods for synthesizing such compounds. Styrene monooxygenase (SMO) can catalyze the epoxidation of styrene and its derivatives to produce the corresponding (S)- or (R)-epoxides, but the para-substituted styrene derivatives catalyzed by SMO discovered so far still mainly produce (S)-epoxides (Catalysis Science & Technology, 2021, 11(6): 2195-2201). However, a variety of para-substituted styrene (R)-epoxides are important pharmaceutical intermediates, such as (R)-2-(4-chlorophenyl) oxirane, (R)-2-(4-(trifluoromethyl)phenyl) oxirane and (R)-4-cyanophenyl oxirane, which can be used to synthesize human β3 adrenergic receptor agonists (Journal of medicinal chemistry, 2008, 51(15): 4804-4822). (R)-2-(4-bromophenyl)oxirane can be used to synthesize H3 receptor agonists (Chemical Communications, 2013, 49(98): 11572-11574). (R)-(4-fluorophenyl)oxirane is an intermediate of non-alkane derivatives for treating cocaine addiction (Bioorganic & Medicinal Chemistry, 2008, 16(6): 2769-2778).

[0003] In view of the current lack of styrene monooxygenases that can catalyze the conversion of para-substituted styrene compounds to (R)-epoxides, using protein engineering to transform existing enzyme sources is an efficient strategy. Summary of the invention

[0004] The present invention uses styrene monooxygenase SeStyA derived from Streptomyces vilmorinianum as a parent, and adopts a semi-rational design based on structure guidance to perform molecular modification, thereby obtaining a styrene monooxygenase combination mutant with greatly improved enzymatic performance. The present invention also relates to a styrene monooxygenase combination mutant that catalyzes the epoxidation of 4-chlorostyrene (1a) to synthesize (R)-2-(4-chlorophenyl) oxirane (1b); catalyzes the epoxidation of 4-fluorostyrene (2a) to synthesize (R)-(4-fluorophenyl) oxirane (2b); catalyzes the epoxidation of 4-bromostyrene (3a) to synthesize (R)-2-(4-bromophenyl) oxirane (3b); catalyzes the synthesis of (R)-2-(4-(trifluoromethyl)phenyl) oxirane (4b) from 4-trifluoromethylstyrene (4a) and catalyzes the synthesis of (R)-4-cyanophenyl oxirane (5b) from 4-cyanostyrene (5a).

[0005] Specifically, the styrene monooxygenase combined mutant is based on SeStyA (nucleotide sequence is SEQ ID NO.1; amino acid sequence is SEQ ID NO.2) as the starting sequence, and 4 amino acid sites are mutated simultaneously to obtain the following combined mutant.

[0006] Combination mutant Mut4R: phenylalanine at position 57 was mutated to alanine, phenylalanine at position 59 was mutated to methionine, serine at position 178 was mutated to glycine and alanine at position 312 was mutated to phenylalanine.

[0007] According to the common knowledge in the art, vectors and genetically engineered bacteria constructed to express the above mutants also fall within the protection scope of the present invention.

[0008] The mutant of the present invention is obtained as follows:

[0009] First, the enzyme was semi-rationally designed by molecular docking. The protein structure of wild-type SeStyA was molecularly docked with the substrate 4-chlorostyrene. According to the docking results, 10 proposed mutation sites in the active pocket of the enzyme were identified and mutated into one or more other amino acids. Then single-point mutants were constructed respectively, and the biocatalytic results were analyzed, and some single-point mutants with significantly decreased (S)-selectivity or flipped to (R)-selectivity were found. Taking stereoselectivity and activity into consideration, four key sites F57, A59, S178 and A312 were identified. Saturation mutations were performed on these four sites, and multiple single-point mutants with significantly decreased (S)-selectivity or flipped to (R)-selectivity were obtained. After rationally combining some beneficial sites, four mutants with excellent (R)-selectivity (F57A / A59M / S178G / A312F, named Mut4R) were obtained.

[0010] Mut4R has excellent (R)-selectivity for a variety of substrates. The wild-type SeStyA catalyzes the production of 4-chlorostyrene to produce (S)-2-(4-chlorophenyl)oxirane with an enantiomeric excess of 89% ee, while the mutant Mut4R catalyzes the production of 4-chlorostyrene to produce (R)-2-(4-chlorophenyl)oxirane with an enantiomeric excess of 95% ee. The wild-type SeStyA catalyzes the production of 4-fluorostyrene to produce (R)-(4-fluorophenyl)oxirane with an enantiomeric excess of 68% ee, while the mutant Mut4R catalyzes the production of 4-fluorostyrene to produce (R)-(4-fluorophenyl)oxirane with an enantiomeric excess of 97% ee. The wild-type SeStyA catalyzes the production of 4-bromostyrene to produce (S)-2-(4-bromophenyl)oxirane with an enantiomeric excess of 97%ee, while the mutant Mut4R catalyzes the production of 4-bromostyrene to produce (R)-2-(4-bromophenyl)oxirane with an enantiomeric excess of 98%ee. The wild-type SeStyA catalyzes the production of 4-trifluoromethylstyrene to produce (S)-2-(4-(trifluoromethyl)phenyl)oxirane with an enantiomeric excess of >99%ee, while the mutant Mut4R catalyzes the production of 4-trifluoromethylstyrene to produce (R)-2-(4-(trifluoromethyl)phenyl)oxirane with an enantiomeric excess of 99%ee. The wild-type SeStyA catalyzes the production of 4-cyanostyrene to produce (S)-4-cyanophenyl oxirane with an enantiomeric excess of >99% ee; while the mutant Mut4R catalyzes the production of 4-cyanostyrene to produce (R)-4-cyanophenyl oxirane with an enantiomeric excess of 97% ee.

[0011] Advantages of the present invention: The styrene monooxygenase combination mutant of the present invention can catalyze the synthesis of corresponding (R)-epoxides from para-substituted styrene substrates (4-chlorostyrene, 4-fluorostyrene, 4-bromostyrene, 4-trifluoromethylstyrene and 4-cyanostyrene) with an ee value of 95% to 99%. The products obtained by the wild-type SeStyA catalyzing these substrates are mainly (S)-configuration, and only the catalytic product of 4-fluorostyrene is (R)-(4-fluorophenyl) oxirane, but the stereoselectivity is not high, and the enantiomeric excess is 68% ee; the combination mutant Mut4R has excellent (R)-stereoselectivity and is an excellent catalyst for preparing these optically pure compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 , HPLC spectra of the products of 4-chlorostyrene catalyzed by wild-type SeStyA and mutant Mut4R;

[0013] Figure 2 , HPLC spectra of the products of 4-fluorostyrene catalyzed by wild-type SeStyA and mutant Mut4R;

[0014] Figure 3, HPLC spectra of the products of 4-bromostyrene catalyzed by wild-type SeStyA and mutant Mut4R;

[0015] Figure 4 , HPLC spectra of the products of 4-trifluoromethylstyrene catalyzed by wild-type SeStyA and mutant Mut4R;

[0016] Figure 5 , HPLC spectra of the products of 4-cyanostrene catalyzed by wild-type SeStyA and mutant Mut4R. DETAILED DESCRIPTION

[0017] Example 1: Prediction of mutation sites and construction of single-point mutants

[0018] The nucleotide sequence of SeStyA is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The predicted structure of SeStyA was obtained by AlphaFold2 and evaluated by SAVER6.0. SeStyA was molecularly docked with the substrate 4-chlorostyrene using the molecular docking software AutoDock Vina. The docking results were comprehensively analyzed, and the 10 amino acid residues in the active pocket were mutated into one or more other amino acids. These sites are N56, F57, P58, A59, S178, L204, I206, A219, P309 and A312.

[0019] Since the entire catalytic process requires the participation of the expensive cofactor FADH2, the flavin oxidoreductase StyB was introduced to convert FAD + Reduced to FADH2, realizing the coenzyme cycle. The specific method of constructing the co-expression system of SeStyA, linker and StyB refers to the previous study (Catalysis Science & Technology, 2021, 11(6): 2195-2201).

[0020] The wild-type recombinant plasmid was used as a template for site-directed mutagenesis. The primers were designed as shown in Table 1. A total of 12 single-point mutants were constructed. The saturation mutation primers and combined mutant primers for constructing F57, A59, S178 and A312 sites are shown in Table 2.

[0021] Table 1 Site-directed mutagenesis primers

[0022]

[0023] Table 2 Degenerate primers and combined mutation primers

[0024]

[0025]

[0026] The PCR reaction system is shown in Table 3, and amplification was performed according to the following conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 8 min, 20 cycles, extension at 72°C for 10 min, and storage at 4°C for future use.

[0027] Table 3 PCR reaction system for site-directed mutagenesis

[0028]

[0029] Add 1 μL DpnI to the PCR product, incubate at 37°C for 1 hour, then take 10 μL of the treated PCR product, transfer it into E.coli DH5α competent cells by conventional chemical method, spread it on LB plate containing 50 μg / mL kanamycin, and invert and culture at 37°C for 12 hours. Pick a single clone and place it in LB liquid culture medium (containing 50 μg / mL kanamycin), culture it at 37°C for 10 hours, and send the bacterial solution to Beijing Qingke Biological Company for sequencing verification.

[0030] Example 2: Heterologous expression of wild-type SeStyA and mutants

[0031] The wild-type and mutant plasmids were transformed into E. coli BL21 (DE3) competent cells for protein expression. Single clones were picked and transferred to 3 mL LB liquid medium (containing kanamycin 50 μg / mL) and cultured at 37°C, 180 rpm for 12 h. 2 mL (1% V / V) was transferred to 200 mL TB liquid medium for expansion culture. 600 When the pH reached about 0.6, IPTG was added to make the final concentration 0.01 mmol / L, and then the culture was continued for 20 h at 20°C and 180 rpm. The cells were collected by refrigerated centrifugation and then washed twice with an equal volume of 0.9% sodium chloride solution for later use.

[0032] Example 3: Biocatalytic epoxidation reaction of wild-type SeStyA and mutants

[0033] The collected wet bacteria were resuspended in potassium phosphate buffer solution (0.1M, pH 7.0). 5mL of the catalytic system included: bacteria (0.1g / mL), potassium phosphate buffer solution (0.1M, pH 7.0), 2mmol / L substrate (dissolved in isopropanol to prepare 1mol / L mother solution), placed in a 100mL conical flask, placed in an air bath shaker at 30°C, 200rpm for 2h, extracted twice with an equal volume of ethyl acetate, combined the organic phases and dried with anhydrous sodium sulfate, evaporated under reduced pressure, then redissolved with isopropanol, filtered with a 0.22μm filter membrane, and the substrate conversion rate was detected by GC, and the product ee value was detected by HPLC.

[0034] GC determination of conversion rate: Agilent 7890 was used to detect the conversion rate, and the chromatographic column was CHIRASIL-DEX CB column (Agilent, USA). Product detection procedure: temperature was maintained at 140°C for 10 min, carrier gas (nitrogen) flow rate was 3 mL / min, and split ratio was 20:1.

[0035] The ee values ​​of the products were detected by HPLC with a column temperature of 30°C and a detection wavelength of 210 nm. The separation conditions of the five products are shown in Table 4.

[0036] Table 4 Chiral epoxide separation conditions

[0037]

[0038] Using 4-chlorostyrene as the model substrate for the catalytic reaction, the relative conversion rates and ee values ​​of the single-point mutants in Table 1 show that the ee of F57A is (S) The value is lower, S178G is reversed to R configuration, and the activities of A59G and A312G are higher. Therefore, it is believed that these four sites affect the activity and ee (R) key locations.

[0039] Saturation mutations were performed on these four sites to further identify beneficial mutation sites. Taking activity and selectivity into consideration, multiple combination strategies were performed on beneficial single-point mutants. After heterologous expression of enzymes and product detection after biocatalysis, F57A / A59M / S178G / A312F (named Mut4R) had the highest conversion rate and ee value. The conversion rate and ee value of the combined mutant Mut4R catalyzing the epoxidation of para-substituted styrene substrates are shown in Table 5. They all have excellent R-selectivity and ee. (R) The value is 95-99%, which is a highly stereoselective catalyst for obtaining these compounds.

[0040] Table 5 Epoxidation of para-substituted styrene substrates catalyzed by wild-type and combined mutant Mut4R

[0041]

[0042]

Claims

1. A styrene monooxygenase combined mutant, characterized in that Taking the amino acid sequence SEQ ID NO.2 of styrene monooxygenase SeStyA as the starting sequence, the characteristics after mutation are as follows: the phenylalanine at position 57 is mutated to alanine, the alanine at position 59 is mutated to methionine, the serine at position 178 is mutated to glycine, and the alanine at position 312 is mutated to phenylalanine.

2. Use of the mutant according to claim 1 in catalyzing the epoxidation of 4-chlorostyrene to synthesize (R)-2-(4-chlorophenyl)oxirane.

3. Use of the mutant according to claim 1 in catalyzing the epoxidation of 4-fluorostyrene to synthesize (R)-(4-fluorophenyl)oxirane.

4. Use of the mutant according to claim 1 in catalyzing the epoxidation of 4-bromostyrene to synthesize (R)-2-(4-bromophenyl)oxirane.

5. Use of the mutant according to claim 1 in catalyzing the epoxidation of 4-trifluoromethylstyrene to synthesize (R)-2-(4-(trifluoromethyl)phenyl)oxirane.

6. Use of the mutant according to claim 1 in catalyzing the epoxidation of 4-cyanostyrene to synthesize (R)-4-cyanophenyloxirane.

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