Styrene monooxygenase mutant and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly discloses a styrene monooxygenase mutant with high R-enantioselectivity. The mutant enzyme A59W can catalyze epoxidation of meta-substituted 3-fluorostyrene, 3-chlorostyrene and 3-bromostyrene to synthesize corresponding (R)-epoxide, the enantiomeric excess is more than 99%, and the mutant enzyme A59W 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 mutant catalyzing the meta-halogen substitution of a styrene compound to obtain an optically pure (R)-configuration epoxide. Background Art

[0002] As a key intermediate for synthesizing optically pure bioactive compounds with more complex structures, styrene epoxide has been widely concerned by researchers. (R)-configuration epoxides substituted with meta-halogens are key intermediates for the synthesis of many drugs. For example, (R)-3-chlorophenyl oxirane is a key intermediate for the synthesis of β3 adrenergic receptor agonist FR165914, which can be used to treat diabetes and obesity (Bioorganic & Medicinal Chemistry Letters, 1995, 5 (23): 2821-2824). (R)-3-bromophenyl oxirane is a key intermediate for the synthesis of 1-aryl-2-(1H-1,2,4-triazol-1-yl)ethanol derivatives, which have antifungal activity and can be used to treat fungal infections (Chinese Journal of Organic Chemistry, 2010, 30 (02): 285).

[0003] A variety of methods have been established for the asymmetric epoxidation of olefins, but the asymmetric epoxidation of terminal unactivated olefins remains a major challenge. For example, the enantiomeric excess of (R)-epoxybenzene ethane obtained by Jacobsen-Katsuki epoxidation is only 57% ee, and 86% ee (R)-epoxybenzene ethane can be obtained at -78°C (Journal of the American Chemical Society, 116 (20), 497-504.). A similar modified Shi epoxidation can obtain 71-85% ee (R)-epoxybenzene ethane (Organic Letters, 2001, 3 (12): 1929-31.). It is difficult to obtain high (R)-enantioselective epoxidation products by chemical methods.

[0004] Biocatalytic methods have some significant advantages, such as mild reaction conditions, environmental friendliness, and high stereoselectivity. Styrene monooxygenase (SMO) can catalyze the epoxidation of styrene and its derivatives to produce the corresponding (S)- or (R)-type epoxides, but the stereoselectivity of the currently discovered R-selective SMO cannot meet industrial needs. Therefore, the development of highly (R)-selective styrene epoxidase has certain challenges and important value. Summary of the invention

[0005] 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 mutant with greatly improved enzymatic performance. The present invention also relates to a styrene monooxygenase mutant that catalyzes the epoxidation of 3-fluorostyrene to synthesize (R)-3-fluorophenyl oxirane, catalyzes the epoxidation of 3-chlorostyrene to synthesize (R)-3-chlorophenyl oxirane, and catalyzes the epoxidation of 3-bromostyrene to synthesize (R)-3-bromophenyl oxirane.

[0006] Specifically, the styrene monooxygenase 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 one amino acid site is mutated to obtain the following single point mutant.

[0007] A59W: Alanine at position 59 was mutated to tryptophan.

[0008] 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.

[0009] The mutant of the present invention is obtained as follows:

[0010] First, the enzyme was semi-rationally designed by molecular docking. The protein structure of wild-type SeStyA was molecularly docked with the substrate 3-fluorostyrene. Based on the docking results and virtual mutation, the mutant A59W was rationally designed. Then a single-point mutant was constructed and heterologously expressed in Escherichia coli. The whole cell was used as a catalyst to convert meta-halogen-substituted styrene substrates (3-fluorostyrene, 3-chlorostyrene and 3-bromostyrene) to verify the activity and stereoselectivity of the mutant. It catalyzed the above substrates with excellent stereoselectivity, with an enantiomeric excess of 99% ee.

[0011] Advantages of the present invention: The styrene monooxygenase SeStyA mutant of the present invention can catalyze meta-halogen-substituted styrene compounds (3-fluorostyrene, 3-chlorostyrene and 3-bromostyrene) with high stereoselectivity to generate corresponding (R)-epoxides with an enantiomeric excess of 99%ee. The wild type has low stereoselectivity for catalyzing these substrates and cannot meet actual needs. Therefore, the mutant of the present invention is an excellent catalyst for preparing chiral compounds such as (R)-3-fluorophenyl oxirane, (R)-3-chlorophenyl oxirane, (R)-3-bromophenyl oxirane and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A , wild-type SeStyA and mutant A59W catalyze 3-fluorostyrene,

[0013] Figure 1B , 3-chlorostyrene,

[0014] Figure 1C , HPLC chart of 3-bromostyrene epoxidation. DETAILED DESCRIPTION

[0015] Example 1: Prediction of mutation sites and construction of single-point mutants

[0016] 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 3-fluorostyrene using the molecular docking software AutoDock Vina. The binding mode was analyzed based on the docking results. It was found that the steric hindrance change of A59 would regulate its prochiral conformation. A virtual mutation was performed on it, and it was found that A59W would strengthen the prochiral conformation of the (R)-type epoxy product. Therefore, it was mutated to tryptophan.

[0017] The catalytic process of styrene monooxygenase requires the participation of expensive cofactor FADH2, so the flavin oxidoreductase StyB is introduced into the catalytic system for coenzyme recycling to convert FAD + Reduced to FADH2. The specific method for constructing the co-expression system of SeStyA, linker and StyB refers to previous studies (Catalysis Science & Technology, 2021, 11(6): 2195-2201).

[0018] The wild-type recombinant plasmid was used as a template for site-directed mutagenesis. The primers were designed as shown in Table 1 , and a single-point mutant was rationally constructed.

[0019] Table 1 Site-directed mutagenesis primers

[0020]

[0021] The PCR reaction system is shown in Table 2. The amplification conditions are as follows: 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.

[0022] Table 2 PCR reaction system for site-directed mutagenesis

[0023]

[0024] Add 1 μL DpnI to the PCR product, incubate at 37°C for 1 hour to remove the mother plasmid, then take 10 μL and 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.

[0025] Example 2: Heterologous expression of wild-type SeStyA and mutants

[0026] 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. The cells were transferred to 200 mL TB liquid medium at a 1% inoculum (V / V) 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 washed twice with an equal volume of 0.9% sodium chloride solution for later use.

[0027] Example 3: Biocatalytic epoxidation reaction of wild-type SeStyA and mutants

[0028] The collected wet bacteria were resuspended in potassium phosphate buffer solution (0.1M, pH 7.0). 5mL of the catalytic system includes: bacteria (0.1g / mL), potassium phosphate buffer (0.1M, pH 7.0), 4mmol / 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, and then extracted twice with an equal volume of ethyl acetate, the organic phases were combined and dried with anhydrous sodium sulfate, vacuum evaporated, and 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.

[0029] GC determination of conversion rate: Agilent 7890 detector, chromatographic column is CHIRASIL-DEX CB column (Agilent, USA). Detection procedure for 3-chlorophenyl oxirane and 3-bromophenyl oxirane: temperature 140°C for 10 min, carrier gas (nitrogen) flow rate 3 mL / min, split ratio 20:1. Detection procedure for 3-fluorophenyl oxirane: temperature 100°C for 10 min, carrier gas (nitrogen) flow rate 3 mL / min, split ratio 20:1.

[0030] The ee value of the product was detected by HPLC: the chromatographic column was Chiralpak AS-H, the detection wavelength was 210 nm, the column temperature was 30 °C, the mobile phase was n-hexane:isopropanol 99.5:0.5, and the flow rate was 0.5 mL / min.

[0031] Biocatalytic epoxidation: Wild-type SeStyA catalyzes the synthesis of (R)-3-fluorophenyl oxirane from 3-fluorostyrene with a conversion rate of 82% and an enantiomeric excess of 94% ee; while mutant A59W catalyzes the conversion rate of the substrate to 67% and an enantiomeric excess of >99% ee (see the instruction manual). Figure 1A ). The wild-type SeStyA catalyzes the synthesis of (R)-3-chlorophenyl oxirane from 3-chlorostyrene with a conversion rate of 83% and an enantiomeric excess of 96% ee; the mutant A59W catalyzes the conversion rate of the substrate to 58% and an enantiomeric excess of 99% ee (see the instruction manual). Figure 1B ). The wild-type SeStyA catalyzes the synthesis of (R)-3-bromophenyl oxirane from 3-bromostyrene with a conversion rate of 69% and an enantiomeric excess of 96% ee; the mutant A59W catalyzes the conversion rate of the substrate to 36% and an enantiomeric excess of 99% ee (see the instruction manual). Figure 1C ). Therefore, the mutant has excellent stereoselectivity for the above three substrates.

Claims

1. A styrene monooxygenase mutant, characterized in that The amino acid sequence SEQ ID NO.2 of styrene monooxygenase SeStyA was used as the starting sequence, and the characteristics after mutation were as follows: the alanine at position 59 was mutated to tryptophan.

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

3. Use of the mutant according to claim 1 in catalyzing the epoxidation of 3-chlorostyrene to synthesize (R)-3-chlorophenyl oxirane.

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

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