Styrene monooxygenase mutant and application thereof in catalyzing R-limonene to synthesize trans-limonene-1, 2-epoxide

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly discloses a styrene monooxygenated mutant which is used for catalyzing R-limonene to synthesize trans-limonene-1, 2-epoxide and has excellent selectivity. The compound can catalyze R-limonene to obtain trans-limonene-1, 2-epoxide, the diastereomer ratio value reaches dr 99: 1 at most, and the compound has good activity and is an excellent catalyst for preparing the compound.

Description

Technical Field

[0001] The invention belongs to the technical fields of gene engineering and enzyme engineering, and particularly relates to a styrene monooxygenase mutant and application thereof in catalyzing R-limonene to synthesize trans-limonene-1,2-epoxide. Background Art

[0002] In the modern pharmaceutical and fine chemical industries, the demand for efficient, green, and sustainable synthetic methods is increasingly urgent. Biocatalysis, in particular, has attracted considerable attention due to its unique advantages in the synthesis of chiral drug intermediates. Compared to traditional chemical synthesis, biocatalysis typically operates under mild conditions and offers advantages such as high efficiency, environmental friendliness, and high stereoselectivity, significantly reducing energy consumption and the generation of hazardous waste.

[0003] Limonene-1,2-epoxide is the primary product of the epoxidation of R-limonene and a fundamental monomer in polymer synthesis. Studies have reported that only the trans isomer of limonene-1,2-epoxide can polymerize with CO2 to form polycarbonates or cyclic carbonates. However, commercially available limonene oxide is complex, consisting of only 57% trans-limonene oxide. Therefore, the development of an economically viable, environmentally friendly, and highly trans-selective R-limonene epoxidation method is imperative.

[0004] At present, a variety of biocatalytic methods have been reported for the epoxidation of limonene, but most methods still face challenges such as insufficient selectivity and complex products. For example, Aguila et al. found that chloroperoxidase CPO can catalyze R-limonene with high selectivity, but the product is cis-1,2-epoxide, which is ultimately converted into diols (Green Chem., 2008, 10(6), 647-653). Brandolese et al. explored the application of lipase Novozym 435 to catalyze monoterpenes. Although the reaction conditions are mild and it also shows high selectivity for 3-carene, it catalyzes R-limonene to obtain a mixture and cannot effectively control the formation of trans isomers (Acs Sustain Chem Eng., 2023, 11(12), 4885-4893). In addition, Biondi et al. used oat peroxidase to catalyze the epoxidation of R-limonene. Although it had a high trans selectivity (dr>99:1), the reaction product also produced about 15% of the by-product hydroxylation (Antioxidants, 2021, 10(9), 1462). Therefore, the development of a biocatalytic method that can efficiently and selectively synthesize trans-limonene-1,2-epoxide has important application value. Summary of the Invention

[0005] This invention uses the styrene monooxygenase SgStyA from Streptomyces gardneri strain NBRC 12865 as the parent enzyme and employs sequence- and structure-based semi-rational design to generate molecularly modified styrene monooxygenase mutants with significantly improved enzymatic properties. The invention also relates to the use of these mutants in catalyzing the epoxidation of R-limonene to produce trans-limonene-1,2-epoxide.

[0006] Specifically, the styrene monooxygenase mutant is a single point mutant or a combination mutant obtained by mutating one or more amino acid sites based on SgStyA (nucleotide sequence is SEQ ID NO.1; amino acid sequence is SEQ ID NO.2).

[0007] F194M: Phenylalanine at position 194 is mutated to methionine.

[0008] F50W: Phenylalanine at position 50 mutates to tryptophan.

[0009] V209C: Valine at position 209 was mutated to cysteine.

[0010] Combined mutant SgStyA1: Phenylalanine at position 194 was mutated to methionine and Phenylalanine at position 50 was mutated to tryptophan.

[0011] Combined mutant SgStyA2: Phenylalanine at position 194 was mutated to methionine and valine at position 209 was mutated to cysteine.

[0012] Combination mutant SgStyA3: Phenylalanine at position 194 was mutated to methionine, Phenylalanine at position 50 was mutated to tryptophan, and Valine at position 209 was mutated to cysteine.

[0013] According to the common knowledge in this field, vectors and genetically engineered bacteria that can express the above mutants also fall within the scope of protection of the present invention.

[0014] The mutant of the present invention is obtained as follows:

[0015] First, the structural model of SgStyA was constructed using AlphaFold 2.0 and superimposed with the VpIndA1 / FAD complex structure (PDB: 7Z94) to locate the cofactor FAD and optimize its position. Then, R-limonene was docked into the active cavity of SgStyA. Based on the docking conformation, the active pocket of SgStyA was finally selected. Five potential sites within the substrate near the 1,2 carbon-carbon double bond were identified: F50, G97, F194, T196, and V209. These five potential sites were mutated into three amino acid groups representing varying degrees of steric hindrance (A / G; L / V / C / I / M; F / Y / W) to construct single-point mutants. Biocatalytic validation identified three mutants with significantly improved trans-diastereoselectivity: F194M, F50W, and V209C. Further combinations of favorable sites yielded three superior combination mutants: SgStyA1, SgStyA2, and SgStyA3. Wild-type SgStyA achieved 90% conversion of 4 mmol / L R-limonene within 2 hours, with a selectivity of 79:21 for the product trans-limonene-1,2-epoxide. The conversion rate of the combined mutant SgStyA1 was 50%, and the trans-diastereoselectivity of the product was dr>99:1; the conversion rate of SgStyA2 was 75%, and the trans-diastereoselectivity of the product was dr>99:1; the conversion rate of SgStyA3 was 50%, and the trans-diastereoselectivity of the product was dr>99:1.

[0016] Advantages of the present invention: The styrene monooxygenase SgStyA mutant can catalyze the conversion of R-limonene to produce trans-limonene-1,2-epoxide, marking the first time this enzyme catalyzes the conversion of R-limonene to produce trans-limonene-1,2-epoxide. The mutant achieves a 75% conversion within 2 hours of 4 mmol / L of the substrate R-limonene, with a trans-diastereomer dr value exceeding 99:1, significantly exceeding the diastereoselectivity of the wild-type. This makes it a highly efficient and selective catalyst for the preparation of trans-limonene-1,2-epoxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 GC-MS images of the products of R-limonene conversion by SgStyA. GC: Two products (peak 1 and peak 2) are produced by the epoxidation of R-limonene using SgStyA as the substrate. MS: The mass spectrum of peak 1 is consistent with that of the standard (+)-cis-limonene-1,2-epoxide (SI 96), while the mass spectrum of peak 2 is consistent with that of the standard (+)-trans-limonene-1,2-epoxide (SI 95). DETAILED DESCRIPTION

[0018] Example 1: Prediction of mutation sites and construction of single-point mutants

[0019] The nucleotide sequence of SgStyA is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The SgStyA structure was simulated using AlphaFold2 and the model structure was evaluated using SAVES 6.0. The substrate R-limonene was molecularly docked with the protein SgStyA using AutoDuck 4.0 molecular docking software. Based on the molecular docking results, the active pocket of SgStyA was finally selected. Single-point mutations were performed on five residues within the enzyme: F50, G97, F194, T196, and V209. The present invention primarily aims to improve chemical selectivity by reshaping the enzyme active pocket from a steric perspective, so these five amino acids were subjected to half-saturation mutations (A / G; L / V / C / I / M; F / Y / W).

[0020] Since the entire catalytic process requires the participation of the cofactor FADH2, which is relatively expensive, the participation of StyB (which reduces FAD to FADH2) is required. The connection method of SgStyA and PaStyB can be found in previous studies (Catalysis Science & Technology, 2021, 11(6): 2195-2201). Finally, SgStyA and PaStyB were constructed in the pET-28a(+) vector with NdeⅠ and HindⅢ restriction sites.

[0021] Site-directed mutagenesis was performed using the wild-type recombinant plasmid as a template, and degenerate primers (WDK and SBS degenerate codons) were used to reduce the workload. The designed mutagenesis primers are shown in Table 1. A total of 47 single-point mutants were constructed.

[0022] Table 1 Site-directed mutagenesis primers

[0023]

[0024] The PCR reaction system is shown in Table 2 , and amplification was performed according to the following amplification conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 68°C for 7.5 min, for a total of 18 cycles.

[0025] Table 2 PCR reaction system for site-directed mutagenesis

[0026]

[0027]

[0028] The PCR product was treated with 1 μL of DpnI at 37°C for 1 hour. Then, 10 μL was transferred into E. coli DH5α competent cells using standard chemical methods. The cells were then plated onto LB plates containing 100 μg / mL kanamycin and cultured overnight at 37°C. A single colony was selected for liquid culture, and the plasmid was extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0029] Example 2: Heterologous expression of wild-type SgStyAB and mutants

[0030] According to literature reports, molecular chaperones can participate in the protein folding process, help proteins fold correctly to improve protein soluble expression, so we will SgStyAB wild type and its mutant plasmids and molecular chaperone PGro7 co-transformed into E. coli BL21 (DE3) competent cells for protein expression. Pick a single clone to 4mL LB medium (containing kanamycin 100μg / mL) and culture overnight at 37℃, 180r / min. Take 2mL (1%, V / V) and transfer to 200mL TB medium for expansion culture; wait until OD 600 When the pH reached about 0.8, IPTG (0.5 mM) and arabinose (2 mg / mL) were added, and the culture was continued at 20°C and 180 rpm for 20 h. The cells were collected by refrigerated centrifugation and washed twice with an equal volume of 0.9% saline for later use.

[0031] Example 3: Biocatalysis of wild-type SgStyA and mutants

[0032] Catalytic Epoxidation: Wet microbial cells after centrifugation were resuspended in potassium phosphate buffer (0.1 M, pH 7.0). A 5 mL reaction system containing potassium phosphate buffer (0.1 M, pH 7.0), microbial cells (0.1 g / mL), and the substrate R-limonene (dissolved in isopropanol, final concentration 4 mmol / L) was placed in a 100 mL Erlenmeyer flask and incubated at 30°C in a shaker (200 rpm) for 2 h. The mixture was then extracted twice with equal volumes of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and used directly for GC and GC-MS analysis.

[0033] Conversion and diastereomeric selectivity were determined by GC using a MEGA-DEX DAC chiral column (30 m × 0.25 mm, 0.25 μm) and an Agilent 7890 gas chromatograph. The thermostat was maintained at 100°C for 10 min, with a nitrogen carrier gas flow rate of 3 mL / min and a pressure of 49.5 kPa, and a split ratio of 10:1.

[0034] GC-MS product identification and analysis: SH-WAX column (30 m × 0.25 mm, 0.25 μm), nitrogen (N2) carrier gas, column flow rate 1.0 mL / min, column temperature: 80°C for 1 min, then increased to 110°C at a rate of 2°C / min and held for 10 min. Split injection, split ratio 20:1, 3 μL injection. Injection port temperature 230°C, ion source temperature 230°C, interface temperature 250°C.

[0035] Qualitative analysis of mass spectra: Data retrieval is performed by computer retrieval and comparison with standard mass spectra in the spectral library (NIST20). Compound screening is performed based on the principle of SI (similarity) greater than 90% to identify the substance.

[0036] As the instruction manual Figure 1 As shown, wild-type SgStyA can catalyze the epoxidation of R-limonene, and two products are detected by GC. These two products (peak 1 and peak 2) are consistent with the standard mass spectra of (+)-cis-limonene-1,2-epoxide and (+)-trans-limonene-1,2-epoxide. Figure 1 Therefore, peak 1 was identified as (+)-cis-limonene-1,2-epoxide, and peak 2 was identified as (+)-trans-limonene-1,2-epoxide.

[0037] GC determination showed that the conversion of R-limonene by wild-type SgStyA was 90% with a diastereoselectivity of 79:21.

[0038] Mutant Biocatalysis: All mutants constructed in Example 1 were tested for their conversion and diastereoselectivity (dr) for R-limonene. Compared to the wild-type, the conversion rates of F50W, F194M, and V209C whole cells, catalyzing 4 mmol / L of substrate at 30°C for 2 h, were comparable to those of wild-type SgStyA, at 92%, 85%, and 94%, respectively. Furthermore, the selectivity for the product (+)-trans-limonene-1,2-epoxide was significantly improved. F194M exhibited the best (trans-) selectivity, with a dr value of 98:2. F50W and V209C followed closely behind, each with a dr value of 93:7.

[0039] Example 4: Biocatalysis of Combinatorial Mutagen

[0040] First, V209C and F50W were integrated into F194M, respectively, to construct two combined mutants, SgStyA1 and SgStyA2. These three mutants, V209C, F50W, and F194M, were then combined to construct the combined mutant SgStyA3. The whole-cell catalysis of the combined mutants was the same as in Example 3. As shown in Table 3, the trans-diastereoselectivity of the products of the three mutants, SgStyA1, SgStyA2, and SgStyA3, for the conversion of R-limonene was significantly improved, with dr values > 99:1. The conversion rate of SgStyA1 and SgStyA3 for R-limonene was 50%. The conversion rate of the combined mutant SgStyA2 for R-limonene was 75%, which was not much different from that of the wild-type SgStyA.

[0041] Table 3 Biocatalysis of wild-type and mutant SgStyA

[0042]

[0043]

Claims

1. A styrene monooxygenase mutant, characterized in that: Taking the amino acid sequence SEQ ID NO.2 of styrene monooxygenase SgStyA as the starting sequence, the phenylalanine at position 194 was mutated to methionine.

2. A styrene monooxygenase mutant, characterized in that: The phenylalanine at position 50 of the mutant according to claim 1 is mutated to tryptophan.

3. A styrene monooxygenase mutant, characterized in that: The valine at position 209 of the mutant according to claim 1 is mutated to cysteine.

4. A styrene monooxygenase mutant, characterized in that: The valine at position 209 of the mutant according to claim 2 is mutated to cysteine.

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