Cytochrome p450 monooxygenase p450pl2 mutant and application thereof

Abstract

The application discloses a cytochrome P450 monooxygenase P450PL2 mutant and application thereof in the field of biotechnology. The P450PL2 enzyme derived from the doderlein bacillus DS-1 is subjected to multiple rounds of directional modification through the technical means of iterative saturation mutation, and a P450PL2 mutant with improved asymmetric hydroxylation activity is obtained, and the corresponding amino acid sequence is shown as SEQ ID No. 3. The P450PL2 mutant can be applied to catalyze the asymmetric hydroxylation of aryl ketone compounds to synthesize S configuration chiral alpha-hydroxy ketone compounds. The reaction has strict regioselectivity and stereoselectivity, and the operation process is simple and efficient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cytochrome P450 monooxygenase P450PL2 mutant and its applications. Background Technology

[0002] Cytochrome P450 monooxygenases (CYPs) are a superfamily of heme-thiolate proteins. Their reduced forms, upon binding with CO, form complexes that exhibit a characteristic absorption peak at 450 nm. As one of the largest protein superfamilies, genes encoding P450 enzymes have been discovered in animals, plants, fungi, bacteria, and protozoa. In mammals, P450 enzymes primarily participate in the degradation of heterologous substances, drug metabolism, and the synthesis of endogenous compounds, while in plants and microorganisms they are used for the biosynthesis of secondary metabolites. Under mild conditions, P450 enzymes can exert biocatalytic effects on substrates of various structural types, including hydroxylation, epoxidation, and dealkylation reactions. Currently, with the development of technologies such as directed evolution, an increasing number of P450 enzymes are being used as important biocatalysts in fields such as drug synthesis.

[0003] Chiral α-hydroxy ketones are ketone structural units containing a chiral hydroxyl group, widely found in natural product molecules and drug molecules. Furthermore, chiral α-hydroxy ketones can serve as important intermediates in the synthesis of various drug molecules or active compounds, such as the antidepressant (S)-bupropion, the antifungal drug SCH 42427, α4β2-nAChR antagonists used for smoking cessation, and hypoxia-inducible factor-1α inhibitors used for antitumor therapy. The asymmetric hydroxylation of the carbonyl α-position catalyzed by P450 enzymes is an important green alternative for the chemical synthesis of chiral α-hydroxy ketones, avoiding the use of expensive heavy metal catalysts, harsh reaction conditions, and large amounts of organic solvents. Currently, only a few research teams have reported on the asymmetric hydroxylation of carbonyl α-position catalyzed by P450 enzymes, such as the P450BM3 mutant (Nature Chemistry 2011, 3:738; Journal of Organic Chemistry 2015, 80:950) and P450 154C2 (Biochemical and Biophysical Research Communications 2020, 522:355). However, these enzymes suffer from problems such as low regioselectivity, narrow substrate spectrum, and low catalytic efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a cytochrome P450 monooxygenase P450PL2 mutant and its applications. This invention utilizes iterative saturation mutagenesis to perform multiple rounds of targeted modification on the P450PL2 enzyme derived from *Clostridium difficile* DS-1, obtaining a P450PL2 mutant with enhanced asymmetric hydroxylation activity: V99L / L109Y / A124W / G134T / T179P / M182L / T259G / P299S / L303L. This P450PL2 mutant can be used to catalyze the asymmetric hydroxylation of aryl ketone compounds to synthesize S-configuration chiral α-hydroxy ketone compounds, exhibiting strict regioselectivity and stereoselectivity, and the operation process is simple and efficient.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A mutant of cytochrome P450 monooxygenase P450PL2, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] Furthermore, the mutant is a multi-site mutation, which includes: V99L, L109Y, A124W, G134T, T179P, M182L, T259G, P299S and L303L.

[0008] A second objective of this invention is to provide a vector containing a gene encoding the aforementioned P450PL2 mutant.

[0009] A third objective of this invention is to provide a genetically engineered bacterium containing the vector described above.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned P450PL2 mutant, or the above-mentioned vector, or the above-mentioned genetically engineered bacteria in the catalytic asymmetric hydroxylation reaction of aryl ketone compounds.

[0011] Furthermore, the asymmetric hydroxylation is asymmetric hydroxylation at the carbonyl α-position.

[0012] Furthermore, the general chemical formula of the aryl ketone compound is as follows:

[0013]

[0014] Where Ar represents an aryl group that is optionally substituted or unsubstituted, and R represents a hydrogen atom, a halogen atom, a nitro group, a nitrile group, a methyl group, or a methoxy group.

[0015] The working principle and beneficial effects of the present invention: The P450PL2 mutant is named P450PL2-2 D11, and its amino acid sequence is as shown in SEQ ID NO.1. All amino acids in the amino acid sequence of the P450PL2 enzyme at positions 99, 109, 124, 134, 179, 182, 259, 299 and 303 are mutated, and its amino acid sequence is shown in SEQ ID NO.3.

[0016] ① Compared with the parent enzyme P450PL2, a cytochrome P450 monooxygenase, the P450PL2 mutant provided by this invention exhibits significantly enhanced catalytic activity. It can efficiently catalyze the asymmetric hydroxylation of the carbonyl α-position of the template substrate phenylacetone to generate the chiral product (S)-2-hydroxy-1-phenyl-1-propanone, with an ee value of 98% and a yield of 79%. ② The P450PL2 mutant can catalyze the asymmetric hydroxylation reaction of the carbonyl α-position of various aryl ketone substrates, exhibiting excellent stereoselectivity and high yield. Attached Figure Description

[0017] Figure 1 To synthesize the S-configuration chiral α-hydroxy ketone product (II) from aryl ketone compound (I) catalyzed by the P450PL2 mutant.

[0018] Figure 2 SDS-PAGE analysis of P450PL2 mutant expression. Electrophoresis band 1: recombinant E. coli BL21(DE3) containing P450PL2 enzyme; Electrophoresis band 2: recombinant E. coli BL21(DE3) containing P450PL2 mutant; Electrophoresis band M: standard molecular weight protein.

[0019] Figure 3 The 1H NMR spectrum of the S-configuration chiral α-hydroxy ketone compound II-1 prepared by the reaction.

[0020] Figure 4 The carbon NMR spectrum of the S-configuration chiral α-hydroxy ketone compound II-1 prepared by the reaction.

[0021] Figure 5 Liquid phase analysis spectrum of S-configuration chiral α-hydroxy ketone compound II-1 prepared by the reaction. Detailed Implementation

[0022] The following detailed description illustrates the specific implementation method:

[0023] Example 1: Mutation and Screening of P450 Monooxygenases

[0024] This invention uses an expression plasmid containing the parental P450PL2 monooxygenase gene as a template, designs and synthesizes corresponding PCR primers (as shown in Table 1), uses site-directed saturation mutagenesis PCR technology to perform single-point saturation mutagenesis, transforms the amplification product into host cells, and constructs a site-directed saturation mutagenesis library.

[0025] Table 1. P450PL2 Iterative Saturation Mutant Primers

[0026]

[0027]

[0028] The plasmid extraction was performed according to the instructions of the Axygen AP-MN-P-50 plasmid miniaturization kit.

[0029] Site-directed saturation mutagenesis involves inserting a simplified codon NNK (N = A / T / G / C, K = T / G) at the target site, which randomly replaces 20 amino acids at that site.

[0030] The following PCR amplification system and cycling conditions were used for PCR.

[0031] The PCR amplification system is shown in Table 2:

[0032] Table 2 HS PCR system

[0033]

[0034] Perform PCR according to the cycling conditions in Table 3.

[0035] Table 3. Iterative saturation mutant PCR reaction program for P450PL2

[0036]

[0037]

[0038] In the PCR reaction program, when setting the annealing temperature in step 3, the annealing temperature can be determined by setting a gradient temperature program. The gradient program can be set ±5℃ based on the annealing temperature value provided by Shanghai Sangon Biotech Co., Ltd. after synthesizing the primers.

[0039] Agarose gel electrophoresis for PCR product detection: Prepare a 0.9% agarose gel, using 1×TAE as the electrophoresis gel medium and a 1kb ladder DNA marker as a control. Mix 5μL of PCR amplification product with 1μL of 6×RNA / DNA loading buffer and load the gel. Electrophoresis is performed at 110V for 40 min. Remove the gel and check for the target band at approximately 6000bp under UV light. If a band is found, proceed with further steps.

[0040] Digestion and purification of PCR products: ① Digestion: After electrophoresis, the remaining PCR products were mixed with 1.8 μL FastDigestDpn I and 5.2 μL 10×FastDigest buffer and then incubated at 37℃ for 5 h for digestion; ② Purification: The PCR digestion products were purified according to the instructions of the Axygen PCR Purification Kit.

[0041] Heat shock plasmid transformation: E. coli BL21(DE3)-pCDF-duet-1-FdR-Fdx2 competent cells were removed from a -80℃ freezer and placed in an ice bath to thaw. 5 μL of purified PCR product was slowly added to the E. coli BL21(DE3)-pCDF-duet-1-FdR-Fdx2 competent cells. After incubating on ice for 30 min, the cells were immediately transferred to a 42℃ water bath for heat shock for 90 s, and then placed in an ice bath for 2 min. 600 μL of TB liquid culture medium was added to a clean bench, and the cells were incubated at 37℃ and 150 rpm in a shaker for 1 h. Then, 200 μL of the incubated culture was spread onto LB agar plates containing Kan-Str resistance and cultured at 37℃ for 16–18 h.

[0042] After inducing mutant expression, preliminary screening of reaction results was conducted. Mutants exhibiting good catalytic activity in synthesizing chiral (S)-2-hydroxy-1-phenyl-1-propanone from the acetone template substrate were selected for secondary screening. High-performance liquid chromatography (HPLC) analysis confirmed the improved catalytic activity of these mutants. Based on this, using these superior mutants as templates, site-directed saturation mutagenesis was performed at other sites, repeating the same screening strategy to ultimately obtain the cytochrome P450 monooxygenase P450PL2 mutant with even higher activity. As shown in Table 4, in the P450 monooxygenase mutation and screening examples, the superior P450 monooxygenase mutant P450PL2-2D11 was obtained. Its optical purity in synthesizing chiral 2-hydroxy-1-phenyl-1-propanone from acetone increased from 9% ee(R) to 98% ee(S), and the yield increased from 55% to 79%.

[0043] Table 4. Experimental results of P450PL2 and its mutants catalyzing the conversion of acetone to 2-hydroxy-1-phenyl-1-propanone.

[0044]

[0045] Example 2: Expression of the P450 monooxygenase mutant P450PL2-2 D11 engineered strain

[0046] Activated P450 monooxygenase mutant P450PL2-2 D11 engineered bacteria were picked from LB solid agar plates and inoculated into 50 mL of TB liquid medium containing Kan-Str resistance (final concentration 50 μg / mL). The culture was incubated at 37°C and 250 rpm for 8 h. The bacterial culture was then transferred at a 3% volume ratio to the corresponding large-scale TB medium (Kan-Str resistance, final concentration 50 μg / mL) and incubated at 37°C and 250 rpm for approximately 1.5 h (OD). 600 After adding 0.8–0.9 mM IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.12 mM, the cells were cultured at 25 °C and 250 rpm for 12 h to induce enzyme protein expression. The culture was then collected using a centrifuge (4 °C, 9000 rpm, 3 min) to obtain recombinant whole cells containing the P450 monooxygenase mutant P450PL2-2 D11 protein, which were used for subsequent biocatalytic reactions.

[0047] Example 3: P450 monooxygenase mutant P450PL2-2 D11 recombinant cells catalyze the synthesis of chiral (S)-2-hydroxy-1-phenyl-1-propanone (Formula 1) from phenylacetone.

[0048] Formula 1:

[0049] Add 5 mL of phosphate buffer (0.01 M, pH 7.5) containing P450 monooxygenase mutant P450PL2-2 D11 recombinant cells (10 g cdw / L) to a 25 mL Erlenmeyer flask with a stopper and ground glass. Simultaneously add acetone to a final concentration of 2 mM. Incubate the flask at 15 °C and 250 rpm with shaking for 2 h. After the reaction is complete, add 5 mL of ethyl acetate containing an internal standard to fully extract the reaction mixture. Centrifuge the mixture at 9000 rpm and 15 °C until separation occurs. Transfer 1 mL of the organic phase to a 1.5 mL EP tube containing an appropriate amount of anhydrous sodium sulfate. Filter the dried organic phase through a 0.22 μm microporous membrane. Transfer the filtrate to a clean liquid chromatography sample vial. HPLC analysis showed that the yield of (S)-2-hydroxy-1-phenyl-1-propanone was 97%, with an ee value of 99%. In this embodiment, the HPLC analysis used the Shimadzu LC-20A system and Chiralcel IH packed column. The mobile phase was hexane-isopropanol (98:2 v / v) gradient elution for 44 min. The detection wavelength was 220 nm and the flow rate was 0.7 mL / min.

[0050] Take 30–40 25 mL Erlenmeyer flasks with ground-glass stoppers, and add 5 mL of phosphate-buffered saline (0.01 M, pH 7.5) containing 10 g cdw / L recombinant cells (P450PL2-2 D11, a P450 monooxygenase mutant). Simultaneously, add acetone to a final concentration of 2 mM. Incubate the flasks at 15 °C and 250 rpm with shaking for 2 h. After the reaction is complete, combine the reaction mixtures and extract thoroughly with an equal volume of ethyl acetate. Centrifuge the mixture at 9000 rpm and 15 °C until separation occurs. Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure and purify by silica gel column chromatography to obtain the target product (S)-2-hydroxy-1-phenyl-1-propanone. NMR characterization data: 1 H NMR (400MHz, CDCl3) δ7.85(d,J=7.2Hz,2H),7.54(m,1H),7.42(t,J=7.8Hz,2H),5.09(q,J=7.0Hz,1H),3.65(br s,1H),1.37(d,J=7.0Hz,3H); 13 CNMR (100MHz, CDCl3) δ 201.35, 133.96, 133.28, 128.84, 128.63, 69.27, 22.24. High-resolution mass spectrometry characterization: HRMS-ESI (m / z): calcd. for C9H 11 O2[M+H] + 151.0753, found 151.0751.

[0051] Example 4: P450 monooxygenase mutant P450PL2-2 D11 recombinant cells catalyze the synthesis of chiral (S)-2-hydroxy-1-(4′-fluorophenyl)-1-propanone (Formula 2) from 4-fluorophenylacetone.

[0052] Formula 2:

[0053] Add 5 mL of phosphate buffer (0.01 M, pH 7.5) containing P450 monooxygenase mutant P450PL2-2 D11 recombinant cells (10 g cdw / L) to a 25 mL Erlenmeyer flask with a stopper and ground glass. Simultaneously add 2 mM of 4-fluorophenylacetone. Incubate the flask at 15 °C and 250 rpm with shaking for 2 h. After the reaction is complete, extract the reaction mixture thoroughly with 5 mL of ethyl acetate containing the internal standard. Centrifuge the mixture at 9000 rpm and 15 °C until separation occurs. Transfer 1 mL of the organic phase to a 1.5 mL EP tube containing an appropriate amount of anhydrous sodium sulfate. Filter the dried organic phase through a 0.22 μm microporous membrane. Transfer the filtrate to a clean liquid chromatography sample vial. The yield of the product (S)-2-hydroxy-1-(4′-fluorophenyl)-1-propanone was 96%, with an ee value of 98%. In this embodiment, the HPLC analysis used the Shimadzu LC-20A system and Chiralcel OD-H packed column. The mobile phase was hexane-isopropanol (97:3 v / v) gradient elution for 14 min. The detection wavelength was 254 nm and the flow rate was 1.0 mL / min.

[0054] Take 25–40 25 mL Erlenmeyer flasks with ground-glass stoppers, and add 5 mL of phosphate-buffered saline (0.01 M, pH 7.5) containing 10 g cdw / L of P450 PL2-2 D11 recombinant cells (P450 monooxygenase mutant). Simultaneously, add 2 mM of 4-fluorophenylacetone. Incubate the flasks at 15 °C and 250 rpm with shaking for 2 h. After the reaction, combine the reaction mixtures and extract thoroughly with an equal volume of ethyl acetate. Centrifuge the mixture at 9000 rpm and 15 °C until separation occurs. Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure and purify by silica gel column chromatography to obtain the target product (S)-2-hydroxy-1-(4′-fluorophenyl)-1-propanone. NMR characterization data: 1 H NMR (400MHz, CDCl3) δ7.93-7.90(m,2H),7.2(t,J=8.4Hz,2H),5.08(q,J=7.0Hz,1H),3.80(br s,1H),1.39(d,J=7.0Hz,3H); 13C9H NMR (100MHz, CDCl3) δ 200.73, 166.07 (d, J = 255.2 Hz, 1C), 131.34 (d, J = 9.4 Hz, 1C), 129.70 (d, J = 3.0 Hz, 1C), 116.05 (d, J = 21.8 Hz, 1C), 69.18, 22.16. High-resolution mass spectrometry characterization: HRMS-ESI (m / z): calcd. for C9H 10 FO2[M+H] + 169.0659, found 169.0659.

[0055] Example 5: P450 monooxygenase mutant P450PL2-2 D11 recombinant cells catalyze the synthesis of chiral (S)-2-hydroxy-1-(2′-thienyl)-1-propionone (Formula 3) from 2-propionylthiophene.

[0056] Formula 3:

[0057] Add 5 mL of phosphate buffer (0.01 M, pH 7.5) containing P450 monooxygenase mutant P450PL2-2 D11 recombinant cells (10 g cdw / L) to a 25 mL Erlenmeyer flask with a stopper and ground glass to a final concentration of 2 mM of 2-propionylthiophene. Incubate the flask at 15 °C and 250 rpm with shaking for 2 h. After the reaction is complete, extract the reaction mixture thoroughly with 5 mL of ethyl acetate containing the internal standard. Centrifuge the mixture at 9000 rpm and 15 °C until the layers separate. Transfer 1 mL of the organic phase to a 1.5 mL EP tube containing an appropriate amount of anhydrous sodium sulfate. Filter the dried organic phase through a 0.22 μm microporous membrane. Transfer the filtrate to a clean liquid chromatography sample vial. The yield of the product (S)-2-hydroxy-1-(2′-thienyl)-1-propanone was 75%, with an ee value of 99%. In this embodiment, the HPLC analysis used the Shimadzu LC-20A system and Chiralcel IG packed column. The mobile phase was hexane-isopropanol (90:10 v / v) gradient elution for 20 min. The detection wavelength was 254 nm and the flow rate was 1.0 mL / min.

[0058] Take 25–40 25 mL Erlenmeyer flasks with ground-glass stoppers, and add 5 mL of phosphate-buffered saline (0.01 M, pH 7.5) containing 10 g cdw / L recombinant cells (P450PL2-2 D11, a P450 monooxygenase mutant). Simultaneously, add 2 mM 2-propionylthiophene. Incubate the flasks at 15 °C and 250 rpm with shaking for 2 h. After the reaction is complete, combine the reaction mixtures and extract thoroughly with an equal volume of ethyl acetate. Centrifuge the mixture at 9000 rpm and 15 °C until separation occurs. Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure and purify by silica gel column chromatography to obtain the target product (S)-2-hydroxy-1-(2′-thienyl)-1-propanone. NMR characterization data: 1 H NMR (400MHz, CDCl3) δ7.76(dd,J=3.8,1.0Hz,1H),7.74(dd,J=4.9,1.0Hz,1H),7.18(dd,J=4.9,3.8Hz,1H),4.97(q,J=7.0Hz,1H),3.14(br s,1H),1.53(d,J=7.0Hz,3H); 13 C10 NMR (100MHz, CDCl3) δ 194.94, 139.53, 134.81, 133.08, 128.38, 70.16, 23.09. High-resolution mass spectrometry characterization: HRMS-ESI (m / z): calcd. for C7H9O2S[M+H] + 157.0317, found 157.0316.

Claims

1. A cytochrome P450 monooxygenase P450PL2 mutant, characterized in that: The amino acid sequence of the P450PL2 mutant is shown in SEQ ID NO.

3.

2. The coding gene of the P450PL2 mutant according to claim 1, characterized in that: The nucleic acid sequence is shown in SEQ ID No.

4.

3. A vector containing the coding gene of the P450PL2 mutant as described in claim 2.

4. Genetically engineered bacteria containing the vector described in claim 3.

5. The cytochrome P450 monooxygenase P450PL2 mutant according to claim 1, characterized in that: The application of the P450PL2 mutant in the catalytic asymmetric hydroxylation reaction of aryl ketone compounds, wherein the aryl ketone compounds are phenylacetone and 4-fluorophenylacetone.

6. The cytochrome P450 monooxygenase P450PL2 mutant according to claim 5, characterized in that: The asymmetric hydroxylation is asymmetric hydroxylation at the α-position of the carbonyl group.

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