Fatty acid decarboxylase P450BS beta mutant, preparation method and application

Abstract

The invention belongs to the field of protein engineering, and particularly relates to a fatty acid decarboxylase P450BS beta mutant as well as a preparation method and application thereof.The fatty acid decarboxylase P450BS beta mutant is obtained by mutating the 78th site, the 85th site, the 86th site, the 170th site, the 173rd site, the 246th site and the 290th site of an amino acid sequence of wild type carboxylic acid beta-hydroxylase P450BS beta, the mutant can catalyze C-H hydroxylation of a benzyl position of a non-carboxylic acid substrate phenyl methyl ester derivative at a high substrate conversion rate through H2O2 which is low in cost and easy to obtain, hydroxylation products are treated by trifluoroacetic acid and then cyclized into corresponding gamma-phenyl lactone and delta-phenyl lactone, the substrate spectrum of P450BS beta enzyme is widened, and the mutant is successfully used for asymmetric synthesis of gamma-phenyl lactone and delta-phenyl lactone. The application value of the biocatalyst as a broad-spectrum biocatalyst is further improved.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and in particular relates to a fatty acid decarboxylase P450BSβ mutant, a preparation method and an application thereof. Background Art

[0002] Lactones are biological lipid metabolites, formed by the dehydration condensation of hydroxyl and carboxyl groups within their molecules under certain acid-base conditions. Lactones and their derivatives are widely used in drug development and intermediate synthesis due to their biochemical activity. In addition, the chiral carbon atoms at the hydroxyl position also divide lactones with the same number of rings into R-type and S-type. For example, some γ- and δ-phenyl lactones carrying benzene rings, such as γ-phenyl-γ-butyrolactone, can inhibit cytochrome enzymes CYP1A2 and CYP2A6. and CYP2B6; γ-aryloxymethyl-α-methylene-γ-phenyl-γ-butyrolactone is an anticancer drug, and EFBL has anticonvulsant and hypnotic effects; among the δ-phenyl lactones, 6-phenyl-dihydro-pyran-2,4-dione is effective against Biomphalaria glabrata, one of the main intermediate hosts of deadly schistosomiasis. ata) has bactericidal properties and can be used to eliminate Schistosoma mansoni; (6S)-6-(Bromomethyl)teerahydro-3-methylene-6-phenyl-2H-pyran-2-one ((6S)-6-(Bromomethyl)teerahydro-3-methylene-6-phenyl-2H-pyran-2-one) has certain anti-tumor activity, and the S-type has higher anti-tumor activity than the R-type; γ-halo-δ-lactones are a class of artificially synthesized γ-halogenated δ-phenyl lactones with multiple antibacterial and anti-cancer functions.

[0003] Chiral lactones are the building blocks of many natural compounds and bioactive molecules, exhibiting diverse pharmacological activities such as antibacterial, antiviral, and antitumor activities. Over the past few years, various chemical methods have been developed to synthesize chiral lactones, most of which are based on metal-catalyzed pathways such as copper-catalyzed carbonyl esterification or oxidative [3 + 2] cycloaddition, chiral noble metal- or transition metal-catalyzed hydrogenation, and hydroacylation. However, most of these methods require harsh conditions and complex reaction steps, which can lead to environmental concerns. Therefore, finding efficient and environmentally friendly methods to synthesize enantiomerically enriched lactones is highly desirable.

[0004] Biocatalytic methods have the advantages of high efficiency, good selectivity, and environmental friendliness, and have become an important supplement to traditional organic synthesis. Currently, the main methods for the biocatalytic synthesis of chiral lactones include: oxidation of cyclic ketones catalyzed by Baeyer-Villiger monooxygenase (BVMO) achieved by hydrolase-catalyzed resolution, hydroxylation catalyzed by nonspecific peroxygenase (UPO) or cytochrome P450 (CYP), and asymmetric reduction of aromatic ketoesters to the corresponding hydroxyesters and lactones using alcohol dehydrogenases (ADH) from different sources. However, since aromatic ketoesters with a "bulky (aryl)-bulky (ester)" structure are not easily accessible to the active site of the enzyme, the biocatalytic reduction of aromatic ketoesters still faces the challenges of low enantioselectivity and a limited range of aromatic lactones.

[0005] The catalytic mechanism of P450BSβ relies on a salt bridge formed by the interaction between the carboxyl group of the fatty acid substrate and the guanidinium group at the active site Arg242. This is key to generating the highly oxidative intermediate Compound I and initiating the oxidation cycle. Therefore, the fatty acid substrate itself acts as an initiator and activator for the reaction. This indicates that molecules without carboxylic acid groups cannot initiate the oxidation cycle, resulting in P450BSβ having little catalytic activity toward non-carboxylic acid substrates. Currently, high substrate-specific enzymes can be catalyzed toward non-carboxylic acid substrates by adding decoy molecules, but this approach still suffers from problems such as a narrow substrate range, difficulty obtaining decoy molecules, and competitive inhibition. To overcome the enzyme's restriction toward carboxylic acid substrates, the present invention introduces an acidic functional group into a specific site within the protein framework. This replaces the substrate and forms a salt bridge with the conserved guanidinium group at Arg242 through electrostatic interaction, ensuring that the enzyme itself forms Compound I during the catalytic reaction of non-carboxylic acid substrates. Based on this, we successfully obtained a mutant that does not require a fatty acid substrate to initiate the oxidation cycle, breaking the limitation of P450BSβ that it can only catalyze natural substrates with carboxylic acid groups.

[0006] The present invention uses a P450BSβ mutant that is independent of substrate carboxylic acid to catalyze the benzylic hydroxylation reaction of a phenyl methyl ester substrate, and then realizes a lactonization reaction through spontaneous intramolecular condensation, thereby asymmetric synthesis of γ- and δ-phenyl lactone products. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention provides a fatty acid decarboxylase P450BSβ mutant, preparation method, and application. By introducing an acidic functional group into the protein framework, a mutant is successfully obtained that does not require a fatty acid substrate to initiate the oxidation cycle, breaking the limitation of P450BSβ, which can only catalyze natural substrates with carboxylic acid groups. This mutant can oxidize a range of non-carboxylic acid substrates with high conversion rates using inexpensive and readily available H2O2, broadening the application prospects of P450BSβ.

[0008] The technical solution provided by the present invention is as follows:

[0009] The present invention provides a fatty acid decarboxylase P450BSβ mutant. The amino acid sequence of the fatty acid decarboxylase P450BSβ mutant is shown in SEQ ID NO: 1.

[0010] The present invention also provides a nucleotide sequence, which encodes the fatty acid decarboxylase P450BSβ mutant according to claim 1, and the nucleotide sequence is shown in SEQ ID NO: 2.

[0011] The present invention also provides a method for preparing the fatty acid decarboxylase P450BSβ mutant as described above, characterized in that the following mutations are performed on the basis of the wild-type fatty acid decarboxylase P450BSβ: valine at position 170 is mutated into glutamic acid, glycine at position 290 is mutated into isoleucine, alanine at position 246 is mutated into glycine, glutamine at position 85 is mutated into histidine, leucine at position 78 is mutated into methionine, phenylalanine at position 173 is mutated into aspartic acid, and glycine at position 86 is mutated into valine; the nucleotide sequence of the wild-type fatty acid decarboxylase P450BSβ is shown in SEQ ID NO: 3.

[0012] Further, the following steps are included: S1 Construction of a plasmid expressing wild-type fatty acid decarboxylase P450BSβ; S2 constructed a saturation mutation library through seven rounds of site-directed saturation mutagenesis; S3 The constructed plasmid expressing the fatty acid decarboxylase P450BSβ mutant was transformed into Escherichia coli; Expression and purification of the S4 fatty acid decarboxylase P450BSβ mutant.

[0013] Furthermore, the expression and purification of the fatty acid decarboxylase P450BSβ mutant comprises the following steps: S41: The strain constructed in S3 was cultured overnight in LB liquid medium, expanded in TB liquid medium, and induced by adding heme precursor δ-aminolevulinic acid and IPTG during the logarithmic growth phase, and then the bacteria were harvested by centrifugation; S42 The obtained bacteria were resuspended in a buffer solution, the cells were lysed by ultrasonication, and centrifuged to obtain the supernatant; S43 adds the supernatant to a nickel ion affinity column, allows the target protein with the His tag to bind to the nickel ions, removes impurities, elutes the target protein, and obtains a fatty acid decarboxylase P450BSβ mutant.

[0014] The present invention also provides a method for asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone, wherein a non-natural carboxylic acid is used as a substrate, hydrogen peroxide is used as an oxidant, and the fatty acid decarboxylase P450BSβ mutant is used as a catalyst to carry out a catalytic reaction, wherein the non-natural carboxylic acid is a derivative of phenyl methyl ester.

[0015] Furthermore, the γ-phenyl lactone is S-type γ-phenyl lactone, and the δ-phenyl lactone is S-type δ-phenyl lactone.

[0016] Furthermore, the derivatives of the phenyl methyl ester include methyl 4-phenylbutyrate, methyl 5-phenylvalerate, methyl 4-fluorophenylbutyrate, methyl 4-fluorophenylvalerate, methyl 4-chlorophenylbutyrate, methyl 4-chlorophenylvalerate, methyl 4-methoxyphenylbutyrate, methyl 4-methoxyphenylvalerate, methyl 2-thiophenebutyrate, methyl 2-thiophenevalerate, methyl 4-methoxycarbonylphenylbutyrate, and methyl 4-methoxycarbonylphenylvalerate.

[0017] Furthermore, the catalytic reaction is carried out at room temperature, the reaction system is an aqueous buffer system, and Triton X-100 and isopropanol are used as cosolvents.

[0018] The present invention also provides the use of the fatty acid decarboxylase P450BSβ mutant in the asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone.

[0019] Beneficial effects

[0020] 1. The biocatalyst constructed by the present invention successfully breaks away from its dependence on carboxylic acid substrates by using a protein directed evolution strategy. Instead, it uses cheap and readily available hydrogen peroxide to catalyze the synthesis of the corresponding (S)-γ-phenyllactone and (S)-δ-phenyllactone from the non-carboxylic acid substrate phenyllactone, thus broadening its catalytic substrate range.

[0021] 2. The fatty acid decarboxylase P450BSβ mutant of the present invention catalyzes the benzylic CH hydroxylation of non-carboxylic acid substrates, phenyl methyl ester derivatives, and the hydroxylated products are cyclized to the corresponding γ- and δ-phenyl lactones after treatment with trifluoroacetic acid. The high-substrate-specific enzyme P450BSβ mutant of the present invention is obtained by mutating the amino acid sequence of the wild-type P450BSβ at positions 78, 85, 86, 170, 173, 246, and 290. Using a protein directed evolution strategy, it successfully frees itself from its dependence on carboxylic acid substrates. Based on a novel oxidative cycle mechanism, directed evolution enables it to utilize inexpensive and readily available H2O2 to catalyze the asymmetric synthesis of γ- and δ-phenyl lactones from non-natural substrates, thereby enhancing its synthetic application value in the field of biosynthesis.

[0022] 3. Most biocatalytic syntheses of chiral lactones are primarily achieved through hydrolytic enzyme-catalyzed resolution, such as oxidation of cyclic ketones catalyzed by Baeyer-Villiger monooxygenase (BVMO), or hydroxylation catalyzed by nonspecific peroxygenases (UPOs) or cytochrome P450 (CYPs). However, most P450BSβs, due to their highly conserved Arg242 guanidinium group, require electrostatic interactions with the carboxyl group of carboxylic acid substrates to activate H2O2. This highly specific substrate recognition mechanism results in little catalytic activity for non-carboxylic acid substrates. This P450BSβ mutant catalyzes the hydroxylation of non-carboxylic acid substrates, phenyl lactone derivatives, leading to the asymmetric synthesis of γ-phenyl and δ-phenyl lactones.

[0023] 4. The reaction process is mild and is carried out in aqueous solution at room temperature, with high synthesis efficiency, regioselectivity and enantioselectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a catalytic example diagram of P450BSB-V170E / G290I / A246G / Q85H / L78M / FI73N / G86V;

[0025] Figure 2 This is a diagram of a catalytic example of P450BSB-V170E / G290I / A246G / Q85H / L78M / FI73N / G86V. DETAILED DESCRIPTION

[0026] The present invention is further described below. The following examples are intended only to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, all compounds and reagents below were purchased from Sigma-Aldrich, EMD Millipore, Tokyo Chemical Industry, Macklin Biochemical, Bidepharm Biochemical, and New England Biolabs.

[0027] Example 1 Construction of Fatty Acid Decarboxylase P450BSβ Gene Expression Vector

[0028] A fully synthesized wild-type P450 BSβ fragment (sequence shown in SEQ ID NO: 3, synthesized by Anshengda Biotechnology Co., Ltd.) was double-digested with the restriction endonucleases NcoI and XhoI (New England Biolabs) according to the manufacturer's instructions and ligated into the expression vector pET28a, which had been double-digested with NcoI and XhoI, using T4 ligase (New England Biolabs). The ligation product was transformed into competent Escherichia coli DH5α cells (Tiangen Biochemical Technology Co., Ltd.). Successfully transformed single colonies were picked from solid LB medium plates containing 50 μg / ml kanamycin and cultured overnight in LB liquid medium containing the same concentration of kanamycin at 37°C with a shaker at 220 rpm / min. The recombinant plasmid was extracted from the overnight culture using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) according to the manufacturer's instructions, and the extracted plasmid was sent for sequencing verification (Anshengda Biotechnology Co., Ltd.).

[0029] Example 2 Preparation of fatty acid decarboxylase P450BSβ mutant

[0030] (1) Construction and expression of the P450BSβ mutant library: The plasmid successfully sequenced above was used as a PCR template. Based on the crystal structure of the P450BSβ protein (PDB ID: 1IZO), a P450BSβ mutant library was established. Seven rounds of saturation mutation library screening were performed. In each round, overlapping saturation mutagenesis was performed using the single mutant with the best performance in the previous round as a template. Excellent P450BSβ mutants that catalyze the asymmetric synthesis of γ-phenyl lactone from phenylmethyl ester were screened and sent for sequencing identification. Preparation of the cell-free lysate used for screening: The overnight culture seed solution was expanded into 40 mL of TB liquid medium (containing 1000× kanamycin and chloramphenicol) and cultured in a shaker at 37°C and 220 rpm. When the OD600 reached 0.3, 0.5 μM δ-aminolevulinic acid was added to the culture solution. When the OD600 reached 0.6, the culture solution was ice-bathed to below 10°C, and IPTG was added to the culture solution at a final concentration of 1 μM. The culture was induced overnight at 27°C and 220 rpm. Centrifuge the overnight induced bacterial suspension at 5000 rpm for 10 minutes, discard the supernatant, and perform subsequent operations on ice. Resuspend the suspension in 40 mL of Buffer A containing 10% glycerol and centrifuge at 5000 rpm for 10 minutes at 4°C, discard the supernatant, and resuspend the suspension in 3.4 mL of Buffer A containing 10% glycerol. Disrupt the cells using an ultrasonic disruptor at 25% power, a Ø2 amp, for 6 minutes, with 2-second bursts and 4-second intervals. Take 1.4 mL of the disrupted bacterial suspension and centrifuge at 12000 rpm for 15 minutes at 4°C to obtain the cell-free supernatant.

[0031] (2) Screening of fatty acid decarboxylase P450BSβ mutant plasmids: 930 μL of cell-free lysate supernatant was added with substrate (0.5 mM) and H2O2 (5 mM) in sequence, and the mixture was reacted at 26°C and 180 rpm for 45 min. After the reaction, 2 mL of ethyl acetate was added and the mixture was thoroughly mixed using a vortex mixer. After centrifugation at 3500 rpm for 3 min, 800 μL of the supernatant was removed with an appropriate amount of anhydrous MgSO4, filtered, and the contents of γ- and δ-lactone and their racemic sample standards were determined by gas chromatography and used as reference. Variants with higher C-γ and C-δ selectivity than the parent were selected.

[0032] Example 3 Large-scale expression and purification of fatty acid decarboxylase P450BSβ mutant protein

[0033] Expression and purification of the highly substrate-specific enzyme P450BSβ: The strain obtained in Example 2 was inoculated into 40 mL of LB liquid medium (Kan / Cam) and cultured overnight at 37°C. The overnight culture was then inoculated into 500 mL of TB liquid medium (Kan / Cam) and expanded at 37°C with a shaker at 220 rpm. Induction was initiated by adding the heme precursor δ-aminolevulinic acid and IPTG in the late logarithmic phase. The culture was shaken at 25°C with a shaker at 220 rpm for 16 hours.

[0034] Harvest the cells by centrifugation at 5000 rpm for 10 minutes. Resuspend the cells in buffer A and disrupt the cells by sonication. Centrifuge at 10000 rpm for 45 minutes at 4°C to obtain the cell lysate supernatant. Add the supernatant containing the target protein obtained in step 4 to a pretreated nickel affinity column to allow the His-tagged target protein to bind to the nickel column. Remove contaminants with buffer A containing 35 mM imidazole, and then elute the target protein with buffer B containing 250 mM imidazole.

[0035] The eluted target protein was dialyzed against buffer A to remove imidazole. The purified protein was concentrated and stored at -80°C. The protein concentration was determined by the P450-CO method.

[0036] Example 4: Identification and analysis of fatty acid deacidase P450BSβ mutant products

[0037] Product content was determined using a TraceISQ gas chromatography-mass spectrometer (GC-MS). The chromatographic column was TG-5MS (30 m × 0.25 mm, 0.25 μm film). Detection conditions included: injection volume, 1 μL; injection temperature, 250°C; carrier gas, N₂, splitless. Detection procedure: 40°C, 4 min; then, ramping from 10°C min⁻¹ to 250°C, 5 min, for a total of 30 min.

[0038] Chiral analysis of the products was performed using a Fuli GC9790Ⅱ gas chromatograph. The column was a CP-ChirasilDex CB (25 m × 0.25 mm, 0.25 μm film). Detection conditions included: injection volume 1 μL; carrier gas: N2; injection temperature: 200°C; split ratio: 1:2. The detection procedure was: hold at 100°C for 1 min, then increase the temperature to 150°C at a rate of 10°C min-1 and hold for 1 min; then increase the temperature to 180°C at a rate of 2°C min-1 and hold for 7 min; and finally increase the temperature to 200°C at a rate of 25°C min-1 and hold for 8 min, for a total of 37.8 min.

[0039] NMR: recorded on an Agilent DD2400 MHz NMR spectrometer 1 H-NMR spectrum.

[0040] Example 5: Asymmetric Synthesis of γ-Phenyl Lactone and δ-Phenyl Lactone

[0041] A 100 mL reaction system containing Buffer A (0.1 M KPi, 0.3 M KCl, pH 7.0) and a non-carboxylic acid substrate, a phenyl methyl ester derivative (4 mM), was used as a substrate cosolvent in 0.125% v / v Triton X-100 and 5% v / v isopropanol. Purified P450BSβ mutant protein (10 or 15 μM) was reacted in Buffer A and Buffer B containing 10% glycerol, and H₂O₂ (4 mM x 2, 3 min intervals) was added. The reaction was incubated at 26°C for 10 min in a final volume of 100 mL. The organic layer, extracted with ethyl acetate, was dried over anhydrous magnesium sulfate, vacuum filtered, and concentrated. The 0.5 mmol enzyme extract was reconstituted in 10 mL of dichloromethane and precooled in an ice-water bath with stirring at 0°C. Subsequently, 50 μL of trifluoroacetic acid was slowly added at room temperature and the reaction was continued for 4 h under a nitrogen atmosphere. After the reaction was completed, saturated NaHCO₃ was used to terminate the reaction and neutralize the trifluoroacetic acid. The reaction solution was washed three times with water and once with saturated brine to obtain the lower dichloromethane organic phase, which was dried over anhydrous magnesium sulfate, vacuum filtered, and concentrated in vacuo. The products γ-lactone and δ-lactone were purified by silica gel column chromatography (EtOAc / Hexanes = 10:90-20:80). The products were identified by thin layer chromatography analysis. The purified products were identified by nuclear magnetic resonance spectroscopy, and chiral separation was analyzed by GC chiral column. The results are as follows. Figure 1 and Figure 2 As shown. Figure 1 As shown, methyl 4-phenylbutyrate is obtained as Figure 1 The product (1) shown in FIG; obtained from methyl 4-fluorophenylbutyrate as shown in FIG. Figure 1 The product (2) shown in FIG; obtained from methyl 4-chlorophenylbutyrate as shown in FIG. Figure 1 The product (3) shown in FIG; obtained from methyl 4-methoxyphenylbutyrate as shown in FIG. Figure 1 The product (4) shown in FIG; obtained from 2-thiophenebutyric acid methyl ester as shown in FIG. Figure 1 The product (5) shown in FIG; obtained from methyl 4-methoxycarbonylphenylbutyrate as shown in FIG. Figure 1 The product shown in (6); Figure 2 As shown, 5-phenylpentanoic acid methyl ester is obtained as Figure 2The product (7) shown in FIG; obtained from methyl 4-fluorophenylvalerate as shown in FIG. Figure 2 The product (8) shown in FIG; obtained from methyl 4-chlorophenylvalerate as shown in FIG. Figure 2 The product (9) shown in FIG; obtained from methyl 4-methoxyphenylvalerate as shown in FIG. Figure 2 The product (10) shown in FIG; obtained from 2-thiophene valeric acid methyl ester as shown in FIG. Figure 2 The product (11) shown in FIG; obtained from methyl 4-methoxycarbonylphenylvalerate as shown in FIG. Figure 2 The product shown in (12).

[0042] The biocatalyst constructed in this study successfully transcends its dependence on carboxylic acid substrates through directed evolution. Based on a novel oxidative cycle mechanism, directed evolution enabled the biocatalyst to utilize inexpensive and readily available H₂O₂ to catalyze the asymmetric synthesis of γ- and δ-phenyl lactones from the non-natural substrate phenyl methyl ester, leading to the asymmetric synthesis of γ- and δ-phenyl lactones. The catalytic potential of this whole-cell catalytic system was also explored. This approach offers the following advantages: 1) It overcomes the limitation of P450 BSβ, which can only catalyze natural substrates bearing carboxylic acid groups. 2) The mutant can more specifically catalyze non-natural substrates using inexpensive and readily available H₂O₂. 3) It provides a sound theoretical and experimental foundation for broadening the substrate spectrum of the P450 enzyme family, enhancing its synthetic application value in biosynthesis.

[0043] SEQ ID NO: 1 (amino acid sequence of fatty acid decarboxylase P450BSβ mutant): MetAspGluGlnIleProHisAspLysSerLeuAspAsnSerLeuThrLeuLeuLysGlu GlyTyrLeuPheIleLysAsnArgThrGluArgTyrAsnSerAspLeuPheGlnAlaArg LeuLeuGlyLysAsnPheIleCysMetThrGlyAlaGluAlaAlaLysValPheTyrAsp ThrAspArgPheGlnArgGlnAsnAlaLeuProLysArgValGlnLysSerMetPheGly ValAsnAlaIleHisValMetAspGlySerAlaHisIleHisArgLysMetLeuPheLeu SerLeuMetThrProProHisGlnLysArgLeuAlaGluLeuMetThrGluGluTrpLys AlaAlaVlaThrArgTrpGluLysAlaAspGluValValLeuPheGluGluAlaLysGlu IleLeuCysArgValAlaCysTyrTrpAlaGlyValProLeuLysGluThrGluValLys GluArgAlaAspAspPheIleAspMetGluAspAlaAsnGlyAlaValGlyProArgHis TrpLysGlyArgArgAlaArgProArgAlaGluGluTrpIleGluValMetIleGluAsp AlaArgAlaGlyLeuLeuLysThrThrSerGlyThrAlaLeuHisGluMetAlaPheHis ThrGlnGluAspGlySerGlnLeuAspSerArgMetAlaAlaIleGluLeuILEAsnVal LeuArgProIleValGlyIleSerTyrPheLeuValPheSerAlaLeuAlaLeuHisGlu HisProLysTyrLysGluTrpLeuArgSerGlyAsnSerArgGluArgGluMetPheVal GlnGluValArgArgTyrTyrProPheIleProPheLeuGlyAlaLeuValLysLysAsp PheValTrpAsnAsnCysGluPheLysLysGlyThrSerValLeuLeuAspLeuTyrGly ThrAsnHisAsoProArgLeuTrpAspHisProAspGluPheArgProGluArgPheAla GluArgGluGluAsnLeuPheAspMetIleProGlnGlyGlyGlyHisAlaGluLysGly HisArgCysProGlyGluGlyIleThrIleGluValMetLysAlaSerLeuAspPheLeu ValHisGlnIleGluTyrAspValProGluGlnSerLeuHisTyrSerLeuAlaArgMet ProSerLeuProGluSerGlyPheValMetSerGlyIleArgArgLysSer SEQ ID NO: 2 (nucleotide sequence of fatty acid decarboxylase P450BSβ mutant): SEQ ID NO: 3 (nucleotide sequence of wild-type fatty acid decarboxylase P450BSβ):

Claims

1. A fatty acid decarboxylase P450BSβ mutant, characterized in that The amino acid sequence of the fatty acid decarboxylase P450BSβ mutant is shown in SEQ ID NO:

1.

2. A nucleotide sequence, characterized in that The nucleotide sequence encodes the fatty acid decarboxylase P450BSβ mutant according to claim 1, and the nucleotide sequence is shown in SEQ ID NO:

2.

3. A method for preparing the fatty acid decarboxylase P450BSβ mutant according to claim 1, characterized in that: The following mutations were performed on the wild-type fatty acid decarboxylase P450BSβ: the valine at position 170 was mutated into glutamic acid, the glycine at position 290 was mutated into isoleucine, the alanine at position 246 was mutated into glycine, the glutamine at position 85 was mutated into histidine, the leucine at position 78 was mutated into methionine, the phenylalanine at position 173 was mutated into aspartic acid, and the glycine at position 86 was mutated into valine; the nucleotide sequence of the wild-type fatty acid decarboxylase P450BSβ is shown in SEQ ID NO:

3.

4. The method for preparing the fatty acid decarboxylase P450BSβ mutant according to claim 3, wherein The following steps are involved: S1 Construction of a plasmid expressing wild-type fatty acid decarboxylase P450BSβ; S2 constructed a saturation mutation library through seven rounds of site-directed saturation mutagenesis; S3 The constructed plasmid expressing the fatty acid decarboxylase P450BSβ mutant was transformed into Escherichia coli; Expression and purification of the S4 fatty acid decarboxylase P450BSβ mutant.

5. The method for preparing the fatty acid decarboxylase P450BSβ mutant according to claim 4, wherein The expression and purification of the fatty acid decarboxylase P450BSβ mutant comprises the following steps: S41: The strain constructed in S3 was cultured overnight in LB liquid medium, expanded in TB liquid medium, and induced by adding heme precursor δ-aminolevulinic acid and IPTG during the logarithmic growth phase, and then the bacteria were harvested by centrifugation; S42 The obtained bacteria were resuspended in a buffer solution, the cells were lysed by ultrasonication, and centrifuged to obtain the supernatant; S43 adds the supernatant to a nickel ion affinity column, allows the target protein with the His tag to bind to the nickel ions, removes impurities, elutes the target protein, and obtains a fatty acid decarboxylase P450BSβ mutant.

6. A method for asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone, characterized in that: The catalytic reaction is carried out using a derivative of a non-natural carboxylic acid phenyl methyl ester as a substrate, hydrogen peroxide as an oxidant, and the fatty acid decarboxylase P450BSβ mutant according to claim 1 as a catalyst.

7. The method for asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone according to claim 6, characterized in that: The γ-phenyl lactone is an S-type γ-phenyl lactone, and the δ-phenyl lactone is an S-type δ-phenyl lactone.

8. The method for asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone according to claim 6, characterized in that: The derivatives of the phenyl methyl ester include methyl 4-phenylbutyrate, methyl 5-phenylvalerate, methyl 4-fluorophenylbutyrate, methyl 4-fluorophenylvalerate, methyl 4-chlorophenylbutyrate, methyl 4-chlorophenylvalerate, methyl 4-methoxyphenylbutyrate, methyl 4-methoxyphenylvalerate, methyl 2-thiophenebutyrate, methyl 2-thiophenevalerate, methyl 4-methoxycarbonylphenylbutyrate, and methyl 4-methoxycarbonylphenylvalerate.

9. The method for asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone according to claim 6, characterized in that: The catalytic reaction is carried out at room temperature, and the reaction system is an aqueous buffer system with Triton X-100 and isopropanol as cosolvents.

10. Use of the fatty acid decarboxylase P450BSβ mutant according to claim 1 in the asymmetric synthesis of γ-phenyl lactone and δ-phenyl lactone.

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