Use of p450 enzymes in catalyzing squalene oxidation

Abstract

This invention belongs to the field of bioengineering technology and provides the application of P450 enzyme in catalyzing the oxidation of squalene. Several cytochrome P450 genes were cloned from the full-length transcriptome data of the medicinal plant *Alisma plantago-aquatica*. Preliminary screening of the gene sequences corresponding to the P450 enzyme was conducted using bioinformatics. Recombinant vectors were prepared using the screened genes and transformed into squalene-producing yeast for gene function verification. This demonstrated the oxidation effect of the P450 enzyme encoded by the AoCYP_3836 gene on squalene, providing a novel reaction mode different from the traditional 2,3-epoxidation reaction of squalene. The results show that the P450 enzyme encoded by the AoCYP_3836 gene can catalyze the oxidation of squalene to produce two new products: squalene-1,1'-diol and squalene-1-carboxylic acid-24-ol, providing a new pathway for the production of these squalene oxides.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of P450 enzyme in the catalytic oxidation of squalene. Background Technology

[0002] Squalene is a natural linear triterpenoid compound widely found in plants and animals, and is an important precursor in the biosynthesis of cholesterol, steroid hormones, and various triterpenoids. Squalene possesses antioxidant, anti-inflammatory, and immunomodulatory properties, and exhibits excellent biocompatibility. Its core applications are concentrated in three major areas: pharmaceuticals, cosmetics, and health supplements, while its applications are expanding into food and animal feed. In pharmaceuticals, squalene is a key adjuvant in vaccines against influenza and COVID-19, and can also assist in the treatment of tumors and cardiovascular diseases. It can also serve as a lipid-soluble drug delivery carrier. In cosmetics, squalene is commonly used to moisturize and repair the skin barrier, provide antioxidant and anti-aging benefits, and promote the penetration and absorption of other active ingredients, making it a core ingredient in high-end skincare. In health supplements, squalene is mainly used for immune regulation, liver and cardiovascular protection, and fatigue relief, and is a common ingredient in daily and sports nutrition supplements. Furthermore, squalene can also be used as a high-end food antioxidant preservative, an aquaculture feed additive, and a precursor for high-quality biofuels, demonstrating diverse application potential. The global squalene market reached $151.4 million in 2025 and is projected to grow to $232.9 million by 2035. Of this, the animal-derived squalene market grew from $180 million in 2021 to $260 million in 2025, representing a CAGR of 7.8%, demonstrating steady growth. However, squalene exhibits poor stability and solubility, necessitating research into its oxidation products to achieve better stability and solubility. Currently, the only commonly available naturally extracted squalene oxidation product is Squalen-1-ol (Nat Prod Res. 2022,36:2154-2157), while chemical synthesis usually requires the use of multiple heavy metal catalysts and has poor selectivity and many byproducts. Therefore, it is necessary to use biosynthetic technology to achieve squalene oxidation. The most common squalene oxidation product is 2,3-epoxysqualene, which is a key intermediate in the downstream biosynthetic pathways of steroids (such as ergosterol and cholesterol) and triterpenoids (such as ginsenosides and oleanolic acid). Xin Song et al. overexpressed Ganoderma lucidum CYP505D13 in Saccharomyces cerevisiae YL-T3 and isolated and identified three oxides from the fermentation extract: 2,3;22,23-diepoxysqualene, 8-hydroxy-2,3;22,23-diepoxysqualene, and 4,8-dihydroxy-22,23-oxysqualene (Bioresour. Bioprocess. 2019, 6:19). However, the dimethyl oxidation reaction of squalene has not been reported.

[0003] Therefore, to address the above problems, this invention screens a novel, efficient, and highly selective P450 enzyme that can specifically oxidize the terminal of squalene to form corresponding alcohol and acid structures. Based on this, it provides the application of P450 enzyme in catalytic squalene oxidation, which can specifically and rapidly convert squalene into specific oxidation products and has good catalytic performance and stability, providing a new solution for the green and sustainable production of squalene oxidation products. Summary of the Invention

[0004] The purpose of this invention is to provide the application of P450 enzyme in catalytic oxidation of squalene, utilizing the heterologous expression of specific P450 enzyme by microbial strains to catalyze squalene, thus providing a new approach and means for preparing novel squalene oxidation products.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] This invention provides the application of P450 enzyme in the preparation of squalene oxidation products. The P450 enzyme is derived from the root of Alisma plantago-aquatica. The P450 enzyme directly catalyzes the formation of squalene oxidation products from squalene. The squalene oxidation products are two products: squalene C-1, 24-hydroxylated and squalene C-1-carboxylated and C-24-hydroxylated.

[0007] Preferably, the encoding gene corresponding to the P450 enzyme is AoCYP_3836, which belongs to the CYP86 subfamily. The gene sequence of AoCYP_3836 is shown in SEQ ID NO: 1, and the amino acid sequence of the P450 enzyme is shown in SEQ ID NO: 2.

[0008] Preferably, the two products of squalene C-1,24-hydroxylation and squalene C-1-carboxylation and C-24-hydroxylation are named squalene-1,1'-diol and squalene-1-carboxylic acid-24-ol.

[0009] Preferably, the source of the P450 enzyme is:

[0010] (1) The roots of Alisma plantago-aquatica were sent for complete full-length transcriptome sequencing to screen for gene sequences corresponding to P450 enzymes.

[0011] (2) Prepare recombinant vectors from the selected sequences;

[0012] (3) The recombinant vector was transformed into Saccharomyces cerevisiae, and the effective P450 enzyme sequence was screened for catalytic squalene oxidation, and the function of the P450 enzyme was identified.

[0013] The beneficial effects of this invention are:

[0014] This invention discovers and identifies an enzyme capable of catalyzing the terminal oxidation of squalene, providing a novel reaction mode different from the traditional 2,3-epoxidation reaction of squalene. Based on the discovered enzyme element, a method for heterologous production of squalene oxidation products using microbial strains is provided. The results show that the P450 enzyme encoded by the AoCYP_3836 gene can catalyze the oxidation of squalene to generate two new products, namely squalene-1,1'-diol and squalene-1-carboxylic acid-24-ol, providing a new route for the production of these squalene oxides. Attached Figure Description

[0015] Figure 1 These are the LC-MS detection results from this invention;

[0016] Figure 2 This is a structural diagram of squalene-1,1'-diol in this invention;

[0017] Figure 3 This is a structural diagram of squalene-1-carboxylic acid-24-ol in this invention;

[0018] Figure 4 This is the pathway by which squalene is catalyzed by the P450 enzyme encoded by the AoCYP_3836 gene to generate squalene-1,1'-diol and squalene-1-carboxylic acid-24-ol in this invention. Detailed Implementation

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] Example

[0023] 1 Experimental Methods

[0024] 1.1 RNA extraction and sequencing from Alisma plantago-aquatica roots

[0025] The roots of the plant Alisma plantago-aquatica (Fushun, Liaoning) were sent to a company (BenaGene) for full-length transcriptome sequencing (third-generation sequencing) to obtain its transcriptome data.

[0026] 1.1.1 Experimental Procedures for Plant RNA Extraction and Library Construction

[0027] (1) After grinding the plant leaves with liquid nitrogen, collect ≤100 mg of plant sample into a centrifuge tube, add 500 µL RBBuffer / 10 µL β-mercaptoethanol, and immediately vortex to mix.

[0028] (2) Insert the gDNA Filter column into a 2 mL collection tube, transfer the mixture to the gDNA Filter column, and centrifuge at 14,000×g for 5 min at room temperature.

[0029] (3) Transfer the filtrate to a new 1.5 mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, vortex at high speed for 20 seconds to mix well. If a precipitate forms, pipette it 10-15 times.

[0030] (4) Insert the HiBind® RNA Mini binding column into a 2 mL collection tube, transfer 700 µL of the mixture from step 3 into the HiBind® RNA Mini binding column, centrifuge at 12,000×g for 1 min at room temperature, and discard the filtrate;

[0031] (5) Repeat step 4 until all the mixture has been transferred through the column;

[0032] (6) Place the HiBind® RNA Mini binding column into the same 2 mL collection tube, add 400 µL RWFWash Buffer, centrifuge at 10,000×g for 30 s at room temperature, and discard the filtrate;

[0033] (7) Place the HiBind® RNA Mini binding column into the same 2 mL collection tube, add 500 µL RNAWash Buffer II (diluted with anhydrous ethanol beforehand), centrifuge at 10,000×g for 30 s at room temperature, and discard the filtrate;

[0034] (8) Repeat step 7;

[0035] (9) Insert the HiBind® RNA Mini binding column into the same 2 mL collection tube, centrifuge at room temperature at maximum speed for 2 min, and then spin dry the HiBind® RNA Mini binding column matrix;

[0036] (10) Place the HiBind® RNA Mini binding column into a new 1.5 mL centrifuge tube, take 50-100 µL of DEPC Water, accurately add it to the center of the HiBind® RNA Mini binding column membrane, incubate at room temperature for 2 min, centrifuge at 10,000×g at room temperature for 1 min, and elute the RNA.

[0037] 1.1.2 Sequencing Experimental Procedure

[0038] (1) Full-length cDNA of mRNA was synthesized using the Clontech SMARTer PCR cDNA Synthesis Kit. Eukaryotic mRNA has a polyA tail at the 3' end. Primers with Oligo dT were used to pair with polyA bases to form AT bases, which were then used as primers for reverse synthesis of cDNA. Primers were also added to the ends of the reverse synthesized full-length cDNA. The obtained full-length cDNA was amplified by PCR, and the product was purified using PB magnetic beads and quantified using Qubit 3.0.

[0039] (2) Use PB magnetic beads to purify the amplified full-length cDNA and remove some small cDNA fragments less than 1kb.

[0040] (3) End repair of full-length cDNA and ligation of SMRT dumbbell adapter.

[0041] (4) The fragments without ligated adapters were digested with exonuclease and purified again using PB magnetic beads to obtain the sequencing library.

[0042] (5) After the library is constructed, the library quality is tested. Sequencing can only be performed after the test results meet the requirements.

[0043] 1.2 P450 enzyme verification

[0044] Plasmid construction and sequence validation: The attB sequence was added to both sides of the open reading frame (ORF) of P450 with complete data by polymerase chain reaction (PCR), and then constructed into the entry vector pDONR221 by BP reaction. After that, it was constructed into the expression vector pAG416GPD-ccdB by LR reaction. The cytochrome P450 reductase AoCPR derived from Alisma plantago-aquatica was similarly constructed into the yeast expression vector (p413TEF). The reaction products were transformed into chemically competent Escherichia coli DH10B and plated on LB plates with appropriate antibiotics. Positive clones were verified by Sanger sequencing, covering the full-length ORF and the linker region.

[0045] 1.3 Yeast Transformation and Strain Validation

[0046] Yeast transformation was performed using the Frozen-EZ Yeast Transformation II (Zymo Research, T2001) kit, following the instructions in principle, with minor adjustments made for multi-plasmid co-transformation.

[0047] Preparation of competent yeast cells: 10 mL of JCR27 YPD medium was incubated at 30 °C until mid-log phase (OD600 0.8–1.0). Cells were collected by centrifugation at 500 × g for 4 min and washed once with 10 mL of Frozen-EZ Yeast Solution 1. The pellet was resuspended in 1 mL of Frozen-EZ Yeast Solution 2 to obtain competent cells. Competent cells can be used immediately or stored at ≤-70 °C.

[0048] Yeast transformation: For each transformation, take 50 µL of competent cells and mix them with 0.2–1 µg of plasmid 1 (pAG416GPD-AoCYP3836) DNA + plasmid 2 (p413TEF-AoCPR) DNA (total DNA volume <5 µL). Add 500 µL of Frozen-EZ Yeast Solution 3 and mix thoroughly. Incubate at 30°C for 45 min, mixing vigorously 2–3 times during incubation. Spread 50–150 µL of the transformation mixture directly onto the corresponding SD deficient plate and incubate at 30°C for 2–4 days until colonies appear.

[0049] Colony isolation and validation: After 2–4 days of plate culture, three single colonies were picked and cultured overnight (18 h) in SD liquid medium on a shaker (30℃, 220 rpm). Then, 60 µL was transferred to 3 mL of SD auxotrophic liquid medium and cultured for 24 h on a shaker (30℃, 220 rpm). The bacterial culture was then centrifuged at 3000 rpm / min for 2 min to collect the cells. 3 mL of SD auxotrophic liquid medium containing 2% galactose and 2% raffinose was added and cultured for 24 h on a shaker (30℃, 220 rpm). The culture was then centrifuged at 4500 rpm / min for 5 min to collect the cells. 400 µL of methanol and 100 µL of acetone were added, and the mixture was extracted using a homogenizer. The extract was evaporated to dryness in a concentrator, and then reconstituted with 40 µL of methanol. The mixture was sonicated for 5 min and then centrifuged at 13000 rpm / min for 5 min. After a period of time, the supernatant was collected into a sample vial, and its components were then analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0050] 2. Experimental Results

[0051] Based on the complete sequencing results of Alisma plantago-aquatica roots, 38 gene sequences corresponding to P450 enzymes were screened out. These genes were then constructed into pAG416GPD-ccdB to prepare recombinant vectors. These recombinant vectors were then transformed into yeast cells, and the cells were collected, extracted, concentrated, and analyzed by LC-MS. It was found that only one P450 gene (AoCYP_3836) produced squalene oxidation products after transformation into yeast. Figure 1Compounds 1 and 2 were obtained by silica gel column chromatography and semi-preparative separation. Their structures were identified by NMR 600M as oxidation products of squalene. Figure 2 and Figure 3 The NMR data are as follows:

[0052] Compound 1 ( Figure 2 ): 1 H NMR (600 MHz, Chloroform-d) δ 5.39 (tq, J = 7.0, 1.4Hz, 2H, H-3, 3'), 5.16 – 5.14 (m, 2H, H-7, 7'), 5.14 – 5.10 (m, 2H, H-11,11'), 3.99 (d, J = 1.4 Hz, 4H, H-1, 1'), 2.13 (q, J = 7.4 Hz, 4H, H-4, 4'), 2.08 (q, J = 7.3 Hz, 4H, H-8, 8'), 2.00 (ddt, J = 15.4, 10.0, 5.2 Hz, 12H, H-5, 5', 9, 9', 12, 12'), 1.67 (d, J = 1.4 Hz, 6H, H-13, 13'), 1.60 (d, J = 1.5Hz, 12H, H-14, 14', 15, 15').

[0053] 13 C NMR (151 MHz, CDCl3) δ 135.21 (C-2, 2'), 134.84 (C-10, 10'), 134.69 (C-6, 6'), 126.35 (C-3, 3'), 124.72 (C-7, 7'), 124.49 (C-11, 11'),69.23 (C-1, 1'), 39.87 (C-9, 9'), 39.45 (C-5, 5'), 28.41 (C-12, 12'), 26.78(C-8, 8'), 26.40 (C-4, 4'), 16.20 (C-14, 14'), 16.14 (C-15, 15'), 13.84 (C-13, 13').

[0054] Compound 2 ( Figure 3 ): 1H NMR (600 MHz, Chloroform-d) δ 6.84 (t, J = 7.4 Hz,1H, H-3), 5.42 – 5.36 (m, 1H, H-22), 5.17 – 5.10 (m, 4H, H-7, 11, 14, 18), 4.00 (s, 2H, H-24), 2.28 (q, J = 7.6 Hz, 2H, H-4), 2.16 – 2.05 (m, 8H, H-8,16, 17, 21), 2.04 – 1.96 (m, 10H, H-5, 9, 12, 13, 20), 1.83 (s, 3H, H-25),1.66 (s, 3H, H-30), 1.61 (s, 3H, H-26), 1.60 (s, 9H, H-27~29).

[0055] 13 C NMR (151 MHz, CDCl3) δ 172.46 (C-1), 144.40 (C-3), 135.17 (C-23), 135.09 (C-6), 134.72 (C-10), 134.65 (C-19), 133.83 (C-15), 127.20 (C-2),126.43 (C-22), 125.40 (C-7), 124.74 (C-11), 124.58 (C-14), 124.50 (C-18), 69.20 (C-24), 39.86 (C-5), 39.76 (C-9), 39.42 (C-20), 38.26 (C-13, 16), 28.39(C-12), 27.66 (C-4), 26.73 (C-17, 21), 26.36 (C-8), 16.17 (C-27~29), 16.12(C-26), 13.84 (C-30), 12.34 (C-25).

[0056] Table 1. Compound 2 1 H NMR (600 MHz), 13 C NMR (151 MHz), COSY and HMBC data

[0057]

[0058] Based on the structural identification results, compound 1 was named squalene-1,1'-diol ( Figure 2Compound 2 was named squalene-1-carboxylic acid-24-ol. Figure 3 This recombinant vector was named pAG416GPD-AoCYP3836, the gene was named AoCYP_3836, and the gene sequence was SEQ ID NO: 1.

[0059]

[0060] The amino acid sequence of the P450 enzyme encoded by this gene is SEQ ID NO: 2: MTPFNSTGSLTAESLPPLPAAAAETLTFFVHKLLHELHAVELFLALLLFITIHSLRQRKPQGLPSWPLVGmLPSLIFALNGDNIYEWLTEVLRRRGGTFTFRGPAFTSLHCVLTADDPRNLEHILKTRFPSFPKGPYFR DAVRDLLGEGIFGVDGEAWRRQRRAASLEFHSAGFRAMTAESLNELVHGRLIPVLDGVCETKEAVDLQDVLLRLTFDNVCMIAFGADPGCLSPGLPNIPFAKAFEEATEATVVRFVTPTAVWRALRFLRLGHERKLRDCLRDV DEFAYGVIQTRKKELQSADPTVADEDSASNPSWKRNKSDLLTVFLKGRDEDGREYGEEFLRDVCVNFILAGRDTSSVALAWFFWLLDSHREVEQEVLRELCGILAQRNDKETEQGEVLGSADHREVVFRPEELKKMEYLQAAL SEALRLYPSVPVDHKEVMEDEVFPDGTVLKKGTKVIYAIYAMGRMESIWGDDCLEFRPNRWLKDGRFVSESAYRFSAFNGGPRLCLGKDFAYYQMKFVAAAmLLRYRVRVVPGHPVAPKLALTMYMKHGLRVTLEKREAGELAR

[0061] The process of squalene oxidation by the P450 enzyme encoded by AoCYP_3836 is as follows: Figure 4 As shown, the methyl group at C-1 and 24 of squalene is first oxidized to generate an alcohol (squalene-1,1'-diol), and then the hydroxyl group at C-1 is continuously oxidized to generate a second carboxyl group (squalene-1-carboxylic acid-24-ol).

[0062] In summary, this invention verifies the oxidation effect of the P450 enzyme on squalene by screening the gene sequence corresponding to the P450 enzyme and preparing a recombinant vector from the screened gene and transferring it into yeast. The results show that the P450 enzyme can catalyze the oxidation of squalene to generate two novel products, providing a new approach for the production of squalene oxide.

[0063] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of P450 enzyme in the preparation of squalene oxidation products, characterized in that, The P450 enzyme is derived from the roots of Alisma plantago-aquatica. The P450 enzyme directly catalyzes the formation of squalene oxidation products, which are two products: squalene C-1, 24-hydroxylated and squalene C-1-carboxylated and C-24-hydroxylated.

2. The application according to claim 1, characterized in that, The encoding gene for the P450 enzyme is AoCYP_3836, which belongs to the CYP86 subfamily. The gene sequence of AoCYP_3836 is shown in SEQ ID NO: 1, and the amino acid sequence of the P450 enzyme is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The two products of squalene C-1, 24-hydroxylation and squalene C-1-carboxylation and C-24-hydroxylation are named squalene-1,1'-diol and squalene-1-carboxylic acid-24-ol.

4. The application according to claim 1, characterized in that, The specific source of the P450 enzyme is as follows: (1) The roots of Alisma plantago-aquatica were sent for complete full-length transcriptome sequencing to screen for gene sequences corresponding to P450 enzymes. (2) Prepare recombinant vectors from the selected sequences; (3) The recombinant vector was transformed into Saccharomyces cerevisiae, and the effective P450 enzyme sequence was screened for catalytic squalene oxidation, and the function of the P450 enzyme was identified.

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