Cytochrome p450 enzyme mutant and method for catalyzing synthesis of betulinic acid by the mutant
By mutating the amino acid of the rosemary-derived cytochrome P450 enzyme RoCYP01, its efficiency in catalyzing the conversion of betulinol to betulinic acid was improved, solving the problem of low betulinic acid synthesis efficiency in existing technologies and achieving a highly efficient biosynthesis effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2025-01-23
- Publication Date
- 2026-06-02
AI Technical Summary
The efficiency and yield of microbial heterologous synthesis of betulinic acid in the existing technology are low, mainly because the rate-limiting enzyme lupeol C28 oxidase (cytochrome P450) has low catalytic activity.
By protein engineering the RoCYP01 enzyme derived from rosemary, the first 30 amino acids were truncated, and mutations were made at leucine position 78 and/or tryptophan position 106 to obtain the mutant RoCYP01-W106A/L78A, thereby enhancing its catalytic activity.
The mutant RoCYP01-W106A/L78A significantly improved the efficiency of betulinic acid production from betulinol, with a relative increase in enzyme activity of 198%, meeting the needs of industrial production.
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Figure CN119709665B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a cytochrome P450 enzyme mutant and a method for catalyzing the synthesis of betulinic acid, belonging to the field of biocatalytic synthesis technology. Background Technology
[0002] Betulinic acid is a naturally occurring pentacyclic triterpenoid compound widely found in the bark of birch trees. Due to its significant biological activity, betulinic acid has become an important target compound in drug development and phytochemical research. Studies have shown that betulinic acid possesses anticancer, antiviral, anti-inflammatory, antioxidant, and antimalarial biological activities, thus attracting attention from the pharmaceutical industry. Although betulinic acid can be extracted from birch bark, its low abundance in the plant and high extraction costs prevent it from meeting market demand. Therefore, developing efficient and sustainable methods for the synthesis of betulinic acid is of great significance.
[0003] With the rapid development of synthetic biology and metabolic engineering, the biosynthesis of betulinic acid using microorganisms or plant cell factories has attracted widespread attention from the scientific and industrial communities. Microbial fermentation has advantages such as mild reaction conditions, low cost, and low pollution. The heterologous synthesis of betulinic acid by microorganisms begins with the key terpene synthesis precursor IPP / DMAPP, and is catalyzed by ERG20 / 9 / 1, LUPS, and CYP / CPR to obtain betulinic acid. Although previous studies have achieved certain results in the synthesis of betulinic acid in microorganisms such as yeast using the natural MVA / MEP pathway, the synthesis efficiency and yield are still low and cannot meet the needs of industrial production. The main reason is the low catalytic activity of its rate-limiting enzyme, lupeol C28 oxidase (cytochrome P450). Summary of the Invention
[0004] To address the aforementioned problems, the present invention aims to provide a mutant of lupeol C28 oxidase (cytochrome P450) RoCYP01 for betulinic acid biosynthesis.
[0005] This invention provides a cytochrome P450 mutant, which uses RoCYP01 derived from rosemary as the parent, shortens the first 30 amino acids, and mutates leucine at position 78 and / or tryptophan at position 106.
[0006] In one embodiment, the mutation is to mutate leucine at position 78 to alanine, and / or to mutate tryptophan at position 106 to alanine.
[0007] In one embodiment, the amino acid sequence of the cytochrome P450 enzyme RoCYP01 parent is shown in SEQ ID NO.1.
[0008] In one embodiment, the amino acid sequence of the RoCYP01 mutant W106A / L78A is shown in SEQ ID NO.3.
[0009] The present invention also provides a gene encoding the above-mentioned cytochrome P450 enzyme RoCYP01 mutant.
[0010] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.4.
[0011] The present invention also provides an expression vector carrying the RoCYP01 mutant gene of the cytochrome P450 enzyme.
[0012] In one embodiment, the vector includes, but is not limited to, the pESC-URA plasmid.
[0013] The present invention also provides recombinant bacteria expressing the above-mentioned cytochrome P450 enzyme RoCYP01 mutant.
[0014] In one embodiment, the recombinant bacteria uses Saccharomyces cerevisiae WAT11 as the host and pESC-URA plasmid as the vector to express the cytochrome P450 enzyme RoCYP01 mutant.
[0015] The present invention also provides a catalyst containing the RoCYP01 mutant of the cytochrome P450 enzyme.
[0016] In one embodiment, the catalyst includes, but is not limited to, the recombinant bacterial cell or its cell lysate, and microsomes containing the cytochrome P450 enzyme RoCYP01 mutant.
[0017] The present invention also provides a method for catalytic synthesis of betulinic acid, wherein the cytochrome P450 enzyme RoCYP01 mutant or the catalyst is used to carry out the catalytic reaction in a system containing betulinol.
[0018] In one embodiment, the reaction system contains betulin, NADPH, and microsomes containing the RoCYP01 mutant of the cytochrome P450 enzyme.
[0019] In one embodiment, the pH of the reaction system is 6.0-9.0.
[0020] In one embodiment, the concentration of betulin in the reaction system is 2-4 mM, and the concentration of NADPH is 1-3 mM.
[0021] In one embodiment, the betulin is dissolved in DMSO at a concentration of 5-10%.
[0022] In one embodiment, the volume ratio of anhydrous ethanol: Tween 80: betulin solution in the reaction system is 8:1:2.25.
[0023] This invention also protects the use of the above-mentioned cytochrome P450 enzyme RoCYP01, the above-mentioned gene, the above-mentioned recombinant bacteria, or the above-mentioned method in the preparation of products containing betulinic acid.
[0024] Beneficial effects:
[0025] This invention modifies the RoCYP01 protein from rosemary to obtain the optimal mutant RoCYP01-W106A / L78A. This mutant significantly improves the efficiency of catalyzing the conversion of betulinol to betulinic acid, with a relative enzyme activity 198% higher than that of the wild type. It has great potential in the bioconversion of betulinol to betulinic acid or in the fermentation synthesis of betulinic acid using glucose as a substrate. Attached Figure Description
[0026] Figure 1 This is the reaction formula for the synthesis of betulinic acid from betulinol catalyzed by cytochrome P450 enzyme (lupinol C28 oxidase) in this invention.
[0027] Figure 2 HPLC chromatograms of the synthesis of betulinic acid from betulinol oxidase catalyzed by wild-type and mutant lupeol C28 oxidase.
[0028] Figure 3 The standard curve for betulinic acid is shown (the horizontal axis represents the betulinic acid solubility in mg / L, and the vertical axis represents the response value).
[0029] Figure 4 Comparison of the activities of wild-type and mutant lupeol C28 oxidase in catalyzing the synthesis of betulinic acid from betulinol. Detailed Implementation
[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.
[0032] Saccharomyces cerevisiae WAT11 is a commercial strain, and pESC-URA is a commercial plasmid.
[0033] The culture media involved in the following examples:
[0034] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder.
[0035] SD-URA liquid medium: 20% glucose, yeast basic nitrogen source (YNB) 1.7 g / L ammonium sulfate, L-arginine 0.1 g / L, L-cysteine 0.1 g / L, L-lysine 0.1 g / L, L-threonine 0.1 g / L, L-asparagine 0.05 g / L, L-isoleucine 0.05 g / L, L-phenylalanine 0.05 g / L, L-proline 0.05 g / L, L-serine 0.05 g / L, L-tyrosine 0.05 g / L, L-valine 0.05 g / L, L-methionine 0.05 g / L, L-tryptophan 0.1 g / L, L-histidine 0.1 g / L, L-leucine 0.1 g / L, adenine 0.1 g / L.
[0036] YPGE medium: 10 g / L yeast extract, 10 g / L peptone, 5 g / L glucose and 3% (v / v) ethanol.
[0037] Detection method of betulinic acid and betulinol: HPLC analysis was performed using a CORTECS C18 1.6μm column (2.1×50mm). The HPLC conditions were: 70% acetonitrile + 0.1% formic acid, 30% water + 0.1% formic acid for 35 min, flow rate 0.3 mL / min; column temperature 35℃; UV detection wavelength 203 nm.
[0038] Enzyme catalytic efficiency is defined as the ratio of the amount of betulinic acid produced by the enzyme or mutant catalyzing betulinol under the same catalytic conditions.
[0039] Example 1: Gene acquisition and expression vector construction of cytochrome P450 enzyme (lupinol C28 oxidase)
[0040] The protein sequence of rosemary-derived cytochrome P450 enzyme (lupinol C28 oxidase) was downloaded from Genbank (Genbank accession number: MK592864.1), the first 30 amino acids were truncated, and codon preference optimization and gene synthesis of Saccharomyces cerevisiae were performed by Yixin Biotechnology Co., Ltd. The nucleotide sequence of the synthesized gene is shown in SEQ ID NO.2, and it was ligated to the GAL1 / 10 site of the vector pESC-URA to obtain the recombinant plasmid PESC-RoCYP01.
[0041] Using recombinant plasmid PESC-RoCYP01 as a template, full plasmid PCR was performed sequentially using primers L78A-1 / L78A-2 and W106A-1 / W106A-2 (primers are shown in Table 1). Leucine at position 78 of RoCYP01 was mutated to alanine, and tryptophan at position 106 was mutated to alanine to construct the recombinant plasmid PESC-RoCYP01-W106A / L78A expressing the mutant shown in SEQ ID NO.3.
[0042] Table 1 Primer names and primer sequences
[0043]
[0044]
[0045] Example 2: Construction of recombinant strains, induction of target protein expression, and microsomal construction
[0046] The recombinant plasmids PESC-RoCYP01 and PESC-RoCYP01-W106A / L78A constructed in Example 1 were sequenced and verified, and then transformed into Saccharomyces cerevisiae WAT11 competent cells, respectively. The transformants were verified in a deficient medium, and the correctly verified strains were named Saccharomyces cerevisiae PESC-RoCYP01 and Saccharomyces cerevisiae PESC-RoCYP01-W106A / L78A, respectively.
[0047] 1) Inoculate about 4 mL of the activated single colony on the SD deficient plate into liquid SD deficient medium and grow it in an incubator at 28℃ and 250 rpm for 48 h.
[0048] 2) Take 2 ml and inoculate it into 500 ml of LYPGE medium, and incubate overnight at 28℃ and 250 rpm for 12 h.
[0049] 3) Add galactose to a final concentration of 2% (v / v) to induce incubation, and culture for 12 h.
[0050] 4) When galactose induction is complete, centrifuge at 5000g for 5 minutes to obtain cells.
[0051] 5) Resuspend the cell particles in 0.5 g / mL TEK buffer (50 mM Tris HCl, 1 mM EDTA, 100 mM KCl, pH 7.4) and incubate at room temperature for 5 minutes.
[0052] 6) Centrifuge the cells at 5000g for 5 minutes and resuspend them in 50mL of ice-cold TES B buffer (50mM TrisHCl, 1mM EDTA, 600mM sorbitol, pH 7.4).
[0053] All of the following steps are performed at 4°C or on ice.
[0054] 7) Disrupt cells and collect the lysate using a high-pressure homogenizer. The pressure in the high-pressure homogenizer should be 900-1000 bar. Then centrifuge the lysate at 18000g at 4°C for 10 minutes to form large cell fragments. Aspirate the supernatant and discard the precipitate.
[0055] 8) Add 150 mM NaCl to the final volume and 0.1 g / mL polyethylene glycol (PEG)-4000 to the final concentration to precipitate microsomes. After incubation on ice for about 1 h with periodic mixing, collect the microsomal protein fraction by centrifugation at 10000 g for 10 min at 4 °C.
[0056] 9) Discard the supernatant and resuspend the microparticles in 1 mL of ice-cold TEG storage buffer (50 mM TrisHCl, 1 mM EDTA, 20% (v / v) glycerol, pH 7.4).
[0057] Example 3: The reaction of cytochrome P450 enzyme RoCYP01 and its mutants catalyzing the synthesis of betulinic acid from betulinol.
[0058] The RoCYP01 and its mutant obtained in Example 2 were used in the synthesis of betulinic acid from betulinol. The catalytic reaction system (500 μL) is shown in Table 2.
[0059] Table 2 500μL reaction system
[0060]
[0061]
[0062] The system described in Table 2 was reacted at 30℃ and 220 rpm for 24 h. Immediately after the reaction was completed, 800 μL of ice-cold methanol was added to terminate the reaction. Then, the mixture was centrifuged at 20000×g for 5 min, and the supernatant was filtered through a 0.22 μm organic filter before being used for HPLC analysis.
[0063] Liquid phase analysis revealed the formation of a novel product in the reaction system with the same elution time as betulinol. Figure 2 This demonstrates that the cytochrome P450 enzyme RoCYP01 can catalyze the conversion of betulinol to betulinic acid. Furthermore, the betulinol yield was calculated using the peak area in the liquid chromatography phase (standard curve shown in Figure 1). Figure 3As shown in the figure, the catalytic efficiency of the mutant RoCYP01-W106A / L78A is increased by 198% compared with the wild type. Figure 4 The catalytic activity is greatly improved.
[0064] Comparative Example 1:
[0065] The specific implementation method is the same as in Examples 1 to 3, except that the mutants shown in Table 3 were also constructed, and the results are shown in Table 3.
[0066] Table 3 Catalytic efficiency of different mutants relative to WT
[0067] mutant relative catalytic efficiency WT 1.00 W106A 1.67 L78A 1.66 F359A 1.22 I286A 1.05 L209A 0.75 I210A 0.91 L355A 0.86 P460A 0.93 W106A / L78A 1.98 W106A / F359A 1.01 L78A / F359A 0.98 L78A / F359A / W106A 1.15
[0068] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A cytochrome P450 mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.
1. Ro Using CYP01 as the parent, leucine at position 78 is mutated to alanine, and / or tryptophan at position 106 is mutated to alanine; and the first 30 amino acids are truncated.
2. The gene encoding the cytochrome P450 mutant of claim 1.
3. An expression vector carrying the gene of claim 2.
4. Recombinant bacteria expressing the cytochrome P450 mutant of claim 1.
5. A recombinant brewing yeast, characterized in that, Using Saccharomyces cerevisiae WAT11 as the host and pESC--URA plasmid as the vector, the cytochrome P450 mutant described in claim 1 was expressed.
6. A catalyst containing the cytochrome P450 mutant of claim 1.
7. The catalyst according to claim 6, characterized in that, It includes the recombinant bacterial cells or their cell lysates as described in claim 4, or microsomes containing the cytochrome P450 mutant as described in claim 1.
8. A method for catalytic synthesis of betulinic acid, characterized in that, The catalytic reaction was carried out in a system containing betulin with the cytochrome P450 mutant of claim 1 or the catalyst of claim 6 or 7.
9. The use of the cytochrome P450 mutant of claim 1, or the gene of claim 2, or the recombinant bacteria of claim 4, or the recombinant Saccharomyces cerevisiae of claim 5, or the catalyst of claim 6 or 7, or the method of claim 8 in the preparation of a product containing betulinic acid.