Cytochrome P450 enzyme mutant and method for catalytically synthesizing betulinic acid through cytochrome P450 enzyme mutant

Abstract

The invention discloses a cytochrome P450 enzyme mutant and a method for catalytically synthesizing betulinic acid through the cytochrome P450 enzyme mutant, and relates to the technical field of genetic engineering and biological enzyme catalysis. According to the invention, reported RoCYP01 from rosemary is used as a mutation template, and a substrate channel for a substrate and a product to enter and exit from an enzyme catalysis center is changed through site-specific mutagenesis, so that the mutant with improved efficiency of catalyzing betulinic acid synthesis from betulin is obtained. Under the same catalytic condition, compared with a wild type, the mutant disclosed by the invention has the advantages that the catalytic activity is improved by 198%, and the mutant has important guiding significance for constructing betulinic acid yeast engineering bacteria.

Description

Technical Field

[0001] The invention relates to a cytochrome P450 enzyme mutant and a method for catalytically synthesizing betulinic acid thereof, belonging to the technical field of biocatalytic synthesis. Background Art

[0002] Betulinic acid is a naturally occurring pentacyclic triterpenoid compound that is 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 has anti-cancer, anti-viral, anti-inflammatory, antioxidant, and anti-malarial biological activities, which has attracted the attention of the medical community. Although betulinic acid can be extracted from the bark of birch trees, its low content in plants and high extraction process cost make it unable to meet market demand. Therefore, it is of great significance to develop an efficient and sustainable method for synthesizing betulinic acid.

[0003] With the rapid development of synthetic biology and metabolic engineering, the biosynthesis of betulinic acid using microorganisms or plant cell factories has received widespread attention from the scientific and industrial communities. Microbial fermentation has the advantages of mild reaction conditions, low cost, and less pollution. The pathway for heterologous microbial synthesis of betulinic acid starts with IPP / DMAPP, a key precursor for terpenoid synthesis, and betulinic acid is obtained through the catalysis of ERG20 / 9 / 1, LUPS, and CYP / CPR. Although previous studies have achieved certain results in the synthesis of betulinic acid in microorganisms such as yeast using the natural MVA / MEP pathway, its synthesis efficiency and yield are still relatively low and cannot meet the needs of industrial production. The main reason is that the catalytic activity of its rate-limiting enzyme, lupeol C28 oxidase (cytochrome P450), is low. Summary of the invention

[0004] In order to solve the above problems, the object of the present invention is to provide a mutant of lupeol C28 oxidase (cytochrome P450) RoCYP01 for betulic acid biosynthesis.

[0005] The invention provides a cytochrome P450 mutant, which takes RoCYP01 derived from rosemary as a parent, truncates the first 30 amino acids, and mutates the 78th leucine and / or the 106th tryptophan.

[0006] In one embodiment, the mutation is a mutation of leucine at position 78 to alanine, and / or a mutation of tryptophan at position 106 to alanine.

[0007] In one embodiment, the amino acid sequence of the cytochrome P450 enzyme RoCYP01 parent is shown as 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 cytochrome P450 enzyme RoCYP01 mutant.

[0010] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.4.

[0011] The present invention also provides an expression vector carrying the cytochrome P450 enzyme RoCYP01 mutant gene.

[0012] In one embodiment, the vector includes but is not limited to a pESC-URA plasmid.

[0013] The present invention also provides a recombinant bacterium expressing the above cytochrome P450 enzyme RoCYP01 mutant.

[0014] In one embodiment, the recombinant bacteria uses Saccharomyces cerevisiae WAT11 as a host and pESC-URA plasmid as a vector to express the cytochrome P450 enzyme RoCYP01 mutant.

[0015] The present invention also provides a catalyst containing the cytochrome P450 enzyme RoCYP01 mutant.

[0016] In one embodiment, the catalyst includes but is not limited to the recombinant bacterial cells or their cell lysates, and microsomes containing the cytochrome P450 enzyme RoCYP01 mutant.

[0017] The present invention also provides a method for catalytically synthesizing betulinic acid, wherein the cytochrome P450 enzyme RoCYP01 mutant or the catalyst is used to carry out a catalytic reaction in a system containing betulin.

[0018] In one embodiment, the reaction system contains betulin, NADPH and microsomes containing the cytochrome P450 enzyme RoCYP01 mutant.

[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] The present invention also protects the use of the above cytochrome P450 enzyme RoCYP01 or the above gene or the above recombinant bacteria or the above method in preparing products containing betulinic acid.

[0024] Beneficial effects:

[0025] The present invention obtains the optimal mutant RoCYP01-W106A / L78A by protein engineering of rosemary-derived RoCYP01. The mutant significantly improves the efficiency of catalyzing betulin to produce betulic acid, and the relative enzyme activity is increased by 198% compared with the enzyme activity of the wild type. The mutant has great potential in the biotransformation of betulin to prepare betulic acid or the fermentation synthesis of betulic acid with glucose as a substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the reaction formula for the synthesis of betulinic acid from betulin catalyzed by the cytochrome P450 enzyme (lupeol C28 oxidase) in the present invention.

[0027] Figure 2 HPLC chart of the synthesis of betulinic acid from betulin catalyzed by wild-type and mutant lupeol C28 oxidase.

[0028] Figure 3 This is the standard curve of betulinic acid (the abscissa is the betulinic acid solubility in mg / L, and the ordinate is the response value).

[0029] Figure 4 To compare the activity of wild-type and mutant lupeol C28 oxidase in catalyzing betulinol to betulic acid. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[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 medium involved in the following examples is:

[0034] LB solid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder.

[0035] SD-URA liquid medium: 20% glucose, yeast basal 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.

[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 betulin: HPLC analysis used a CORTECS C18 1.6μm column (2.1×50mm), and the liquid phase conditions were: 70% acetonitrile + 0.1% formic acid, 30% water + 0.1% formic acid for 35min, flow rate 0.3mL / min; column temperature 35°C; UV detection wavelength 203nm.

[0038] Definition of enzyme catalytic efficiency: Under the same catalytic conditions, the ratio of the amount of betulin produced by the enzyme or mutant to betulic acid is used to evaluate the catalytic efficiency of the enzyme.

[0039] Example 1 Gene acquisition of cytochrome P450 enzyme (lupeol C28 oxidase) and construction of expression vector

[0040] The protein sequence of rosemary-derived cytochrome P450 enzyme (lupeol C28 oxidase) was downloaded from Genbank (Genbank accession number: MK592864.1), the first 30 amino acids were truncated, and the Saccharomyces cerevisiae codon preference was optimized and gene synthesis was performed by Yixin Biotechnology Co., Ltd. The nucleotide sequence of the synthesized gene is shown in SEQ ID NO.2 and ligated to the GAL1 / 10 site of the vector pESC-URA to obtain the recombinant plasmid PESC-RoCYP01.

[0041] Using the recombinant plasmid PESC-RoCYP01 as a template, whole plasmid PCR was performed using primers L78A-1 / L78A-2 and W106A-1 / W106A-2 (primers are shown in Table 1) to mutate the leucine at position 78 of RoCYP01 to alanine, and the tryptophan at position 106 to alanine, and 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, induced expression of target protein and microsome construction

[0046] The recombinant plasmids PESC-RoCYP01 and PESC-RoCYP01-W106A / L78A constructed in Example 1 were sequenced and verified and transformed into Saccharomyces cerevisiae WAT11 competent cells, respectively. The transformants were verified in a defective culture medium, and the correctly verified strains were named Saccharomyces cerevisiae PESC-RoCYP01 and Saccharomyces cerevisiae PESC-RoCYP01-W106A / L78A, respectively.

[0047] 1) Inoculate a single colony activated on the SD defective plate into about 4 mL of liquid SD defective medium and grow in an incubator at 28°C and 250 rpm for 48 h.

[0048] 2) Take 2 ml and inoculate into 500 mL YPGE medium, and culture overnight at 28°C and 250 rpm for 12 h.

[0049] 3) Add galactose to a final concentration of 2% (v / v) for induction and culture for 12 h.

[0050] 4) When galactose induction is complete, the cells are harvested by centrifugation at 5000 g for 5 minutes.

[0051] 5) The cell pellet was resuspended in 0.5 g / mL TEK buffer (50 mM Tris HCl, 1 mM EDTA, 100 mM KCl, pH 7.4) and incubated at room temperature for 5 minutes.

[0052] 6) Centrifuge the cells at 5000 g for 5 minutes and resuspend in 50 mL of ice-cold TES B buffer (50 mM TrisHCl, 1 mM EDTA, 600 mM sorbitol, pH 7.4).

[0053] All following steps were performed at 4°C or on ice.

[0054] 7) Use a high-pressure homogenizer to break up the cells and collect the lysate. The pressure of the high-pressure homogenizer is 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) Microsomes were precipitated by adding 150 mM NaCl to the final volume and 0.1 g / mL polyethylene glycol (PEG)-4000 to the final concentration. After incubation on ice for about 1 h with regular mixing, the microsomal protein fraction was collected by centrifugation at 10,000 g for 10 minutes at 4°C.

[0056] 9) Discard the supernatant and resuspend the microsomal pellet 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 Reaction of betulinol to betulic acid catalyzed by cytochrome P450 enzyme RoCYP01 and its mutants

[0058] RoCYP01 and its mutants obtained in Example 2 were used for the reaction of synthesizing betulinic acid from betulin. The catalytic reaction system (500 uL) is shown in Table 2.

[0059] Table 2 500μL reaction system

[0060]

[0061]

[0062] The system described in Table 2 was reacted at 30°C and 220 rpm for 24 hours. After the reaction was completed, 800 μL of ice-cold methanol was immediately added to terminate the reaction, and then centrifuged at 20,000×g for 5 minutes. The supernatant was filtered using a 0.22 μm organic filter and then used for HPLC analysis.

[0063] Liquid phase analysis showed that a new product with the same elution time as betulin was generated in the reaction system ( Figure 2 ), proving that cytochrome P450 enzyme RoCYP01 can catalyze betulin to generate betulic acid. And the betulin yield was calculated by the liquid phase peak area (standard curve as shown in Figure 3As shown), it can be seen that 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] Specific implementation method is the same as Examples 1 to 3, except that the mutants shown in Table 3 are also constructed, and the results are shown in Table 3.

[0066] Table 3 Catalytic efficiency of different mutants relative to WT

[0067] Mutants 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 as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A cytochrome P450 mutant, characterized in that RoCYP01 from rosemary was used as a parent, the first 30 amino acids were truncated, and the leucine at position 78 was mutated to alanine, and / or the tryptophan at position 106 was mutated to alanine.

2. The cytochrome P450 mutant according to claim 1, characterized in that The amino acid sequence of the parent is shown in SEQ ID NO.

1.

3. A gene encoding the cytochrome P450 enzyme RoCYP01 mutant according to claim 1 or 2.

4. An expression vector carrying the gene according to claim 3.

5. A recombinant bacterium expressing the cytochrome P450 enzyme RoCYP01 mutant according to claim 1 or 2.

6. A recombinant Saccharomyces cerevisiae, characterized in that: The cytochrome P450 enzyme RoCYP01 mutant according to claim 1 or 2 is expressed by using Saccharomyces cerevisiae WAT11 as a host and pESC--URA plasmid as a vector.

7. A catalyst comprising the cytochrome P450 enzyme RoCYP01 mutant according to claim 1 or 2.

8. The catalyst according to claim 7, characterized in that Including but not limited to the recombinant bacterial cells or cell lysates thereof according to claim 5, or microsomes containing the cytochrome P450 enzyme RoCYP01 mutant according to any one of claims 1 to 2.

9. A method for catalytic synthesis of betulinic acid, characterized in that: The catalytic reaction is carried out in a system containing betulin using the cytochrome P450 enzyme RoCYP01 mutant according to claim 1 or 2 or the catalyst according to claim 7 or 8.

10. Use of the cytochrome P450 enzyme RoCYP01 according to claim 1 or 2, or the gene according to claim 3, or the recombinant bacteria according to claim 5, or the recombinant Saccharomyces cerevisiae according to claim 6, or the catalyst according to claim 7 or 8, or the method according to claim 8 in the preparation of a product containing betulinic acid.

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