O-methyltransferase AwOMT and application thereof

Abstract

The invention relates to an O-methyltransferase AwOMT and an application of the O-methyltransferase AwOMT. The enzyme shows a function of catalyzing the methylation of emodin and various polyphenol compounds, and can catalyze the methylation of emodin and a wide range of polyphenol compounds to generate emodin-3-methyl ether and corresponding polyphenol methylation products. Therefore, the O-methyltransferase of the present invention can be used to produce emodin-3-methyl ether, for example, a purified O-methyltransferase can be mixed with emodin, or a whole cell of a host expressing the O-methyltransferase or a cell lysate can be mixed with emodin to produce emodin-3-methyl ether. Or carrying out mixed fermentation on a host expressing the O-methyltransferase and a host capable of producing and secreting the emodin to construct a reaction system so as to produce the emoditin-3-methyl ether; the encoding gene of the O-methyltransferase can also be used for constructing an engineering strain to directly produce the emoditin-3-methyl ether.

Description

Technical Field

[0001] The invention belongs to the field of microbial metabolic engineering, and more particularly, relates to a novel O-methyltransferase AwOMT and application thereof in the production of rhamnosin-3-methyl ether. Background Art

[0002] Emodin is an important active ingredient in traditional Chinese medicines such as rhubarb, aloe vera and knotweed. It has multiple biological activities and pharmacological effects such as antibacterial, anti-inflammatory, anti-tumor and antioxidant, and has become a hot topic in research and application. As a derivative of emodin, emodin-3-methyl ether (CAS registration number: 521-61-9) ( Figure 1 ) also shows a wide range of pharmacological and agricultural application prospects. Medical research shows that rhubarb-3-methyl ether can effectively inhibit Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and other pathogens. In addition, the compound has been found to have the potential to prevent and treat depression-related diseases. By inhibiting the activity of 6-phosphate glucose dehydrogenase, rhubarb-3-methyl ether can inhibit the proliferation and spread of cancer cells. In the agricultural field, rhubarb-3-methyl ether has been developed into a biological pesticide for the prevention and control of diseases in green agriculture and organic vegetables due to its low toxicity and environmental protection. The compound has a broad spectrum of insecticidal activity and can effectively inhibit the development and reproduction of a variety of pests, especially in the prevention and control of powdery mildew, downy mildew, gray mold and anthrax. In addition, rhubarb-3-methyl ether, as a veterinary drug, can reduce the mortality rate of native chickens and improve the egg-laying performance and quality of chickens.

[0003] However, the content of rhamnosin and its methyl ether derivatives in plants is extremely low, and plants are restricted by the growth environment, making it difficult to achieve stable mass production, which seriously limits the promotion of the two, especially rhamnosin-3-methyl ether, as fungicides. Therefore, reconstructing the microbial cell factory of rhamnosin-3-methyl ether based on the concept of synthetic biology is of great significance for breaking through the shackles of production capacity and quality and expanding market share. Summary of the invention

[0004] Our team discovered a rhamnosin 3-hydroxy-O-methyltransferase gene through bioinformatics, expressed the gene heterologously, and purified the expression product. Enzyme-catalyzed reaction experiments showed that the O-methyltransferase can catalyze the methylation of rhamnosin and a wide range of polyphenol compounds to obtain rhamnosin-3-methyl ether and methylated polyphenol compounds.

[0005] Based on the above findings, the present invention provides an O-methyltransferase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0006] The present invention also provides a gene encoding the above O-methyltransferase.

[0007] In a specific embodiment, the nucleic acid sequence of the gene is shown in SEQ ID NO:2.

[0008] The present invention also provides the use of the above O-methyltransferase in catalyzing the methylation of anthraquinone compounds or polyphenol compounds.

[0009] In a specific embodiment, the anthraquinone compound is rhein;

[0010] The polyphenol compound is selected from one or more combinations of resveratrol, quercetin, apigenin, naringenin, fisetin, luteolin and genistein.

[0011] In addition to using the expressed O-methyltransferase for in vitro reaction to produce rhamnosin-3-methyl ether, the gene encoding the above O-methyltransferase can be introduced into a strain that can produce rhamnosin in combination with metabolomics methods to construct a rhamnosin-3-methyl ether production strain.

[0012] Therefore, the present invention also provides a method for producing rhein-3-methyl ether, comprising the step of mixing the above O-methyltransferase with rhein.

[0013] In a specific embodiment, the O-methyltransferase is mixed with rhein in vitro or in cells. For example, the purified O-methyltransferase can be mixed with rhein, or the whole cells or cell lysates of a host expressing the O-methyltransferase can be mixed with rhein, or the host expressing the O-methyltransferase can be mixed with a host that can produce and secrete rhein for fermentation to construct a reaction system to produce rhein-3-methyl ether; the gene encoding the above-mentioned O-methyltransferase can also be introduced into a strain that can produce rhein to construct an engineered strain to directly produce rhein-3-methyl ether. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the structural formula of rheumatoid artemisia and rheumatoid artemisia-3-methyl ether.

[0015] Figure 2 The figure shows SDS-PAGE analysis of AwOMT protein expression and purification. M is a protein marker.

[0016] Figure 3 This is the HPLC analysis chart of the reaction products of AwOMT protein catalyzing rhamnosine in vitro.

[0017] Figure 4 This is the HPLC analysis chart of the reaction products of 7 polyphenolic compounds catalyzed by AwOMT protein in vitro.

[0018] Figure 5This is the HR-LCMS analysis of the reaction products of 7 polyphenol compounds catalyzed by AwOMT protein in vitro. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] 1. Gene synthesis and heterologous expression of O-methyltransferase AwOMT

[0021] 1.1 Gene synthesis of O-methyltransferase AwOMT

[0022] Through bioinformatics analysis, we finally screened out a methyltransferase AwOMT from Aspergillus winterii (nucleic acid sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) that may have the activity of catalyzing the conversion of rhamnosin to rhamnosin-3-methyl ether.

[0023] After the gene fragment of the enzyme was synthesized, it was connected to the expression vector pET28b to obtain the AwOMT expression vector.

[0024] 1.2 Inducible expression and protein purification of AwOMT

[0025] The E. coli puncture bacteria containing the AwOMT expression vector were inoculated into 5 ml of LB medium containing a final concentration of 50 μg / ml kanamycin, cultured overnight at 37°C for 18 h, and the plasmid was extracted using the OMEGA plasmid extraction kit (the method refers to the kit manual), and the quality of the plasmid extraction was detected by 1% agarose gel electrophoresis.

[0026] Take 1 μl of plasmid to transform BL21 (DE3), pick the growing transformants on the kanamycin resistance plate and transfer them to 10 ml of liquid LB medium containing 50 μg / ml kanamycin at a final concentration of 50 μg / ml, and culture them at 37°C for 18 h. Then transfer them to 1 L of LB medium containing 50 μg / ml kanamycin at a final concentration of 50 μg / ml and 5% glycerol and culture them at 37°C until the OD reaches 0. 600 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.2 mM at 16°C to induce protein expression for 20 h, and collect the bacteria by centrifugation at 5000×g.

[0027] Resuspend the cells with 20 ml lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 10 mM imidazole, pH 8.0), place under ultrasonic disruptor for 30 min (5 s on / 5 s pause), and then centrifuge at 10000 rpm for 40 min to collect the supernatant. Add 1 ml Ni-NTA Agarose resin (Tiangen Biochemical, Germany) to the supernatant, rotate and shake at 4 ° C for 2 h, then slowly fill Ni-NTA Agarose resin into an empty column and wait for the liquid to flow out. Next, use 100 ml washing buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 20 mM imidazole, pH 8.0) to slowly wash away non-specifically bound proteins. Thereafter, the bound target protein was eluted with 10 ml of elution buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 250 mM imidazole, pH 8.0). Finally, the ultrafiltration concentration and desalting were performed by an Amicon Ultra-15 ultrafiltration tube (Millipore, Ireland) with a molecular weight cut-off of 30 kDa. All of the above purification steps were performed at 4 °C.

[0028] The purification effect was detected by SDS-PAGE. Figure 2 As shown, the protein AwOMT has been successfully expressed and purified with a purity of over 95% and can be used for the next in vitro activity analysis study.

[0029] 2. In vitro reconstitution of AwOMT enzyme activity and product analysis

[0030] 2.1 Enzyme-catalyzed reactions

[0031] The target protein AwOMT purified above was used to construct an enzyme catalytic reaction system to study its catalytic activity with rhamnolide as a substrate. The reaction system is as follows:

[0032]

[0033]

[0034] After reacting at 30°C for 12 h, 100 μl of methanol was added, and the protein was precipitated by centrifugation at 12,000 rpm after vigorous shaking. The supernatant was filtered through a 0.22 μm filter membrane and waited for the next step of analysis.

[0035] 2.2 Product analysis

[0036] The above reaction product was analyzed using Agilent 1260HPLC on an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm×250 mm). The analysis method is as follows:

[0037] Mobile phase A (100% H2O + 0.05% TFA), mobile phase B (100% ACN + 0.05% TFA), gradient elution (0-1min 50% A, 1-20min 50%-0% A, 20-24min 0% A, 24-25min 0%-50% A, 25-30min 50% A), flow rate 1 ml / min, detection wavelength 440 nm.

[0038] The results are as follows Figure 3 , AwOMT can catalyze rhamnosin to produce rhamnosin-3-methyl ether.

[0039] 3. Analysis of the substrate broadness of AwOMT

[0040] 3.1 Enzyme-catalyzed reactions

[0041] The target protein AwOMT purified above was used to construct an enzyme catalytic reaction system to study its catalytic activity with 11 polyphenolic compounds (resveratrol, apigenin, fisetin, daidzein, genistein, epicatechin, quercetin, naringenin, kaempferol, luteolin, dihydroquercetin) as substrates. The reaction system is as follows:

[0042]

[0043] After reacting at 30°C for 12 h, 100 μl of methanol was added, and the protein was precipitated by centrifugation at 12,000 rpm after vigorous shaking. The supernatant was filtered through a 0.22 μm filter membrane and waited for the next step of analysis.

[0044] 3.2 Product analysis

[0045] The extracted compounds were analyzed using an Agilent 1260HPLC on an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm×250 mm) as follows:

[0046] Mobile phase A (100% H2O + 0.05% TFA), mobile phase B (100% ACN + 0.05% TFA), gradient elution (0-1 min 95% A, 1-20 min 95% -0% A, 20-24 min 0% A, 24-25 min 0% -95% A, 25-30 min 95% A), flow rate 1 ml / min, detection wavelength 340 nm.

[0047] The results are as follows Figure 4 As shown, methylated products of 7 polyphenolic compounds could be detected, namely methylated resveratrol, quercetin, apigenin, naringenin, fisetin, luteolin and genistein, while no methylated products were detected for the remaining 4 substrates.

[0048] The extracted compounds were analyzed using Thermo Fisher LC-MS (LC: Vanquish; MS: Orbitrap Exploris 480) on a Waters Symmetry C18 column (5 μm, 2.1 mm×150 mm) as follows:

[0049] Mobile phase A (100% H2O + 0.1% FA), mobile phase B (100% ACN), gradient elution (0-1min 40% A, 1-18min 40%-0% A, 18-24min 0% A, 24-25min 0%-40% A, 25-30min 40% A), flow rate 0.2 ml / min, column temperature 30°C.

[0050] The results are as follows Figure 5 As shown in the figure, the molecular weights of the methylated products of 7 polyphenolic compounds can be detected, while the molecular weights of the methylated products of the remaining 4 compounds cannot be detected. This indicates that AwOMT can catalyze the methylation of compounds such as resveratrol, quercetin, apigenin, naringenin, fisetin, luteolin and genistein.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An O-methyltransferase, characterized in that The amino acid sequence is shown in SEQ ID NO:

2.

2. A gene encoding an O-methyltransferase, characterized in that: Encodes the O-methyltransferase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleic acid sequence is shown in SEQ ID NO:

2.

4. Use of the O-methyltransferase according to claim 1 in catalyzing the methylation of anthraquinone compounds or polyphenol compounds.

5. The use according to claim 4, characterized in that: The anthraquinone compound is rhein; The polyphenol compound is selected from one or more combinations of resveratrol, quercetin, apigenin, naringenin, fisetin, luteolin and genistein.

6. A method for producing rhamnosin-3-methyl ether, characterized in that: The method comprises the step of mixing the O-methyltransferase according to claim 1 with rhein.

7. The method according to claim 6, characterized in that The O-methyltransferase is mixed with rhein in vitro or in cells.

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