Methytransferase catalyzing the production of emodin-3-methyl ether and use thereof

Abstract

The present application relates to a kind of methyltransferase that can catalyze anthraquinone compound methylation, amino acid sequence is as shown in SEQ ID NO:2, also provide the application of this methyltransferase in the production of emodin-3-methyl ether and other methylated anthraquinone compounds.The present application digs a new methyltransferase, which can methylate anthraquinone compound, and can convert emodin into emodin-3-methyl ether.The present application constructs the engineering bacteria that can produce emodin-3-methyl ether using the coding gene of the enzyme, widens the methyltransferase library that can be used for emodin methylation, provides new way for emodin-3-methyl ether cell factory construction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of metabolic engineering, and relates to a methyltransferase capable of catalyzing the methylation of emodin and other anthraquinones, a gene encoding the same, and application thereof. BACKGROUND

[0002] The continuous use of chemical pesticides can cause serious pollution to the environment. Plant-derived pesticides have the advantages of low toxicity to humans and livestock, good environmental compatibility, etc. due to their natural plant origin, and have become a new development hotspot in the global pesticide industry.

[0003] Emodin and its derivative emodin-3-methyl ether (CAS registration number: 521-61-9) have various biological activities such as antibacterial, anti-inflammatory, anticancer, cough-relieving, and free radical scavenging. Medical research shows that emodin-3-methyl ether has good control effect on powdery mildew, downy mildew, gray mold, and anthracnose, and has been developed into a low-toxicity and environmentally friendly biological pesticide product for the control of diseases in green agriculture and organic vegetables. However, the traditional production method of plant-derived compounds has harsh growth conditions and a long cycle, and the complex components increase the cost of separation and purification. Emodin-3-methyl ether has a low content of only 0.1% in plants, and the direct separation and extraction cost is high. At the same time, it is difficult to achieve stable mass production due to the harsh growth conditions, long growth cycle, and influence of environmental factors, which seriously limits the agricultural promotion of emodin-3-methyl ether fungicide and its market share has been low.

[0004] Therefore, it is of great significance to reconstruct the microbial cell factory of emodin-3-methyl ether based on the concept of synthetic biology to break through the constraints of capacity and quality and expand market share. At present, an invention has used genetic engineering strategies to introduce an exogenous emodin 3-OH O-methyltransferase gene to construct a genetically engineered strain that efficiently accumulates emodin-3-methyl ether, and a new method for preparing a plant-derived biological fungicide emodin-3-methyl ether by fermentation has been developed.

[0005] In order to realize the above-mentioned method, it is necessary to explore new methyltransferases. SUMMARY

[0006] We found a new methyltransferase that can methylate the hydroxyl group at the C3 position of anthraquinones to generate methoxyl group during the research process, and its function was verified by in vivo knockout and in vitro enzyme activity, proving that it has methylation function.

[0007] On this basis, the present application provides a methyltransferase capable of catalyzing the methylation of emodin to generate emodin-3-methyl ether, and the amino acid sequence is shown as SEQ ID NO: 2.

[0008] The present application also provides a nucleic acid encoding the above-mentioned methyltransferase.

[0009] In one specific embodiment, the nucleic acid has a sequence as shown in SEQ ID NO: 1.

[0010] The application also provides the use of the above-mentioned methyltransferase in the methylation of anthraquinones.

[0011] The application also provides a method for preparing a methylated anthraquinone, comprising the step of mixing an anthraquinone to be methylated with the above-mentioned methyltransferase and a methyl donor and performing a catalytic reaction.

[0012] In one specific embodiment, the catalytic reaction is performed in vitro.

[0013] In one specific embodiment, the catalytic reaction is performed in vivo by expressing the methyltransferase in a cell containing an anthraquinone to be methylated.

[0014] In one specific embodiment, the methylated anthraquinone is emodin-3-O-methyl ether, and the cell is a cell capable of accumulating emodin.

[0015] In one specific embodiment, the cell is Aspergillus terreus capable of accumulating emodin.

[0016] In one specific embodiment, the Aspergillus terreus has a knocked-out gedA gene.

[0017] In one specific embodiment, the amino acid sequence encoded by the gedA gene is as shown in SEQ ID NO: 3.

[0018] The application also provides a cell capable of producing a methylated anthraquinone prepared by the above-mentioned method.

[0019] The application discovers a new methyltransferase capable of methylating anthraquinones and converting emodin into emodin-3-O-methyl ether. The application uses the gene encoding the enzyme to construct an engineered bacterium capable of producing emodin-3-O-methyl ether, broadens the library of methyltransferases that can be used for emodin methylation, and provides a new approach for constructing an emodin-3-O-methyl ether cell factory. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Genomic PCR verification results of the transformant for knocking out the ngoD gene.

[0021] Figure 2 HPLC analysis results of the mutant strain MKAJ-ΔngoD and the control strain.

[0022] Figure 3SDS-PAGE analysis results of protein NgoD.

[0023] Figure 4 In vitro enzyme activity reaction results of NgoD catalyzing the methylation of compound 2. (I) is a standard sample of compound 1; (II) is a standard sample of compound 2; (III) is a sample of normal reaction; (IV) is a control group without enzyme; (V) is a control group without donor; (VI) is a control group without substrate.

[0024] Figure 5 In vitro enzyme activity reaction results of NgoD catalyzing the methylation of compound 2. (I) is a standard sample of compound 1; (II) is a standard sample of compound 2; (III) is a sample of normal reaction; (IV) is a control group without enzyme; (V) is a control group without donor; (VI) is a control group without substrate.

[0025] Figure 6 Schematic diagram of three expression elements for expressing ngoD while knocking out gedA in Aspergillus terreus.

[0026] Figure 7 Genomic PCR verification results of Aspergillus terreus transformants. A is the genomic PCR verification results of the transformant obtained by transforming expression element UgedA-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04; B is the genomic PCR verification results of the transformant obtained by transforming expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04; C is the genomic PCR verification results of the transformant obtained by transforming expression element UgedA-PgpdAt-ngoD-Tpgk-ptrA-DgedA into Aspergillus terreus HZ04.

[0027] Figure 8 HPLC analysis of fermentation products of three strategy transformants. DETAILED DESCRIPTION

[0028] The principles and features of the present application are described below in conjunction with examples, which are used to explain the present application and are not intended to limit the scope of the present application.

[0029] 1. Function verification of the methyltransferase NgoD in the fungus Spathaspora passalioidea MEFC009

[0030] After analyzing the genomic sequence of Spathaspora passalioidea MEFC009 and analyzing the fermentation products, it was speculated that the gene ngoD (nucleic acid sequence as shown in SEQ ID NO: 1, and the encoded protein sequence as shown in SEQ ID NO: 2) might have the function of methyltransferase. In order to verify its function, the gene was knocked out in the strain MKAJ. The method is as follows:

[0031] Using the genome of wild-type Coleophoma sp. MEFC009 as a template, the upstream sequence U-ngoD (approximately 1201 bp) and the downstream sequence D-ngoD (approximately 1200 bp) of ngoD were amplified. The hygromycin resistance selection fragment hph (2183 bp), the upstream sequence U-ngoD, and the downstream sequence D-ngoD were fused by fusion PCR. Using the fusion product as a template, the 4257 bp knockout expression element UngoD-hph-DngoD was amplified using nested primers U-ngoD-CS-F / D-ngoD-CS-R.

[0032] Using MKAJ as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 mL of the seed culture was inoculated into 50 mL of ME seed medium and cultured in a 250 mL Erlenmeyer flask at 220 rpm and 25°C using a shaker. After 2 days, the mycelium was collected by centrifugation (5000 rpm, 4°C, 5 min). The mycelium was homogenized again, and 2 mL of the seed culture was inoculated into 50 mL of ME seed medium. The culture was then incubated for 1 day under the same conditions. The medium and mycelium were then transferred together into a 50 mL sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed five times with 0.6 M MgSO4. 1 g of mycelium was weighed and added to 10 mL of ME enzymatic hydrolysate (filtered through a 0.22 μm sterile filter for sterilization), and treated at 30°C and 130 rpm for 4 h. The protoplast reaction solution was then filtered through a sterile filter cloth, and the protoplasts were collected by centrifugation at 5000 rpm and 4°C. Wash once with pre-cooled STC, resuspend the protoplasts in pre-cooled STC, and adjust the protoplast concentration to 10 using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0033] Add the UngoD-hph-DngoD fragment to 150 μL of the above protoplast suspension, then add 50 μL of PSTC, mix gently, and incubate on ice for 30 min. Add 1 mL of PSTC, mix well, and incubate at room temperature for 20 min. Finally, mix with 10 mL of top agar and pour onto three 0.8 M PDAS-H plates. Incubate at 30 °C in the dark for 5 days to obtain transformants.

[0034] Transformants with hygromycin resistance were picked from 0.8M PDAS-H transformation selection plates and transferred to PDA-H plates, where they were cultured at 25°C for 5 days. The genomes of each transformant were extracted, and PCR verification was performed using external primers U-ngoD-F / D-ngoD-R. Transformants that amplified a band of 4520 bp were considered positive, while the control strain MKAJ only amplified a band of approximately 2.9 kb. Figure 1 ).

[0035] Transformants were cultured on PDA-H plates, with MKAJ as the control strain, at 25°C for 5 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor. -24) The mycelia were disrupted, and the disrupted mycelia were inoculated into 50 mL of ME seed medium (250 mL Erlenmeyer flask) and cultured at 25℃ and 220 rpm on a shaker for 45-48 h. 5 mL of the above-cultured seed solution was transferred to 50 mL of ME fermentation medium (250 mL Erlenmeyer flask) and cultured at 25℃ and 220 rpm on a shaker for 8 days. Three replicates were set up for each strain. After fermentation, 90 mL of ethyl acetate was added to 50 mL of fermentation broth, and extraction was performed at 25℃ and 220 rpm for 1 h. After standing for 10 min, the upper organic phase was collected and subjected to vacuum distillation. After evaporation to dryness, 2 mL of chromatographic methanol was added to dissolve the crude extract. The sample was then filtered through a 0.22 μm organic filter and analyzed by HPLC.

[0036] The HPLC analysis method was as follows: The liquid chromatography column was an Agilet C-18 reversed-phase column 883975-902 (4.6 × 150 mm, 5 μm); mobile phase A: 0.05% (v / v) trifluoroacetic acid acetonitrile solution, mobile phase B: 0.05% (v / v) trifluoroacetic acid aqueous solution; flow rate: 1 mL / min; UV detection wavelength: 210 nm; temperature: 30 °C; total elution time: 33 min. Gradient elution conditions: 0-25 min, the volume of mobile phase A linearly increased from 5% to 100%; 25-30 min, the volume of mobile phase A remained at 100%; 30.1-33 min, the volume of mobile phase A remained at 5%.

[0037] The results are as follows Figure 2 As shown, knocking out the ngoD gene completely prevents the production of anthraquinone compound 1, but produces an anthraquinone intermediate 2. It is speculated that the ngoD gene is responsible for methylating intermediate 2 to generate the final product 1.

[0038] 2. In vitro enzyme activity verification of methyltransferase NgoD

[0039] The function of the ngoD gene was verified using an in vitro enzymatic assay. First, the ngoD gene was synthesized and ligated into the expression vector pET28a, followed by induced expression and purification.

[0040] E. coli containing the ngoD expression vector were inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C for 18 h. The plasmid was extracted using the OMEGA plasmid extraction kit, and the extraction quality was assessed by 1% agarose gel electrophoresis. 1 μL of the plasmid was transformed into BL21(DE3). Transformants that grew on kanamycin-resistant plates were picked and cultured in 10 mL of liquid LB medium containing 50 μg / mL kanamycin at 37°C for 18 h. The transformed cells were then transferred to 1 L of LB medium containing 50 μg / mL kanamycin and cultured at 37°C until the OD reached 0.6–0.8. 0.2 mM IPTG (isopropyl-β-D-thiogalactoside) was added to induce protein expression at 16°C for 20 h. The cells were collected by centrifugation at 5000 × g.

[0041] The bacterial cells were resuspended in 20 mL of lysis buffer and sonicated for 30 min (5 s on / 5 s pause). The mixture was then centrifuged at 10,000 rpm for 60 min, and the supernatant was collected. 1 mL of Ni-NTA agarose resin was added to the supernatant, and the mixture was incubated at 4°C with vortexing for 2 h. The Ni-NTA agarose resin was then slowly packed into an empty column, and the column was allowed to drain completely. Next, 100 mL of washing buffer was used to slowly wash away non-specifically bound proteins. Then, 10 mL of elution buffer was used to elute the bound target protein. Finally, the solution was concentrated and desalted by ultrafiltration using an Amicon Ultra-15 ultrafiltration tube with a molecular weight cutoff of 30 kDa. All purification steps were performed at 4°C.

[0042] like Figure 3 As shown, SDS-PAGE analysis indicates that the target protein ngoD has been successfully expressed and purified, and can be used for further in vitro activity analysis.

[0043] Using the obtained target protein NgoD, the catalytic activity of the enzyme was studied when its own substrate compound 2 was used as a substrate. The reaction system is shown in Table 1.

[0044] Table 1. In vitro reaction systems using compound 2 as a substrate.

[0045]

[0046]

[0047] After reacting at 30℃ and 160 rpm for 2 h, 400 μL of ethyl acetate was added to stop the reaction. After vigorous shaking for 30 min, the supernatant ethyl acetate phase was collected and dried under nitrogen. Then, 200 μL of methanol was added and vigorously shaken for 10 min to fully dissolve the product. Finally, the mixture was centrifuged at 13000 rpm for 10 min to precipitate impurities. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC.

[0048] The results are as follows Figure 4 As shown, NgoD can catalyze the reaction of compound 2 to produce a compound with the same HPLC retention time as the standard of compound 1, indicating that NgoD has methyltransferase activity.

[0049] Since the structure of compound 2 is similar to that of emodin, in order to verify whether NgoD can methylate emodin in vitro to produce emodin methyl ether, the obtained protein NgoD was used to study the catalytic activity of the enzyme when emodin is used as a substrate. The reaction system is shown in Table 2.

[0050] Table 1. In vitro reaction systems using emodin as a substrate

[0051] System Concentration Substrate: Emodin 100 μΜ Donor: SAM 1 mM Enzyme: NgoD protein 20 μΜ Buffer: Tris-HCl (pH 7.5, supplemented to 100 μL) 20 mM

[0052] After reacting at 30℃ and 160 rpm for 2 h, 400 μL of ethyl acetate was added to stop the reaction. After vigorous shaking for 30 min, the supernatant ethyl acetate phase was collected and dried under nitrogen. Then, 200 μL of methanol was added and vigorously shaken for 10 min to fully dissolve the product. Finally, the mixture was centrifuged at 13000 rpm for 10 min to precipitate impurities. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC.

[0053] The results are as follows Figure 5 As shown, NgoD can catalyze the reaction of emodin to produce a compound with an HPLC retention time consistent with that of the emodin-3-methyl ether standard.

[0054] 3. Heterologous expression of methyltransferase NgoD in Aspergillus terreus

[0055] To verify whether NgoD can methylate emodin in vivo to produce emodin-3-methyl ether, a heterologous expression method was used for verification.

[0056] The highly efficient homologous recombination strain *Aspergillus terrestris* HZ04 was selected as the chassis strain. The gene *ngoD* was inserted into its *gedA* gene (encoding a protein sequence as shown in SEQ ID NO:3, responsible for the methylation modification of the C1 hydroxyl group of emodin). The following method was used... Figure 6The homologous recombination strategy shown uses ptrA as an antibiotic resistance selection tag. Based on this strategy, three expression elements were constructed: UgedA-ngoD-Tpgk-ptrA-DgedA, UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA, and UgedA-PgpdAt-ngoD-Tpgk-ptrA-DgedA. (The nucleic acid sequences of the PgedA promoter are shown in SEQ ID NO:4, the PgpdA promoter in SEQ ID NO:5, and the PgpdAt promoter in SEQ ID NO:6).

[0057] The constructed expression elements were subjected to PCR detection, and the results are as follows: Figure 7 As shown, the amplified expression element UgedA-ngoD-Tpgk-ptrA-DgedA is close to the theoretical size of 5037bp, which is correct; the amplified expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedA is close to the theoretical size of 6172bp, which is correct; and the amplified expression element UgedA-PgpdA-ngoD-Tpgk-ptrA-DgedAt is close to the theoretical size of 5783bp, which is correct.

[0058] The expression elements were purified. Since the amplified target elements all had impurities, gel extraction was used for purification to obtain the purified expression elements.

[0059] Using *Aspergillus terrestris* HZ04 as the starting strain, an appropriate amount of spore suspension was first taken from a 1.2M PDAS plate and inoculated into 50 mL of YPM seed culture medium. The culture was then carried out in a 250 mL Erlenmeyer flask at 220 rpm and 30°C using a shaker. After 2 days, mycelia were collected using a 300-mesh filter. The mycelia were washed five times with 0.6M MgSO4. 1 g of mycelia were weighed and added to 10 mL of AT enzyme digest (filtered through a 0.22 μm sterile filter for sterilization), and treated at 30°C and 140 rpm for 2 h. The protoplast reaction solution was then filtered successively through a 500-mesh filter and a sterile filter cloth, and the protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with pre-chilled STC, resuspended in pre-chilled STC, and the protoplast concentration was adjusted to 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0060] The three purified expression elements were added to 150 μL of the protoplast suspension, followed by 50 μL of PSTC. The mixture was gently incubated on ice for 30 min. Then, 1 mL of PSTC was added, and the mixture was incubated at room temperature for 20 min. Finally, each element was mixed with 10 mL of top agar and poured onto three CD-P plates. Transformants were obtained by incubating at 28°C in the dark for 5 days.

[0061] Transformants with pyrithioneine resistance were picked from CD-P transformation selection plates and transferred to new CD-P plates, where they were cultured at 28°C for another 5 days. The genomes of each transformant were extracted and verified by PCR using external primers U-gedA-F / D-gedA-R. The results are shown below. Figure 8 As shown, multiple positive transformants were obtained for all three expression elements.

[0062] Positive transformants were selected and cultured on 1.2M PDAS plates (28℃, 5 days). After expansion, sterile water was added to wash away the spores, obtaining a spore suspension, which was then inoculated with 10... 7 Each spore was added to 50 mL of LPM fermentation medium (250 mL Erlenmeyer flask) and cultured on a shaker at 28 °C and 220 rpm for 8 days, with three replicates for each strain. After fermentation, 1 mL of fermentation broth was taken and centrifuged at 12000 rpm for 10 min, retaining the bacterial cells. The bacterial cells were added to 10 mL of a methanol:dichloromethane (1:1) mixture for rotary extraction for 2 h. After standing for 10 min, the methanol:dichloromethane (1:1) mixture was separated from the bacterial cells, and the mixture was collected. 500 μL of the mixture was dried under nitrogen, and 500 μL of methanol was added and shaken to dissolve for 10 min. The mixture was then filtered through a 0.22 μm organic filter membrane and analyzed by HPLC. The HPLC method was as follows: the fermentation extract was analyzed using an Agileent 1260 HPLC system on an Agileent Eclips Pluus C18 column (5 μm, 4.6 mm × 250 mm). The analytical method is as follows: mobile phase A (100% H₂O + 0.05% TFA), mobile phase B (100% ACN + 0.05% TFA), gradient elution (0-1 min 50% A, 1-20 min 50%-0% A, 20-24 min 0% A, 24-25 min 0%-50% A, 25-30 min 50% A), flow rate 1 mL / min, detection wavelength 440 nm. Results show:

[0063] The gedA gene is responsible for catalyzing the methylation of the C1 hydroxyl group in rhein. Knocking out the gedA gene prevents this methylation, leading to rhein accumulation. Since the starting strain HZ04 did not have the gedA gene knocked out, no rhein accumulated, so it served as a control. Standards of rhein and rhein-3-methyl ether were also used as controls. The analysis showed that the HZ04-PgedA-ngoD transformant produced rhein and trace amounts of rhein methyl ether, indicating successful gedA gene knockout. The gedA promoter can drive the ngoD gene to function, but the transformation efficiency was low. The HZ04-PgpdA-ngoD transformant accumulated rhein, with almost no rhein methyl ether detected. The HZ04-PgpdAt-ngoD transformant produced both rhein and rhein methyl ether, with a higher accumulation of rhein methyl ether than the former two.

[0064] Therefore, it can be concluded that the methyltransferase NgoD can catalyze the production of emodin-3-methyl ether from emodin in Aspergillus terrestris.

[0065] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A methyltransferase capable of catalyzing the methylation of anthraquinone compounds, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

2. The nucleic acid encoding the methyltransferase of claim 1.

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

1.

4. The use of the methyltransferase of claim 1 in the preparation of methylated anthraquinone compounds, wherein the anthraquinone compound is emodin and the methylated anthraquinone compound is emodin-3-methyl ether.

5. A method for preparing methylated anthraquinone compounds, characterized in that, The method includes the step of mixing the anthraquinone compound to be methylated with the methyltransferase and methyl donor described in claim 1 and carrying out a catalytic reaction; wherein the anthraquinone compound is emodin and the methylated anthraquinone compound is emodin-3-methyl ether.

6. The method according to claim 5, characterized in that, The catalytic reaction is carried out in vitro.

7. The method according to claim 5, characterized in that, The catalytic reaction is carried out in vivo by expressing the methyltransferase in cells containing anthraquinone compounds to be methylated.

8. The method according to claim 7, characterized in that, The methylated anthraquinone compound is emodin-3-methyl ether, and the cells are cells capable of accumulating emodin.

9. The method according to claim 8, characterized in that, The cells are Aspergillus terrestris, which is capable of accumulating emodin.

10. A cell capable of producing methylated anthraquinone compounds, characterized in that, It was prepared by expressing the methyltransferase of claim 1 in cells capable of accumulating emodin.

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