Prothioconazole methyltransferase gene proS32 and its application

Abstract

This invention belongs to the field of environmental biotechnology, and in particular, it relates to the prothioconazole methyltransferase gene. proS32 Its applications. This invention discovered strains through bioinformatics analysis and gene cloning. Sphingomonas The key enzyme in sp. AJ-1 that degrades prothioconazole is prothioconazole methyltransferase. proS32 The gene was obtained and prothioconazole methyltransferase ProS32 was expressed through an expression vector. HPLC and enzymatic detection confirmed its efficient and specific degradation catalytic ability for prothioconazole, laying the foundation for the development of corresponding biodegradation reagents or efficient degrading bacteria.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, and in particular, it relates to the prothioconazole methyltransferase gene ProS32 and its applications. Background Technology

[0002] Prothioconazole is a triazole thione fungicide. Its mechanism of action is to inhibit the demethylation of lanosterol or 24-methylenediaminolanosterol at the 14-position, a precursor of sterols in fungi, thus acting as a demethylation inhibitor. It not only possesses excellent systemic activity, but also superior protective, curative, and eradicative activities, and has a long-lasting effect. Prothioconazole is mainly used to control numerous diseases in cereal crops such as wheat, barley, rapeseed, peanuts, rice, and legumes. It has good control effects on almost all cereal diseases, such as powdery mildew, sheath blight, wilt, leaf spot, rust, sclerotinia rot, net blight, and clouding disease in wheat and barley. It can also control soil-borne diseases of rapeseed and peanuts, such as sclerotinia rot, as well as major foliar diseases, such as gray mold, black spot, brown spot, black shank, sclerotinia rot, and rust. The usual dosage is 200 g a.i. / hm². 2 At this dosage, its activity is superior to or equal to that of conventional fungicides such as flutriafol, tebuconazole, and pyraclostrobin. However, the widespread use of prothioconazole poses a potential threat to the ecological environment and human health. The European Food Safety Authority (EFSA) has confirmed the potential acute risks of prothioconazole and has set maximum residue limits (MRLs) of 0.02-0.1 mg / kg for crops such as corn, sugar beets, potatoes, and peanuts. Canada has set an MRL of 0.2 mg / kg for prothioconazole in sunflowers. Therefore, the degradation treatment of prothioconazole is an important research direction. Junwei Huang et al. published (Science of the Total Environment 851 (2022) 158220, DOI: 10.1016 / j.scitotenv.2022.158220) a microorganism, Sphingomonas AJ-1, which has a degrading effect on prothioconazole. Sphingomonas sp. This bacterium (AJ-1) reduces the toxicity of prothioconazole by methylating the C=S bond of the prothioconazole to generate methylated prothioconazole. However, to further improve enzymatic methods and obtain strains with stronger degradation capabilities, it is necessary to conduct further research and exploration on the methylation-related genes and enzymes involved in the degradation of prothioconazole by these strains. Summary of the Invention

[0003] To address the related problems in the prior art, this invention, through further in-depth research, discovered [the virus] from Sphingomonas AJ-1 (… Sphingomonas sp.The prothioconazole methyltransferase gene in AJ-1 proS32, This invention lays the foundation for obtaining stronger prothioconazole through enzymatic degradation or mutation screening, thus completing the present invention.

[0004] The technical solution of the present invention is as follows:

[0005] In one aspect, this invention discloses a prothioconazole methyltransferase gene. proS32 The gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] In one aspect, the present invention discloses a prothioconazole methyltransferase ProS32. ， Its amino acid sequence is shown in SEQ ID NO.2.

[0007] In one aspect, the present invention discloses an expression vector containing the prothioconazole methyltransferase gene. proS32 Gene. The expression vector can be a prokaryotic expression vector or a eukaryotic expression vector; preferably, the expression vector is a prokaryotic expression vector.

[0008] In one aspect, the present invention discloses a bacterial strain containing an expression vector containing a prothioconazole methyltransferase gene. proS32 Gene.

[0009] In one embodiment, the strain is Escherichia coli.

[0010] In one aspect, the present invention discloses a solution containing prothioconazole methyltransferase. proS32 Application of strains with gene expression vectors in the degradation of prothioconazole.

[0011] In one aspect, the present invention discloses the application of prothioconazole methyltransferase ProS32 in the degradation of prothioconazole.

[0012] In one aspect, the present invention discloses an improvement Sphingomonas A method for enhancing the ability of sp. AJ-1 to degrade prothioconazole, the method comprising: Sphingomonas sp. AJ-1 proS32 Gene expression levels.

[0013] In one aspect, the present invention also discloses a formulation for degrading prothioconazole, said formulation containing a prothioconazole methyltransferase gene. proS32 The strain containing the gene expression vector; or the preparation containing prothioconazole methyltransferase ProS32.

[0014] In one aspect, the present invention also discloses an immobilized enzyme preparation, wherein the immobilized enzyme is prothioconazole methyltransferase ProS32. Beneficial effects

[0015] This invention discovered the strain through bioinformatics analysis and gene cloning. Sphingomonas sp. AJ-1's key enzyme in the degradation of prothioconazole is prothioconazole methyltransferase. proS32 The gene was used to confirm its efficient and specific degradation catalytic ability for prothioconazole, laying the foundation for the development of corresponding biodegradation reagents or efficient degrading bacteria. Attached Figure Description

[0016] Figure 1 The action site and reaction equation for the degradation of prothioconazole (PTC) by strain AJ-1. Strain AJ-1 breaks the double bond between the sulfur and triazole ring in prothioconazole by adding a methyl group to the sulfur atom. Corresponding to the structural formula of prothioconazole, it can be seen that the degradation process of prothioconazole in strain AJ-1 is a methylation process, and the product is methylated prothioconazole (Prothioconazole-S-methyl, PTM).

[0017] Figure 2 Methyltransferase gene proS32 A schematic diagram of the expression strategy and functional verification in BL21(pET-32a(+)).

[0018] Figure 3 Recombinant expression plasmid pET- proS32 PCR detection. Where A represents the value obtained from PCR amplification. proS32 Genetic testing, B is for constructing pET- proS32 Expression plasmid, transformed into E. coli After BL21 (DE3) competent cells are used, the positive clones will be sequenced and the PCR results will be detected.

[0019] Figure 4 , E. coil BL21 (DE3) (pET- proS32 Liquid phase diagram of PTC degradation.

[0020] Figure 5 SDS-PAGE gel image of fusion protein His-ProS32, where M is protein marker; A is crude enzyme solution of fusion protein His-ProS32 (1) and purified ProS32 protein (2).

[0021] Figure 6 ProS32 pairs ( Rac)- / ( S )- / ( R The degradation of )-PTC, where A is the degradation curve; B is the degradation rate and EF value change.

[0022] Figure 7 ProS32 degradation ( S )- / ( R Enzyme kinetics of )-PTC.

[0023] Figure 8 Liquid phase diagram of PTC degradation by ProS32.

[0024] Figure 9 Mass spectra of the products of ProS32 degradation of PTC, where A is the primary mass spectrum and B is the secondary mass spectrum. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Methyltransferase proS32 Gene cloning and expression vector construction

[0027] 1.1 Bioinformatics analysis to determine the methyltransferase gene sequence

[0028] extract Sphingomonas DNA of sp. AJ-1 was extracted and sequenced to obtain its genomic data, which was then analyzed using Uedit32 software. Sphingomonas Using sp. AJ-1 genome sequencing data, gene sequences annotated as methyltransferases were identified. Then, the ORF Finder tool (https: / / www.ncbi.nlm.nih.gov / orffinder / ) was used to perform open reading frame (ORF) analysis on the upstream and downstream sequences to identify potential protein-coding regions. Finally, these sequences were submitted to NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for alignment analysis to further verify their function and homology, and to identify the corresponding methyltransferase genes.

[0029] 1.2 Prothioconazole methyltransferase gene proS32 Cloning

[0030] strain Sphingomonas Using sp. AJ-1 genomic DNA as a template, the 2 × phanta® MaxMaster Mix kit was used to... proS32 Perform PCR amplification (prothioconazole methyltransferase gene) proS32 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. A 50 μL system was used according to the kit instructions, and the components are as follows (Table 1):

[0031] Table 1 PCR amplification system

[0032]

[0033] The primer sequences are as follows:

[0034] proS32 -32a-F (SEQ ID NO.3): 5'-gccatggctgatatcggatccACCGAGGCGATCGAGTGG-3'

[0035] proS32 -32a-R (SEQ ID NO.4): 5'-gtggtggtggtggtgctcgagACCGCGCCCCTCATCCGC-3'

[0036] The amplification procedure is as follows (Table 2):

[0037] Table 2 PCR amplification program

[0038]

[0039] After amplification, the amplification products were detected by gel electrophoresis to confirm whether the target band was a single target band. Subsequently, the agarose gel containing the target band was cut and purified by gel recovery using the FastPure® Gel DNAExtraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.) according to the instructions.

[0040] 1.3 Methyltransferase gene proS32 Construction of exogenous expression vectors

[0041] Methyltransferase gene constructed using homologous recombination. proS32 The exogenous recombinant expression vector, the specific steps are as follows:

[0042] (1) Vector linearization: The pET32a(+) expression vector (preserved in our laboratory) was double-digested with QuickCut™ BamHI and QuickCut™ Xho I (Takara). The digestion was carried out in a water bath at 37 ℃ for 1 h. The digestion system is shown in Table 3.

[0043] Table 3. Double enzyme digestion system of pET-32a (+) expression vector

[0044]

[0045] (2) Enzyme digestion gel recovery and purification: After enzyme digestion, the digestion products were detected using an electrophoresis instrument and gel imaging system. The target plasmid fragments were excised and purified using the FastPure® Gel DNA Extraction Mini Kit. Subsequently, the recovered pET32a (+) expression vector digestion fragments were detected by agarose gel electrophoresis, and the concentration and purity were detected using NanoDrop One.

[0046] (3) Homologous recombination: The recovered pET32a (+) expression vector fragments were digested and digested using the ClonExpress® II One Step Cloning Kit. proS32 Homologous recombination was performed on the gene PCR amplification fragments, and the reaction system is shown in Table 4. After the system was prepared, it was reacted in a water bath at 37 ℃ for 30 min.

[0047] Table 4 proS32 Homologous recombination reaction system of exogenous expression vector

[0048]

[0049] (4) Transformation:

[0050] 1) The recombinant product was mixed with the product taken from -80 °C E. coli BL21 (DE3) competent cells were placed on ice;

[0051] 2) After the competent cells have thawed (about 6 min), add 10 μL of the recombinant product to the competent cells and gently tap the bottom of the tube to mix. Place the tube on ice and let it stand for 30 min.

[0052] 3) Heat stimulated in a 42 ℃ water bath for 45 s, then quickly placed back in ice and kept in ice for 2 min;

[0053] 4) Add 700 μL of antibiotic-free LB medium and mix well;

[0054] 5) After shaking culture in a constant temperature shaker at 37 ℃ for 1 h (220 rpm), centrifuge at 5000 rpm for 1 min, discard 500 μL of supernatant, resuspend the bacterial cells, and take 100 μL and spread evenly on LB agar plates containing Amp.

[0055] 6) Invert the container and incubate overnight at 37 ℃ for 12 h.

[0056] (5) Validation of positive clones

[0057] 1) After the colonies have grown on the transformation plate, pick 3 single clones and place them in 4 mL of AmpLB medium containing 100 mg / L. Place the test tubes in a constant temperature shaker at 37 ℃ and shake for 5 h (220 rpm).

[0058] 2) Take 200 μL of bacterial culture for sequencing verification, and temporarily store the remaining bacterial culture in a 4 ℃ refrigerator;

[0059] 3) After the sequencing results are correctly matched, resuspend the preserved bacterial solution, take 700 μL of the bacterial solution and add an equal volume of 35% sterile glycerol, and store it in a -80 ℃ freezer.

[0060] Example 2: Containing prothioconazole methyltransferase gene proS32 Functional verification of recombinant strains

[0061] The correctly sequenced recombinant expression strain was inoculated into 20 mL of LB liquid medium, and Amp was added to a final concentration of 50 mg / L, along with 40 μM ( ) Rac PTC and 0.5 mM IPTG were incubated at 37 ℃ and 220 rpm for 12 h in a shaker. 600 μL of the culture medium was taken and an equal volume of methanol was added. The mixture was thoroughly mixed by inversion and centrifuged at 12000 rpm for 2 min. The supernatant was filtered through a 0.22 μm nylon filter membrane, and the concentration of PTC in the culture medium was detected by high performance liquid chromatography (HPLC).

[0062] The high-performance liquid chromatography (HPLC) method for detecting PTC was as follows: the chromatographic column was an Agilent Eclipse XDB C18 (4.6 mm × 250 mm; 5 μm); the mobile phase was acetonitrile: 0.1% formic acid aqueous solution = 73:27 (v / v); the detection wavelength was 193 nm; the injection volume was 20 μL; the flow rate was 1 mL / min; and the column temperature was 30℃.

[0063] ( Rac )- / ( S )- / ( RHPLC detection conditions for chiral separation of PTC and its metabolites: chromatographic column: CHIRALCEL ® OD-RH (4.6 mm × 150 mm; 5 μm); mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 60:40 (v / v); detection wavelength: 200 nm; injection volume: 20 μL; flow rate: 0.6 mL / min; column temperature: 25 ℃.

[0064] See Figure 4 , E. coli BL21 (pET- proS32 The culture medium was different from the control group. E. coli Compared to BL21 (pET-32a) culture medium (pET-32a(+) plasmid without the target gene constructed), the peak height of the PTC characteristic peak (RT=5.1 min) decreased by 75%, and a new product peak appeared at 9.3 min, indicating that... E. coli BL21 (pET- proS32 It can degrade PTC and generate products. Therefore, it can be determined that... proS32 It is a functional gene that catalyzes PTC methylation.

[0065] Example 3: Inducible expression, purification, and degradation ability test of prothioconazole methyltransferase ProS32

[0066] 3.1 Obtaining the methyltransferase ProS32 protein

[0067] Five mL of the activated recombinant bacterial culture was inoculated into 500 mL of LB medium containing Amp at a ratio of 1:100, and cultured at 37 °C and 220 rpm until the bacterial culture reached OD. 600 When the value reaches 0.5-0.8, [the following is a direct translation of the original text:] to E. coli BL21 (pET- proS32 IPTG was added to the bacterial culture to a final concentration of 1 mM and incubated in a shaker at 16 ℃ for 12 h. The bacterial pellet was then collected by centrifugation at 12000 rpm for 15 min at 4 ℃. The cells were washed twice with 50 mM Tris-HCl (pH 7.4) buffer, and finally resuspended in 35 mL of the same buffer. After cell disruption by sonication, the supernatant was collected by centrifugation at 8000 rpm for 30 min, which was the crude ProS32 enzyme solution. The crude enzyme solution was then purified using a Ni-TED 6FF pre-packed gravity column in a chromatography cabinet at 4 ℃ to obtain purified prothioconazole methyltransferase ProS32.

[0068] See Figure 5The crude ProS32 enzyme solution was obtained by ultrasonic disruption of the bacterial culture, and then purified using a Ni-NTA pre-packed gravity column to obtain prothioconazole methyltransferase ProS32. SDS-PAGE analysis showed that the purified ProS32 enzyme eluted in 500 mM imidazole buffer, and the bands were all single and around 50 kDa, consistent with the theoretical value of 49.32 kDa.

[0069] 3.2 ProS32 degradation ( Rac )- / ( S )- / ( R Comparison of PTC capabilities

[0070] Add 40 μM (50 ng / μL ProS32) to 500 μL of enzyme reaction solution containing 50 ng / μL ProS32. Rac )-PTC、( S )-PTC、( R The reaction was carried out in a 30 °C water bath with 1 mM PTC and 1 mM SAM. Samples were taken every 20 min until 120 min, at which point an equal volume of acetonitrile was added to terminate the enzyme reaction. The substrate added to the above reaction system was replaced with 20 μM PTC. Rac )-PTC to study the degradation of ProS32 pure enzyme ( Rac Changes in EF value during the )-PTC process. The enzyme reaction solution used to terminate the reaction was vortexed, centrifuged at 12000 rpm for 2 min, and filtered through a 0.22 μm organic phase filter. The concentrations of substrate and product were determined by HPLC. All treatments were performed in triplicate.

[0071] See Figure 6 The results showed that 50 μg / mL of purified ProS32 enzyme had an effect on ( ) within 20 min. R The degradation rate of )-PTC is 100%, while that of ( S The degradation rate of PTC was 47.00%. Figure 6 (A), ProS32 pure enzyme pair ( Rac During the degradation of PTC, the EF value increased from 0.50 to 1.00 within 35 minutes. Figure 6 (B), indicating that ProS32 has degraded ( R The rate of )-PTC is greater than ( S )-PTC.

[0072] 3.3 Determination of Enzymatic Parameters of ProS32

[0073] Will E. coli BL21 (pET- proS32The recombinant strain was inoculated into 500 mL LB liquid medium containing 100 mg / L Amp, and 1 mM IPTG was added. After induction culture at 16 °C and 180 rpm for 12 h, the protein was purified.

[0074] Add 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, and 180 μM of ( ) to a 500 μL Tris-HCl enzymatic reaction system containing 50 μg / mL ProS32. S )- / ( R )-PTC, reacted in a 30°C water bath. ProS32 and ( R )-PTC reaction for 15 min, with ( S )-PTC reaction for 1 h.

[0075] Add an equal volume of acetonitrile to the above reaction solution, mix thoroughly by inverting, and terminate the enzymatic reaction. Centrifuge at 12000 rpm for 2 min, pass the sample supernatant through a 0.22 μm oil-based filter membrane, and determine the concentrations of PTC and product PTM using HPLC.

[0076] The substrate concentration and reaction rate in the reaction were nonlinearly fitted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) to obtain... K m , V max and k cat Values. All processes are set up with three parallel groups.

[0077] See Figure 7 Enzyme kinetic analysis showed that ProS32 has the effect of ( R )-PTC catalytic efficiency ( k cat / K m The value is 0.777*10. -5 min -1 μM -1 ( Figure 7 A), for ( S -PTC k cat / K m It is 0.149*10 -5 min -1 μM -1 ( Figure 7 B), ProS32 pair (R The catalytic efficiency of )-PTC is ( S 5.2 times that of PTC.

[0078] 3.4 Substrate profile of prothioconazole methyltransferase ProS32

[0079] In a 500 μL Tris-HCl enzymatic reaction system (containing 50 μg / mL ProS32 pure enzyme and 1 mM SAM), 10 mg / L of triazole fungicides such as propiconazole, difenoconazole, tebuconazole, tricyclazole, triadimefon, and triadimefon were added respectively, and the reaction was carried out at 30 °C for 48 h. The degradation of each substrate was detected by HPLC.

[0080] HPLC detection method for propiconazole: mobile phase: methanol:water = 65:35 (v / v); detection wavelength: 230 nm; injection volume: 20 μL; flow rate: 1 mL / min; column temperature: 40 ℃.

[0081] HPLC detection method for difenoconazole: mobile phase: acetonitrile:methanol:0.1% phosphoric acid solution = 50:20:30 (v / v); detection wavelength: 236 nm; injection volume: 10 μL; flow rate: 1 mL / min; column temperature: 35 ℃.

[0082] HPLC detection method for tebuconazole: mobile phase: methanol:water = 80:20 (v / v); detection wavelength: 223 nm; injection volume: 10 μL; flow rate: 0.8 mL / min; column temperature: 30 ℃.

[0083] HPLC detection method for tricyclazole: mobile phase: methanol:water = 45:55 (v / v); detection wavelength: 226 nm; injection volume: 20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃.

[0084] HPLC detection method for triazolol and triazolone: ​​mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 85:15 (v / v); detection wavelength: 220 nm; injection volume: 5 μL; flow rate: 0.8 mL / min; column temperature: 25 ℃.

[0085] As shown in Table 5, among the substrates degraded by ProS32, except for PTC, ProS32 could not degrade triazole fungicides such as propiconazole, difenoconazole, tebuconazole, tricyclazole, triazole alcohol, and triadimefon. Based on the structural formulas of each substance, it can be seen that ProS32 is a methyltransferase with selective action site on the substrate, and its action atom is a sulfur atom.

[0086] Table 5. Substrate spectrum of ProS32

[0087]

[0088] Note: "+" indicates that the material has degradation ability; "-" indicates that it does not have degradation ability.

[0089] 3.5 Study on the metabolic pathway of PTC degradation by ProS32

[0090] After terminating the reaction with acetonitrile, the ProS32 enzyme reaction solution was vortexed, centrifuged at 12,000 rpm for 2 min, filtered through a 0.22 μm organic phase filter, and the product was detected by HPLC and UPLC-Q-TOF-MS.

[0091] See Figures 8-9 The HPLC chromatogram shows that ProS32 degrades ( Rac During the PTC process, ( R PTC is preferentially degraded. Figure 8 ProS32 catalyst (Rac The product generated by PTC was detected by mass spectrometry using a primary mass spectrometer. m / z =358.0543(M+CH2+H) + ( Figure 9 (A) corresponds to the relative molecular weight of PTM. Further secondary mass spectrometry analysis was then performed on it (…). Figure 9 (B), and a fragment peak was found. m / z =(M+H) + This is consistent with the PTM molecular weight of the strain AJ-1, which lost one o-chlorobenzyl group. Rac The results are consistent with the identification of the metabolites of )-PTC. These results indicate that ProS32 catalyzes ( Rac -PTC generates PTM.

[0092] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A prothioconazole methyltransferase gene proS32 Genes, characterized by, The proS32 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A prothioconazole methyltransferase ProS32 ， It is characterized by having an amino acid sequence as shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The expression vector contains the prothioconazole methyltransferase according to claim 1. proS32 Gene.

4. A strain, characterized in that, The strain contains an expression vector containing the prothioconazole methyltransferase as described in claim 1. proS32 Gene.

5. Contains prothioconazole methyltransferase proS32 The application of strains with gene expression vectors in the degradation of prothioconazole is characterized by, The proS32 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, The strain in question is Escherichia coli.

7. The application of prothioconazole methyltransferase ProS32 in the degradation of prothioconazole, characterized in that, The amino acid sequence of prothioconazole methyltransferase ProS32 is shown in SEQ ID NO.

2.

8. A formulation for degrading prothioconazole, characterized in that, The formulation contains prothioconazole methyltransferase. proS32 The strain containing the gene expression vector; or the preparation containing prothioconazole methyltransferase ProS32, wherein... proS32 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the prothioconazole methyltransferase ProS32 is shown in SEQ ID NO.

2.

9. An immobilized enzyme preparation, characterized in that, The immobilized enzyme in the immobilized enzyme preparation is prothioconazole methyltransferase ProS32, whose amino acid sequence is shown in SEQ ID NO.2.

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