A p450 enzyme mutant degrading herbicides and application thereof
By constructing P450BSβ-F46A and P450BSβ-F46A-CPR enzyme mutants, the problem of the lack of identification of plant P450 enzymes was solved, and the efficient degradation of pyridine oxycarboxylic acid and aryloxyphenoxypropionic acid herbicides was achieved, promoting the cultivation of resistance in transgenic crops and the environmentally friendly application of herbicides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2026-06-25
- Publication Date
- 2026-07-24
AI Technical Summary
In the current technology, plant-derived P450 enzymes have not been identified as being able to effectively degrade pyridine oxycarboxylic acids and aryloxyphenoxypropionic acids, resulting in difficulty in efficiently degrading residual herbicides in the soil, which affects the implementation of crop rotation systems and the cultivation of herbicide-resistant transgenic crops.
The multifunctional CYP152 peroxygenase P450BSβ and its mutants P450BSβ-F46A and P450BSβ-F46A-CPR were constructed and expressed through genetic engineering to achieve efficient degradation of pyridine oxycarboxylic acids and aryloxyphenoxypropionic acids herbicides, generating fatty acid side chain breakage products.
It achieves highly efficient catalytic conversion rates of pyridine oxycarboxylic acids and aryloxyphenoxypropionic acids, reaching 86% and 89% respectively, with low degradation costs, making it suitable for resistance cultivation in transgenic crops and solving the adverse effects of herbicide residues on subsequent crops.
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Abstract
Description
Technical Field
[0001] This invention belongs to the fields of enzyme engineering, biocatalysis, biochemistry and molecular biology, and relates to a P450 enzyme mutant that degrades herbicides and its application. Background Technology
[0002] Pyridine-oxycarboxylic acid herbicides belong to the auxin class of herbicides, with a core structure consisting of a pyridine heterocycle and a carboxylic acid functional group. Their mechanism of action involves interfering with plant hormone balance, disrupting the regulation of cell growth and development, leading to malformed weed growth and eventual death. As an important class of herbicides, pyridine-oxycarboxylic acid herbicides, mainly including fluroxypyr and fluchloraminopyr, are widely used for weed control and management in agroforestry ecosystems due to their broad-spectrum herbicidal activity, rapid efficacy, and low cost, thus promoting the development of precision agriculture and sustainable plant protection technologies.
[0003] However, while the large-scale use of herbicides has promoted agricultural production, it has also brought about significant environmental problems. The residues of these herbicides in the soil can adversely affect the normal growth of subsequent sensitive crops, severely hindering the proper implementation of crop rotation systems. Currently, chemical hydrolysis and microbial degradation are the two main pathways for removing herbicide residues from the soil. Microbial degradation of herbicides is primarily accomplished through the metabolic action of enzymes produced by the microorganisms. Essentially an enzymatic reaction, it is a complex physiological and biochemical process requiring multiple reactions both intracellularly and extracellularly. Through synergistic action, the herbicide is completely degraded or degraded into smaller, less toxic molecules. The main types of microbial degradation of herbicides include: side chain cleavage, ester bond hydrolysis, dehalogenation, aromatic oxidation, methylation, and ring cleavage. Among these, pyridineoxycarboxylic acid herbicides and aryloxyphenoxypropionic acid herbicides, similar to phenoxycarboxylic acid herbicides, require the removal of fatty acid side chains to generate phenolic products with significantly reduced toxicity. This has been shown in some plants to be mediated by P450 enzymes, and this degradation pathway is also considered a herbicide resistance mechanism in some plants. However, plant-derived P450 enzymes with this catalytic function have not yet been identified. Furthermore, the search for genes capable of degrading or resisting herbicides is crucial for developing transgenic crops resistant to pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides.
[0004] The applicant's research found that, if the multifunctional CYP152 peroxygenase P450, with its known crystal structure, high reactivity, and high catalytic efficiency, is selected... BSβ Using Bacillus subtilis and its mutants as candidate enzymes, we screen for highly efficient enzymes that can degrade pyridine oxycarboxylic acid herbicides or aryloxyphenoxypropionic acid herbicides. This holds promise for achieving the low-cost and efficient degradation of various pyridine oxycarboxylic acid herbicides or aryloxyphenoxypropionic acid herbicides, generating their fatty acid side-chain cleavage products, phenols, and corresponding aldehydes or keto acids.Bacillus subtilis P450 BSβ Its mutants can utilize H2O2 as the sole electron and oxygen donor to catalyze different reactions on the substrate. A literature and patent search revealed that no domestic or international literature currently employs P450. BSβ Reports on the degradation of pyridine oxycarboxylic acid herbicides or aryloxyphenoxypropionic acid herbicides by their mutants. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a class of P450 enzyme mutants capable of degrading pyridine oxycarboxylic acids or aryloxyphenoxypropionic acids and their applications.
[0006] The technical solution provided by this invention is as follows: a P450 enzyme mutant for degrading herbicides, wherein the P450 enzyme mutant is P450. BSβ -F46A, this mutant is formed by the mutation of phenylalanine to alanine at the 46th amino acid position of the P450 enzyme, as shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the herbicide is a pyridineoxycarboxylic acid herbicide or an aryloxyphenoxypropionic acid herbicide.
[0007] In some preferred embodiments, the P450 enzyme mutant is P450. BSβ -F46A-CPR, this mutant is derived from P450 BSβ -F46A is fused with the redox chaperone protein CPR to form the amino acid sequence shown in SEQ ID NO.4.
[0008] This invention provides a recombinant expression vector for expressing the P450 enzyme mutant, characterized in that: the recombinant expression vectors are pET28b- P450 BSβ -F46A、 pET28b- P450 BSβ -F46A-CPR Or pET30a- P450 BSβ -F46A、 pET30a- P450 BSβ -F46A-CPR .
[0009] This invention provides genetically engineered bacteria expressing the P450 enzyme mutant, wherein the genetically engineered bacteria are respectively the recombinant expression vector pET28b- of the above-mentioned P450 enzyme mutant. P450 BSβ -F46A、 pET28b- P450 BSβ -F46A-CPROr pET30a- P450 BSβ -F46A、 pET30a- P450 BSβ -F46A-CPR E. coli that were transformed separately.
[0010] In some preferred embodiments, the genetically engineered bacteria are preferably BL21(DE3)-pET28b-P450. BSβ -F46A、BL21(DE3)-pET28b-P450 BSβ -F46A-CPR.
[0011] The present invention also provides the application of the P450 enzyme mutant, the recombinant expression vector, and the genetically engineered bacteria in any of the following: Preparation of compositions for degrading pyridine oxycarboxylic acids or aryloxyphenoxypropionic acids as herbicides; Degrades pyridine oxycarboxylic acids or aryloxyphenoxypropionic acids as herbicides.
[0012] The present invention also provides the application of the gene of the P450 enzyme mutant in the cultivation of transgenic crops resistant to pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides; wherein the crops refer to cotton, soybean, peanut, rice, wheat, corn, millet or sugarcane.
[0013] Furthermore, the present invention also provides a composition for degrading pyridineoxycarboxylic acid or aryloxyphenoxypropionic acid herbicides, the composition comprising the P450 enzyme mutant.
[0014] Furthermore, the present invention also provides a method for degrading pyridineoxycarboxylic acid or aryloxyphenoxypropionic acid herbicides, by reacting the P450 enzyme mutant or the composition with a pyridineoxycarboxylic acid or aryloxyphenoxypropionic acid herbicide substrate.
[0015] This invention discloses a group of P450 enzyme mutants capable of degrading pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides, their application in degrading pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides, and the application of related genes in breeding transgenic crops resistant to pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides. This application screens P450. BSβ The mutants yielded a group of mutants that efficiently degrade pyridine oxycarboxylic acids or aryloxyphenoxypropionic acids: P450 BSβ -F46A, P450 BSβ-F46A-CPR. Experiments have confirmed that its catalytic conversion rate for degrading pyridine oxycarboxylic acid herbicides reaches up to 86%, and the highest conversion number for degrading the herbicide fluchlorfon reaches 7443; the catalytic conversion rate for degrading aryloxyphenoxypropionic acid herbicides reaches up to 89%. The P450 enzyme mutant described in this invention has the advantages of high catalytic efficiency, high protein expression level, and low industrial cost. The entire herbicide degradation process is simple, mature, and inexpensive. It is expected that the P450 enzyme mutant for degrading pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides provided by this invention has broad application prospects in herbicide degradation and in the cultivation of transgenic crops resistant to pyridine oxycarboxylic acid or aryloxyphenoxypropionic acid herbicides, and its application in the field of pesticide degradation is promising. Attached Figure Description
[0016] Figure 1 The chemical structural formula for the CO bond breaking and degradation of pyridine oxycarboxylic acid herbicides provided by this invention; Figure 2 P450 provided for this invention BSβ HPLC chromatogram of fluroxypyr degradation by -F46A; Figure 3 P450 provided for this invention BSβ HPLC chromatogram of F46A degradation of fluchloraminopyr; Figure 4 P450 provided for this invention BSβ Catalytic activity analysis of F46A-CPR in the degradation of fluchloraminopyr; Figure 5 The chemical structural formula for the CO bond breaking and degradation of aryloxyphenoxypropionic acid herbicides provided by this invention; Figure 6 P450 provided for this invention BSβ HPLC chromatogram of F46A degrading highly effective flupyradifurone; Figure 7 P450 provided for this invention BSβ HPLC chromatogram of F46A degrading cyhalofop-butyl. Detailed Implementation
[0017] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.
[0018] Unless otherwise specified, all materials, reagents, plasmids, strains, kits, etc. used in the following examples were obtained commercially.
[0019] Among them, the strains used in the embodiments of the present invention Escherichia coli BL21(DE3) competent cells were purchased from Beijing Qingke, and the *E. coli* expression vector pET28b used was purchased from Invitrogen. The P450 gene... BSβ The nucleotide sequence is shown in SEQ ID NO. 5.
[0020] Culture medium used in the examples: LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; TB medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 40 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L.
[0021] Protein purification buffer used in the examples: Lysis buffer (pH=8.0): NaH2PO4 6 g / L, NaCl 17.532 g / L, glycerol 100 g / L, imidazole 0.6808 g / L.
[0022] Washing buffer (pH=8.0): NaH2PO4 6 g / L, NaCl 17.532 g / L, glycerol 100 g / L, imidazole 1.3616 g / L.
[0023] Elution buffer (pH=8.0): NaH2PO4 6 g / L, NaCl 17.532 g / L, glycerol 100 g / L, imidazole 17.02 g / L.
[0024] Desalting buffer (pH=7.4): NaH2PO4 6 g / L, glycerol 100 g / L.
[0025] Reagents used in the examples: The one-step cloning kit used in the embodiments of this invention was purchased from Nanjing Novizan Pharmaceutical Co., Ltd.; the agarose gel DNA recovery kit and plasmid extraction kit were both purchased from Omega Pharmaceutical Co., Ltd.; the high-fidelity DNA polymerase was purchased from Takara; the restriction endonuclease was purchased from Thermo Fisher Scientific; PCR primer synthesis and DNA sequencing were performed by Shanghai Sangon Biotech Co., Ltd.; fluroxypyr, haloxyfop-R-methyl, cyhalofop-butyl and NADPH were purchased from Sigma, Macklin and Aladdin, respectively.
[0026] Instruments used in the examples: PCR amplification instrument (Eppendorf), high-speed refrigerated centrifuge (Eppendorf), agarose gel electrophoresis system (BIO-RAD), agarose gel imaging system (Shanghai Tianneng Technology Co., Ltd.), constant temperature shaker (Jingqi), SpectraMAX M2 multi-functional microplate reader (Molecular Devices), Agilent high performance liquid chromatograph (Agilent Technologies).
[0027] Example 1 P450 BSβ Construction of mutants P450 was found in previous experimental results. BSβ It exhibits higher stability. (Based on P450) BSβ The crystal structure of the complex with the fatty acid palmitic acid substrate (PDB: 1IZO) shows that binding to pyridine oxycarboxylic acid herbicides requires significant spatial constraints. The applicant aims to facilitate substrate entry and binding at the active site by shaping the substrate channel space. The Phe46 mutation in the substrate active pocket was selected to replace the smaller alanine. Using P450... BSβ Using the P450 mutant as a starting template, site-directed mutagenesis was employed to construct the mutant. BSβ -F46A.
[0028] The specific steps are as follows: Using the gene with the nucleotide sequence SEQ ID NO.5 as a template, PCR amplification is performed using F46A-F / F46A-R primers. Alternatively, using the gene with the nucleotide sequence SEQ ID NO.6 as a template, PCR amplification is performed using CPR-F / CPR-R primers. PCR program: 10 μL of 5×PrimeSTAR GXL Buffer, 200 μM dNTPs, 0.3 μM each of forward and reverse primers, appropriate amount of DNA template (10-100 ng), 2.5 U of high-fidelity polymerase (PrimeSTAR GXL DNA polymerase), and ddH2O to a final volume of 50 μL; reaction conditions are: 98°C pre-denaturation for 5 min, 98°C denaturation for 10 s, 60°C annealing for 15 s, 68°C extension for 4 min, 30 cycles, and a final extension at 68°C for 10 min. After the PCR product was purified by a nucleic acid purification kit, the expression plasmid was obtained using a one-step cloning kit and then directly transformed into E. coli BL21(DE3) competent cells to construct the mutant.
[0029] Three to four single colonies were randomly selected and inoculated into 3 mL LB broth containing 50 μg / mL kanamycin and cultured overnight. The selected single colonies were then sequenced, and the P450 values obtained from the sequencing were used to determine the sequence accuracy. BSβ -F46A or P450 BSβAdd an equal volume of sterile 40% glycerol to the -F46A-CPR mutant bacterial culture and freeze at -80°C to complete P450. BSβ Construction of mutants.
[0030] Table 1: Primer sequences used in the examples .
[0031] Example 2 P450 BSβ Fermentation expression, nickel column affinity chromatography purification, and protein concentration determination of mutant proteins The P450 samples from Example 1, which were frozen at -80°C, were respectively... BSβ -F46A or P450 BSβ The -F46A-CPR strain was removed from glycerol tubes and activated by streak plating on kanamycin-resistant LB agar plates, then incubated at 37°C for 12–16 h. Single colonies were picked and inoculated into 50 mL LB liquid medium containing kanamycin, incubated at 37°C. o Culturing at 220 rpm for 12-16 h; transferring the cultured seed culture to 500 mL of TB liquid medium containing kanamycin at a volume ratio of 1:100, and culturing at 37°C and 220 rpm for 3-4 h; waiting for OD 600 When the concentration is 0.8-1.0, add IPTG to a final concentration of 0.15 mM, and add 5-ALA and VB1 to a final concentration of 0.5 mM. At 18°C... o C, 180 rpm induces protein expression for 20-24 h; 6000 rpm, 4 o Centrifuge at C for 10 min and collect P450. BSβ Mutant bacterial cells, frozen at -80°C o C.
[0032] (1) Protein purification From -80 o C retrieved the frozen P450. BSβ After thawing the mutant cells at room temperature, add 35 mL of lysis buffer per 1 L of fermentation broth and resuspend on ice. Perform ultrasonic disruption on ice: 25% power, 5 s operation, 5 s interval, for approximately 30-35 min until the solution becomes clear. The disrupted suspension is then incubated at 4°C. o Centrifuge at 10000 rpm for 60 min at C, collect the supernatant, add 1-2 mL of nickel column (Ni-NTA) resin, and incubate at 4°C. o Incubate at C for 1-2 h; place the mixed solution in a protein separation column, add approximately 5 column volumes of washing buffer for washing until no protein efflux is detected by G250 staining solution; elute the target protein from the nickel column using 5-10 mL of elution buffer and collect; incubate at 4...o At C, the protein was concentrated using a Millipore ultrafiltration tube (50 kDa) at 5500 rpm until the target protein eluent volume was 1-2 mL. The GE Healthcare PD-10 desalting column was first equilibrated with 5x desalting buffer, then the concentrated protein eluent was added to the column. After the sample solution had completely entered the column, desalting buffer was added for elution. The eluted protein was collected, centrifuged, and ultrafiltered to concentrate to 1-2 mL. The concentrated protein was mixed and aliquoted into 100 μL tubes. The purified proteins were named P450. BSβ -F46A or P450 BSβ -F46A-CPR, flash-frozen in liquid nitrogen, stored at -80°C o C.
[0033] (2) Protein concentration determination The corresponding P450 protein concentration was determined using the previously reported CO difference spectroscopy method.
[0034] To prepare the ferrous complex reduced by sodium dithionite to P450 enzyme, 50-80 μL of P450 enzyme mutant protein was diluted to 900 μL with desalting buffer (pH 7.4, 50 mM NaH2PO4, 10% glycerol). CO gas was slowly passed through the sample in a fume hood. The CO-saturated P450 sample was transferred to a cuvette and scanned across the entire wavelength range of 350-500 nm using a spectrophotometer. The cuvette was then removed, and an appropriate amount of sodium hydrosulfite (Na2S2O4) was added. The entire wavelength range was scanned again. Based on the absorbance values measured twice, the P450 activity concentration was calculated using the difference between the absorbance at 450 nm and 490 nm, using the formula: dilution factor × (ΔA450 - ΔA490) / 0.091, in μM.
[0035] Example 3 P450 BSβ Assay of mutant protein activity in degrading pyridineoxycarboxylic acid herbicides The P450 obtained in Example 2 BSβ The mutant protein was reacted in the following 200 μL reaction system: 1 μM P450 enzyme (P450 BSβ -F46A), 500 μM substrate (flufenoxam or flufenoxam), 5 μM AldO (sugar alcohol oxidase), reaction at 30 ± 2℃ for 6 ± 1 h; or 1 μM P450 enzyme (P450 BSβ -F46A-CPR), 500 μM substrate (flufenoxam or flufenoxam), 1 mM NADPH, reaction at 30 ± 2℃ for 6 ± 1 h. An equal volume of acetonitrile was added to terminate the reaction. After high-speed centrifugation, the supernatant was collected for HPLC analysis, and relative quantification was performed based on the amount of substrate consumed.
[0036] P450 BSβ -F46A catalyzes the formation of 4-amino-3,5-dichloro-6-fluoropyridin-2-ol from fluroxypyr or fluroxypyr, with catalytic activities of 14% and 86%, respectively, from fluroxypyr or fluroxypyr. Figures 1-3 ).
[0037] Experiments confirmed that the above method yielded P450. BSβ The mutant can indeed catalyze the degradation of pyridine oxycarboxylic acid herbicide substrates, which means that the P450 enzyme mutant for degrading pyridine oxycarboxylic acid herbicides provided by this invention has great application potential in pesticide degradation.
[0038] Example 4 P450 BSβ Calculation of the total conversion number of mutant protein degrading pyridine oxycarboxylic acid herbicides Total transformation number calculation experiment: To further evaluate the application potential of the P450 enzyme mutant in the degradation of pyridine oxycarboxylic acid herbicides, the P450 enzyme mutant protein was tested in the following 200 μL reaction system: 0.5 μM or 1 μM P450 enzyme (P450 BSβ -F46A), 10 mM flufenoxuron, 5 μM AldO (sugar alcohol oxidase), reacted at 30 ± 2℃ for 12 ± 1 h. An equal volume of acetonitrile was added to terminate the reaction. After high-speed centrifugation, the supernatant was collected for HPLC analysis, and the total conversion number was analyzed based on the amount of substrate consumed.
[0039] The results showed P450 BSβ -F46A can degrade the herbicide fluchlorfon at a maximum conversion number of 7443.
[0040] Example 5, P450 BSβ Activity assay of mutant protein in degrading aryloxyphenoxypropionic acid herbicides The P450 obtained in Example 2 BSβ The mutant protein was reacted in the following 200 μL reaction system: 1 μM P450 enzyme (P450 BSβ -F46A), 500 μM substrate (quizalofop-P-ethyl or cyhalofop-P-ethyl), 5 μM AldO (sugar alcohol oxidase), reaction at 30±2℃ for 6±1 hours; or 1 μM P450 enzyme (P450 BSβ-F46A-CPR), 500 μM substrate (quizalofop-P-ethyl or cyhalofop-butyl), 2 mM NADPH, reaction at 30 ± 2℃ for 6 ± 1 h. An equal volume of acetonitrile was added to terminate the reaction. After high-speed centrifugation, the supernatant was collected for HPLC analysis, and relative quantification was performed based on the amount of substrate consumed.
[0041] P450 BSβ -F46A catalyzes the highly efficient formation of haloxyfop-methyl or cyhalofop-butyl into products with side-chain carboxyl cleavage, namely 4-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)oxy)phenol or 3-fluoro-4-(4-hydroxyphenoxy)benzonitrile, with catalytic activities of 86% and 89%, respectively. Figures 5-7 ).
[0042] Experiments confirmed that the above method yielded P450. BSβ The mutant can indeed catalyze the degradation of aryloxyphenoxypropionic acid herbicide substrates, which means that the P450 enzyme mutant for degrading aryloxyphenoxypropionic acid herbicides provided by this invention has great application potential in pesticide degradation.
[0043] Example 6: Application of the P450 enzyme mutant gene in the breeding of transgenic crops resistant to pyridine oxycarboxylic acid herbicides The target gene (P450) BSβ A gene-editing vector (F46A-CPR) was constructed for expression in plants. After identification, a plant expression vector carrying the target gene was obtained. This vector was then introduced into a stable expression system using Agrobacterium-mediated transformation or direct transformation of exogenous DNA (such as gene gun transformation). After genetic transformation, transgenic positive seedlings were screened using marker genes such as antibiotics and pyridine oxycarboxylic acid herbicides during plant cell culture. PCR was then used for detection and verification to finally identify transgenic monocotyledonous plant seedlings containing the aforementioned target gene and resistant to pyridine oxycarboxylic acid herbicides. The preferred monocotyledonous plant is rice.
[0044] Experiments have confirmed that the transgenic positive seedlings resistant to pyridine oxycarboxylic acids possess the characteristic of resistance to pyridine oxycarboxylic acid herbicides. The P450 enzyme mutant gene (P450) described in this invention... BSβ -F46A or P450 BSβ-F46A-CPR) can be widely used in the breeding of resistant transgenic crops of monocotyledonous plants sensitive to pyridine oxycarboxylic acid herbicides. It can overcome the selectivity problem of herbicides, enable pyridine oxycarboxylic acid herbicides to be used more widely, and will not affect the normal growth of subsequent pyridine oxycarboxylic acid herbicide-resistant transgenic crops, thus having a large market demand.
[0045] Example 7: Application of the P450 enzyme mutant gene in the breeding of transgenic crops resistant to aryloxyphenoxypropionic acid herbicides The target gene (P450) BSβ A gene-editing vector (F46A-CPR) was constructed to express the gene in plants. After identification, the plant expression vector carrying the target gene was obtained. This vector was then introduced into a stable expression system using Agrobacterium-mediated transformation or direct transformation of exogenous DNA (such as gene gun transformation). After genetic transformation, transgenic positive seedlings were screened using marker genes such as antibiotics and aryloxyphenoxypropionic acid herbicides during plant cell culture. PCR was then used for detection and verification to finally identify positive transgenic monocotyledonous plant seedlings containing the aforementioned target gene and resistant to aryloxyphenoxypropionic acid. The preferred monocotyledonous plant is rice.
[0046] Experiments have confirmed that the transgenic positive seedlings resistant to aryloxyphenoxypropionic acid (PAR) possess the characteristic of resistance to PAR herbicides. The P450 enzyme mutant gene (P450) described in this invention... BSβ -F46A or P450 BSβ -F46A-CPR) can be widely used in the breeding of resistant transgenic crops of monocotyledonous plants sensitive to aryloxyphenoxypropionic acid herbicides. It can overcome the selectivity problem of herbicides, enable aryloxyphenoxypropionic acid herbicides to be used more widely, and will not affect the normal growth of subsequent aryloxyphenoxypropionic acid herbicide-resistant transgenic crops, thus having a large market demand.
[0047] Example 8: P450 enzyme mutant P450 BSβ -F46A and its P450 BSβ Preparation method of -F46A-CPR (1) P450 with the nucleotide sequence shown in SEQ ID NO.5 BSβ Using the gene as a template, PCR amplification was performed with F46A-F / F46A-R primers to obtain the gene named P450 BSβ -F46A PCR products; or nucleotide sequences as shown in SEQ ID NO. 6. P450 BSβ -F46A Using the gene as a template, PCR amplification was performed with CPR-F / CPR-R primers to obtain the gene namedP450 BSβ - F46A-CPR The PCR product was then used; subsequently, the gene carrying the PCR product was constructed using the E. coli expression vector pET28b. P450 BSβ -F46A or P450 BSβ -F46A-CPR The expression vector; the constructed expression vector pET28b- P450 BSβ -F46A or P450 BSβ -F46A-CPR The transformants were transformed into E. coli BL21(DE3) chemocompetent cells, and the resulting transformants were named BL21(DE3)-pET28b-P450, respectively. BSβ -F46A、BL21(DE3)-pET28b-P450 BSβ -F46A-CPR; where the nucleotide sequences of the above primers are shown in Table 1.
[0048] (2) The transformants obtained above were inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured overnight at 37 °C and 220 rpm to prepare two fermentation seed cultures; the two fermentation seed cultures were inoculated into TB medium and cultured at 37 °C and 220 rpm until OD. 600 After adjusting the concentration to 1.0 ± 0.1, 0.2 mM IPTG was added to the fermentation broth, and the culture was incubated at 18℃ and 180 rpm for 24 h. The cultured bacterial broths were centrifuged, the supernatant was discarded, and the bacterial cells were collected to obtain five protein-producing bacterial cells, named BL21(DE3)-pET28b-P450. BSβ -F46A、BL21(DE3)-pET28b-P450 BSβ -F46A-CPR, store frozen at -80℃; (3) The protein-producing bacterial cells were ultrasonically disrupted and then purified by nickel column affinity chromatography to obtain the purified protein of the P450 enzyme mutant. The purified protein was named P450. BSβ -F46A, P450 BSβ -F46A-CPR, frozen in liquid nitrogen, stored at -80℃.
[0049] (4) Determination of P450 enzyme protein concentration: Take 50-80 μL of P450 enzyme. BSβ -F46A or P450 BSβDilute -F46A-CPR to 900 μL with desalting buffer, and slowly pass CO gas through the sample in a fume hood; transfer the CO-saturated P450 sample into a cuvette, place it in a spectrophotometer, and perform a full wavelength scan from 350 to 500 nm; remove the cuvette, add an appropriate amount of sodium hydrosulfite (Na2S2O4), and perform another full wavelength scan; based on the absorbance values measured twice, calculate the P450 activity concentration according to the difference in absorbance at 450 nm and 490 nm, using the formula: dilution factor × (ΔA450 - ΔA490) / 0.091, in μM.
Claims
1. A P450 enzyme mutant for degrading herbicides, characterized in that: The P450 enzyme mutant is P450. BSβ -F46A, this mutant is formed by the mutation of phenylalanine to alanine at the 46th amino acid position of the P450 enzyme, as shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the herbicide is a pyridineoxycarboxylic acid herbicide or an aryloxyphenoxypropionic acid herbicide.
2. The P450 enzyme mutant according to claim 1, characterized in that: The P450 enzyme mutant is P450. BSβ -F46A-CPR, this mutant is derived from P450 BSβ -F46A is fused with the redox chaperone protein CPR to form the amino acid sequence shown in SEQ ID NO.
4.
3. A recombinant expression vector for expressing the P450 enzyme mutant as described in claim 1 or 2, characterized in that: The recombinant expression vector is divided into pET28b- P450 BSβ -F46A pET28b- P450 BSβ -F46A-CPR pET30a- P450 BSβ -F46A、 pET30a- P450 BSβ -F46A-CPR Any one of them.
4. A genetically engineered bacterium expressing the P450 enzyme mutant of claim 1 or 2, characterized in that: The genetically engineered bacteria comprises the recombinant expression vector as described in claim 3.
5. The genetically engineered bacterium according to claim 4, characterized in that: Selected from BL21(DE3)-pET28b-P450 BSβ -F46A or BL21(DE3)-pET28b-P450 BSβ -F46A-CPR.
6. The use of the P450 enzyme mutant of claim 1 or 2, the recombinant expression vector of claim 3, and the genetically engineered bacteria of claim 4 or 5 in any of the following: preparing a composition for degrading pyridineoxycarboxylic acid herbicides or aryloxyphenoxypropionic acid herbicides; degrading pyridineoxycarboxylic acid herbicides or aryloxyphenoxypropionic acid herbicides.
7. The application of the gene of the P450 enzyme mutant described in claim 1 or 2 in the breeding of herbicide-resistant transgenic crops; wherein, The crops referred to are cotton, soybeans, peanuts, rice, wheat, corn, millet, or sugarcane.
8. A herbicide composition, characterized in that: The composition contains the P450 enzyme mutant as described in claim 1 or 2.
9. A method for degrading a herbicide, using the P450 enzyme mutant of claim 1 or 2 or the composition of claim 8 with a pyridineoxycarboxylic acid herbicide or an aryloxyphenoxypropionic acid herbicide.