Method for improving activity of 5-enolpyruvylshikimate-3-phosphate synthase, and use thereof

Abstract

A method for improving the activity of a 5-enolpyruvylshikimate-3-phosphate synthase, and the use thereof. The present invention particularly relates to a method for improving the activity of a 5-enolpyruvylshikimate-3-phosphate synthase, a protein having the activity of the 5-enolpyruvylshikimate-3-phosphate synthase, a gene encoding the protein, a recombinant vector containing the gene, a transformant carrying the gene, a method for preparing the 5-enolpyruvylshikimate-3-phosphate synthase, and the use of the 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate resistance. The 5-enolpyruvylshikimate-3-phosphate synthase has high glyphosate resistance which is 3 times of that of wild-type 5-enolpyruvylshikimate-3-phosphate synthases. The present invention endows target plants with higher glyphosate tolerance capacity.

Description

Method for improving activity of 5-enolpyruvylshikimate-3-phosphate synthase and application thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410732088.X, filed on June 6, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biotechnology, in particular, to a method for improving activity of 5-enolpyruvylshikimate-3-phosphate synthase, a protein with 5-enolpyruvylshikimate-3-phosphate synthase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing 5-enolpyruvylshikimate-3-phosphate synthase, and application of 5-enolpyruvylshikimate-3-phosphate synthase in resisting glyphosate. BACKGROUND

[0004] Glyphosate is one of the most widely used herbicide varieties in the world. Studies have found that glyphosate blocks the shikimic acid metabolic pathway by competitively binding EPSPs (5-enolpyruvylshikimate-3-phosphate synthase, EPSP synthase), thereby preventing plants from synthesizing aromatic amino acids and their derivatives, and causing accumulation of shikimic acid, leading to plant death. Because most plants in nature have EPSP synthase that is sensitive to glyphosate, glyphosate herbicide has excellent broad-spectrum properties. However, the diversity of microorganisms in nature leads to the diversity of EPSP synthase in microorganisms, and some microorganisms have EPSP synthase that is not sensitive to glyphosate. By using genetic engineering technology to transfer the EPSP synthase that is not sensitive to glyphosate into target plants to replace the endogenous sensitive EPSP synthase, the plants can obtain glyphosate resistance.

[0005] Research and development of glyphosate-resistant transgenic plants can greatly promote agricultural development. Studies have shown that the expression ability of EPSP synthase coding gene (aroA) introduced into different plants varies, and the recipient plants exhibit different tolerance to glyphosate herbicide. Increasing the diversity of glyphosate-resistant genes can improve the success rate of herbicide-resistant biotechnological breeding, but the excellent glyphosate-resistant gene resources are limited, which cannot meet the current demand of the agricultural market. Therefore, developing 5-enolpyruvylshikimate-3-phosphate synthase with glyphosate resistance has important research value and application prospect in agriculture.

[0006] SUMMARY

[0007] To further meet the needs of practical applications, the present disclosure provides a method for improving the activity of 5-enolpyruvylshikimate-3-phosphate synthase, a protein having 5-enolpyruvylshikimate-3-phosphate synthase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing 5-enolpyruvylshikimate-3-phosphate synthase, and the use of 5-enolpyruvylshikimate-3-phosphate synthase in resisting glyphosate.

[0008] To achieve the above-mentioned object, the present disclosure provides a method for improving the activity of 5-enolpyruvylshikimate-3-phosphate synthase, the method comprising mutating three amino acid residues to be mutated of a wild-type 5-enolpyruvylshikimate-3-phosphate synthase, the amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase being shown as SEQ ID NO. 2, the three amino acid residues to be mutated including a tyrosine residue at position 40, a phenylalanine residue at position 114, and a serine residue at position 358; the mutation being substitution of the amino acid residues.

[0009] Optionally, the mutation comprises mutating the tyrosine residue at position 40 into an isoleucine residue, mutating the phenylalanine residue at position 114 into a serine residue, and mutating the serine residue at position 358 into a threonine residue.

[0010] The present disclosure provides a protein having 5-enolpyruvylshikimate-3-phosphate synthase activity, the protein being obtained by mutating three amino acid residues to be mutated of a wild-type 5-enolpyruvylshikimate-3-phosphate synthase, the amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase being shown as SEQ ID NO. 2, the three amino acid residues to be mutated including a tyrosine residue at position 40, a phenylalanine residue at position 114, and a serine residue at position 358; the mutation being substitution of the amino acid residues.

[0011] Optionally, the amino acid sequence of the protein is shown as SEQ ID NO. 1.

[0012] The present disclosure provides a gene encoding the protein of the second aspect, the gene being a DNA molecule with the nucleotide sequence shown as SEQ ID NO. 3.

[0013] The present disclosure provides a recombinant vector, the recombinant vector being a recombinant expression vector, the recombinant vector into which the gene of the third aspect is inserted.

[0014] The fifth aspect of the present disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; and the gene introduced into the transformant comprises the gene of the third aspect, or the transformant has the recombinant vector of the fourth aspect introduced therein.

[0015] Optionally, the genetically engineered bacterium is selected from one of Escherichia coli, Bacillus subtilis, Rhizobium, Pseudomonas and Azotobacter.

[0016] The sixth aspect of the present disclosure provides a method for preparing 5-enolpyruvylshikimate-3-phosphate synthase, comprising inoculating the transformant of the fifth aspect into a culture medium for culture to obtain a cultured material.

[0017] The seventh aspect of the present disclosure provides an application of 5-enolpyruvylshikimate-3-phosphate synthase in resisting glyphosate, wherein the 5-enolpyruvylshikimate-3-phosphate synthase comprises the protein of the second aspect.

[0018] By means of the above technical solution, the present disclosure provides a method for improving the activity of 5-enolpyruvylshikimate-3-phosphate synthase, a protein having 5-enolpyruvylshikimate-3-phosphate synthase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing 5-enolpyruvylshikimate-3-phosphate synthase, and an application of 5-enolpyruvylshikimate-3-phosphate synthase in resisting glyphosate. The 5-enolpyruvylshikimate-3-phosphate synthase of the present disclosure has strong glyphosate resistance, which is 3 times that of wild-type 5-enolpyruvylshikimate-3-phosphate synthase. The target plant is endowed with higher glyphosate tolerance.

[0019] Other features and advantages of the present disclosure will be described in detail in the following specific embodiments section. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings are included to provide a further understanding of the present disclosure and constitute a part of the specification, and together with the specific embodiments below, serve to explain the present disclosure but do not constitute a limitation thereof. In the drawings:

[0021] Figure 1 is a graph showing the tolerance analysis of mutant G79M3 to glyphosate; wherein (a) is the glyphosate concentration of 0 mM, (b) is the glyphosate concentration of 200 mM, (c) is the glyphosate concentration of 300 mM, and (d) is the glyphosate concentration of 400 mM.

[0022] Figure 2 is a SDS-PAGE electrophoresis diagram of GR79 protein expression and purification.

[0023] Figure 3 is a SDS-PAGE electrophoresis diagram of G79M3 protein expression and purification.

[0024] Figure 4 is a standard curve of inorganic phosphorus. DETAILED DESCRIPTION

[0025] The specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present disclosure, and are not intended to limit the present disclosure.

[0026] The first aspect of the present disclosure provides a method for improving the activity of 5-enolpyruvylshikimate-3-phosphate synthase, the method comprising mutating three amino acid residues to be mutated in a wild-type 5-enolpyruvylshikimate-3-phosphate synthase, the amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase being shown in SEQ ID NO. 2, the three amino acid residues to be mutated including a tyrosine residue at position 40, a phenylalanine residue at position 114, and a serine residue at position 358; and the mutation being a substitution of the amino acid residues.

[0027] The amino acid sequence shown in SEQ ID NO. 2 is shown as follows:

[0028] The nucleotide sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase is shown in SEQ ID NO. 4, and the nucleotide sequence shown in SEQ ID NO. 4 is shown as follows:

[0029] In one embodiment, the mutation includes mutating the tyrosine residue at position 40 to an isoleucine residue, mutating the phenylalanine residue at position 114 to a serine residue, and mutating the serine residue at position 358 to a threonine residue.

[0030] In the present disclosure, the inventors of the present disclosure surprisingly found that the mutant strain (G79M3) with the tyrosine residue at position 40 mutated to an isoleucine residue, the phenylalanine residue at position 114 mutated to a serine residue, and the serine residue at position 358 mutated to a threonine residue has better glyphosate resistance than the wild-type EPSP synthase (GR79), can confer higher glyphosate tolerance to target plants, and G79M3 has a good application prospect in agriculture.

[0031] The second aspect of the present disclosure provides a protein having 5-enolpyruvylshikimate-3-phosphate synthase activity, wherein the protein is a protein having three amino acid residues in a wild-type 5-enolpyruvylshikimate-3-phosphate synthase mutated, the amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase is shown as SEQ ID NO. 2, and the three amino acid residues to be mutated include a tyrosine residue at position 40, a phenylalanine residue at position 114, and a serine residue at position 358; and the mutation is substitution of the amino acid residues.

[0032] According to the present disclosure, the amino acid sequence of the protein is shown as SEQ ID NO. 1.

[0033] The amino acid sequence shown as SEQ ID NO. 1 is shown as follows:

[0034] The third aspect of the present disclosure provides a gene encoding the protein of the second aspect, wherein the gene is a DNA molecule having a nucleotide sequence shown as SEQ ID NO. 3.

[0035] The nucleotide sequence shown as SEQ ID NO. 3 is shown as follows:

[0036] The fourth aspect of the present disclosure provides a recombinant vector, wherein the recombinant vector is a recombinant expression vector, and the recombinant vector is inserted with the gene of the third aspect.

[0037] The fifth aspect of the present disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; and the gene introduced into the transformant includes the gene of the third aspect, or the recombinant vector of the fourth aspect is introduced into the transformant.

[0038] According to the present disclosure, the genetically engineered bacterium can be a wild-type genetically engineered bacterium or an artificially engineered genetically engineered bacterium, and in an embodiment, the genetically engineered bacterium is selected from one of Escherichia coli, Bacillus subtilis, Rhizobium, Pseudomonas, and Azotobacter vinelandii. Preferably, the genetically engineered bacterium can be Escherichia coli BL21 (DE3) competent cells or Escherichia coli ER2799 competent cells. Escherichia coli is an ideal strain for heterologous expression of proteins.

[0039] The sixth aspect of the present disclosure provides a method for preparing 5-enolpyruvylshikimate-3-phosphate synthase, wherein the method comprises inoculating the transformant of the fifth aspect into a culture medium for culture to obtain a cultured material.

[0040] According to the present disclosure, the culture medium inoculated with the transformant can adopt those commonly used by those skilled in the art, for example, M9 culture medium, LB culture medium, YPD culture medium, preferably, IPTG can be added in the culture medium to induce protein expression.

[0041] The seventh aspect of the present disclosure provides an application of 5-enolpyruvylshikimate-3-phosphate synthase in resisting glyphosate, wherein the 5-enolpyruvylshikimate-3-phosphate synthase contains the protein of the second aspect.

[0042] The present disclosure will be further illustrated by examples, but the present disclosure is not limited in any way by the examples.

[0043] In the present disclosure, if no specific experimental conditions are mentioned, the conditions are according to the conventional conditions well known by those skilled in the art or according to the conditions suggested by the manufacturers.

[0044] Example 1

[0045] This example is used to illustrate the construction of recombinant Escherichia coli ER2799pAGR79.

[0046] In this study, the adaptive laboratory evolution (ALE) technology was used to optimize the glyphosate-resistant gene (i.e., wild-type 5-enolpyruvylshikimate-3-phosphate synthase, named GR79, the amino acid sequence of which is shown in SEQ ID NO. 2; the gene is named GR79-aroA, the nucleotide sequence of which is shown in SEQ ID NO. 4, and the enzyme is described in patent CN101429499A) from the metagenome of contaminated soil (Jinzhou pesticide factory in Hebei province).

[0047] The genetically engineered bacteria were Escherichia coli DH5α, and the vector was a constitutive low-copy vector pACYC184.

[0048] The GR79-aroA gene was ligated to the vector pACYC184 digested with Sal I and BamH I. The primer containing the homologous arm of pACYC184 was designed to amplify the gene fragment.

[0049] Forward primer pA GR79-aroA -F:

[0050] Reverse primer pA GR79--aroA -R:

[0051] Then the Sal I and BamH I double enzyme digestion pACYC184 fragment was recombined with the amplified gene fragment by homologous recombination method to construct plasmid pAGR79 and transform into E. coli DH5α competent strain, and then spread on LB plate containing antibiotic chloramphenicol (Cm). Single colonies growing on the plate were picked and used as templates for PCR amplification followed by agarose gel electrophoresis to detect the target band. The size of GR79-aroA gene fragment was 1338 bp. The recombinant vector gene amplification fragment size was consistent with the size of the respective positive control, and met the expected size. The recombinant plasmid verified successfully was sequenced, and the correct recombinant plasmid was selected for subsequent experiments. The correctly constructed recombinant vector was transformed into the aroA gene-deficient E. coli ER2799 competent strain prepared in the laboratory to construct recombinant E. coli ER2799pAGR79 and spread on M9 medium containing Cm antibiotic for adaptive laboratory evolution and glyphosate resistance determination experiment.

[0052] Example 2

[0053] This example is used to illustrate the screening of mutant strain G79M3.

[0054] 1. Draw the growth curve:

[0055] ER2799pAGR79 was spread on M9 solid medium and incubated at 37°C overnight. Three single colonies were picked from each plate and inoculated into M9 liquid medium containing 200, 150 and 0 mM glyphosate (Gly), respectively, and incubated at 37°C, 220 rpm until the growth plateau. The turbidimetric method was used, with the OD600 value of the culture solution at each culture period as the vertical coordinate and the culture time as the horizontal coordinate to plot the growth curve.

[0056] 2. Laboratory evolution, calculate generation time:

[0057] A section of the logarithmic growth phase was taken and the generation time was calculated according to the formula.

[0058] g = t x ln2 / (lnMt-lnMo); where g is the generation time, t is the culture time, Mo is the number of viable bacteria at the beginning, and Mt is the number of viable bacteria after t hours of culture.

[0059] Pick the single colonies verified successfully for adaptive laboratory evolution experiment:

[0060] (1) Pick the verified ER2799pAGR79 and inoculate into M9 liquid medium containing 200 mM glyphosate and incubate at 37°C, 220 rpm in the dark for 20-24 h (each sample has three replicates).

[0061] (2) The cultured strain was inoculated into 50 mL of M9 liquid containing 200 mM glyphosate at 1% and continued to be cultured, and the above operation was repeated.

[0062] The bacterial liquid before and after culture was taken during the inoculation process, and 10 -4 ,10 -6 After gradient dilution, 100 uL of bacterial liquid was spread on M9 solid medium containing Cm, and the number of single colonies was counted after incubation in a 37°C incubator. The generation time was calculated.

[0063] (3) The inoculation was performed every 24 hours thereafter to ensure the nutrients in the culture medium.

[0064] (4) 750 uL of sample was taken before each inoculation and mixed with 25 uL of 75% glycerol, and stored at -80°C.

[0065] (5) The evolved strain was sequenced and analyzed after about 2000 generations under the selection pressure.

[0066] ER2799pAGR79 was subjected to ALE experiment under the selection pressure of glyphosate, and when about 2000 generations were performed, the sample was spread on M9 solid medium containing 250 mM glyphosate and cultured, and single strains with good growth were selected for sequencing analysis. The experimental results show that after 2000 generations of evolution, multiple mutants are obtained, and the recombinant E. coli containing a three-site mutant gene plasmid has outstanding resistance to glyphosate. The mutant enzyme is named G79M3, and the amino acid sequence of G79M3 is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 3.

[0067] Example 3

[0068] This example is used to illustrate the resistance analysis of G79M3 to glyphosate.

[0069] The growth curves of recombinant E. coli ER2799 strains containing pAG79M3 plasmid and pAGR79 plasmid were determined in M9 medium containing different concentrations of glyphosate (0, 200, 300, 400 mM glyphosate), and the tolerance of the mutant enzyme to glyphosate was evaluated.

[0070] ER2799 and recombinant strains containing plasmids pACYC184 (ER184), pAGR79 (ER79) and pAG79M3 (ER79M3), respectively, were spread on M9 solid medium and incubated at 37°C overnight. Three single colonies were picked from each plate and inoculated into M9 liquid medium containing 0, 200, 300, 400 mM Gly, and incubated at 37°C, 220 rpm until the growth plateau. The growth curves were plotted using the OD600 value of each culture as the vertical coordinate and the culture time as the horizontal coordinate, and the results are shown in Figure 1.

[0071] Since ER2799 cells lack aroA gene, they cannot synthesize aromatic amino acids autonomously, and thus cannot grow on M9 medium lacking aromatic amino acids. As can be seen from Figure 1 ((a), (b), (c), (d) are growth curves of the mutant strains in M9 medium containing glyphosate at a content of 0, 200, 300, and 400 mM, respectively), ER2799 and the recombinant strain into which the empty vector ER184 is transformed do not grow in the four media. The recombinant strains into which the GR79-EPSPS (ER79) and G79M3-EPSPS (ER79M3) plasmids are transformed can grow in M9 medium containing 200 mM glyphosate, and ER79M3 grows significantly better than ER79. In M9 medium containing 300 mM glyphosate, only ER79M3 can grow. This shows that G79M3 confers higher glyphosate tolerance to the host cells.

[0072] Example 4

[0073] This example is used to illustrate the construction of the G79M3 expression vector.

[0074] The primers containing homologous arms are designed, and the target fragments are amplified from the pAGR79 and pAG79M3 plasmids as templates, respectively.

[0075] Forward primer:

[0076] Reverse primer:

[0077] The plasmid vector (pET28a) is then double-digested with EcoRI and XhoI. The single fragment homologous recombination method of Novozyme is referred to, and the target fragment and the digested plasmid are recombined. The recombinant plasmids pETGR79 and pETG79M3 are transformed into BL21 E. coli competent cells, and the cell mixture is uniformly coated on solid LB medium with Kan resistance. Single colonies are picked for colony PCR verification and expanded culture. The successfully transformed recombinant strains are expanded and cultured, and are preserved on slant or in low-temperature glycerol tubes.

[0078] Example 5

[0079] This example is used to illustrate the expression and purification of the G79M3 protein.

[0080] The verified above-mentioned transformed strain was streaked on kan-resistant plates, incubated at 37°C overnight, and a single colony was picked and incubated in 20 mL liquid medium at 37°C, 220 rpm overnight. 2% of the volume was transferred into 150 mL LB medium for continued culture to OD600 of about 0.5-0.7, and then IPTG was added (final concentration 0.85 mM) to induce protein expression at 16°C for 16-20 h.

[0081] The specific steps of protein purification are as follows:

[0082] (1) The 150 mL bacterial solution induced overnight at 16°C was transferred to a 50 mL centrifuge tube, centrifuged at 4°C, 4000 x g for 15 min, and the bacterial cells were collected.

[0083] (2) After discarding the supernatant, 10 mL of Lysis buffer cell disruption solution and 10 μL of protease inhibitor were added, the bacterial cells were resuspended and sampled, and the total bacterial protein was recorded.

[0084] (3) The cells were disrupted by an ultrasonic disrupter with a disruption power of 25 W and a frequency of 23 s per interval for 2 s, and the disruption time was 3 min, which could be appropriately extended to clarify the bacterial solution. After the end, the color of the bacterial solution became dark, and the sample was recorded as precipitated protein P.

[0085] (4) The disrupted bacterial solution was added to an EP tube, centrifuged at 4°C, 14000 x g for 15 min, and the supernatant was added to a test tube and placed in ice for purification. Sampling was recorded as supernatant protein S.

[0086] (5) Prepare and equilibrate the affinity chromatography column: add 3 mL of Ni-NTA resin to the chromatography column, stand for 30 min, then elute with 10 times the volume of ddH2O, and then equilibrate with 10 times the volume of the equilibration buffer (i.e. A solution). When used again, still need to elute with ddH2O to remove alcohol.

[0087] (6) Slowly add the centrifuged supernatant to the chromatography column, control the flow rate at the lower end of the column, and let the liquid penetrate the column slowly. After the residual buffer in the column is completely drained, collect the penetrated liquid using a conical flask, take 1 mL of the penetrated liquid and store it at 4°C for testing, and record it as FT. The remaining liquid is poured back into the column for re-chromatography.

[0088] (7) Prepare a final concentration of 500 mM imidazole using A and B solutions as the mother liquor, and then dilute it to concentrations of 0 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM.

[0089] (8) Add different gradient eluent to collect protein, during which, use 500 μL of Coomassie brilliant blue staining solution to detect protein concentration, until the test eluent is no longer blue, then the elution is complete. Each gradient is sampled with 50 mL centrifuge tube.

[0090] (9) Take a small amount of sample and add 10x Loading Buffer, mix and boil in boiling water for 10 min, centrifuge at 12000 x g for 1 min. Take 10 μL for SDS-PAGE electrophoresis, the results are shown in Figures 2 and 3.

[0091] In Figures 2 and 3, M represents Prestained Protein Ladder 1, 1 represents whole bacterial protein, 2 represents precipitated protein P, 3 represents supernatant protein S, 4 represents flow-through FT, 5-13 represent imidazole eluent with concentrations of 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 150 mM, 200 mM, 500 mM, respectively; the red line indicates the induced protein band. As can be seen from Figures 2 and 3, both GR79 and G79M3 proteins are induced to express, with a size of about 45 KDa. The optimal elution imidazole concentration for GR79 is 80 mM (Figure 2), and the optimal elution imidazole concentration for G79M3 is 60 mM (Figure 3). The eluted protein is desalted and purified, and stored at -80°C for later use.

[0092] Example 6

[0093] This example is used to illustrate the enzymatic kinetics analysis of G79M3 mutant.

[0094] The experimental method refers to the literature (He, M., Yang, Z. Y., Nie, Y. F., et al. A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate. Biochim Biophys Acta, 2001. 1568 (1): 1-6), and the specific operation is as follows:

[0095] (1) Inorganic phosphorus standard curve: 10 mM inorganic phosphorus standard solution was diluted 1:10. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μl was taken in a 1.5 ml Eppendorf centrifuge tube, 100 μl of milli-Q water was added, mixed, 0.8 ml of MAT solution was added, mixed, and after timing for 3 minutes, 100 μl of 34% SC solution was quickly mixed, and after standing at room temperature for 20 min, the OD660value was measured. Repeat three times. As shown in Figure 4, the inorganic phosphorus standard curve was obtained by plotting the inorganic phosphorus concentration as the abscissa and the OD660value as the ordinate.

[0096] (2) Enzyme activity determination: the protein quantification of enzyme crude extract used Coomassie brilliant blue G-250 staining method (Bradford, 1976). The following solutions were added in a 1.5 ml Eppendorf centrifuge tube on ice: 2 μl of 10 mM PEP solution, 2 μl of 10 mM S3P solution, 2 μl of 0.5 M HEPES solution, 2 μl of 1 mM (NH4)6MO7O 24 4H2O solution and 12 μl of milli-Q water were mixed, after incubation at 28°C for 5 min, 1 μl of crude enzyme solution was added to each tube sample with an interval of 2 s and timing, after 2 min, 200 μl of MAT solution was added with an interval of 2 s, and after color development for 3 min, 20 μl of 34% SC solution was quickly mixed with an interval of 2 s, and after color development at room temperature for 20 min, the OD660value was measured. The control was the same as the sample tube except that no enzyme solution was added. After subtracting the OD660value of the sample tube from that of the control tube, the molar amount of inorganic phosphorus released by the reaction was obtained by referring to the inorganic phosphorus standard curve, and then divided by the reaction time and enzyme protein amount to obtain the enzyme activity (U / mg) of the enzyme.

[0097] Km(PEP) determination: the concentration of S3P solution was constant at 1 mM, and the enzyme reaction rate was determined under different PEP concentrations (0.05, 0.067, 0.1, 0.2, 0.5, 1.0 mM) according to the above reaction system, and the measured values were plotted according to the V-v / [S] (Eadic-Hofstee) method.

[0098] Ki(glyphosate) determination: the enzyme reaction rate of EPSPS was determined when the PEP concentration was 66.7, 100, 200, 500 μM under different glyphosate concentrations (0, 10, 50, 100 μM). A double logarithmic plot was used to obtain a 1 / V-1 / [S] straight line, and the slope of each straight line was taken as the ordinate and the glyphosate concentration as the abscissa to obtain a new straight line. The intersection of this straight line with the X-axis was the Ki(glyphosate) value.

[0099] The results are shown in Table 1.

[0100] Table 1 Enzyme kinetics parameters of GR79 and G79M3.

[0101] Wherein, IC50 is the half-inhibitory concentration, i.e. IC50, the concentration of the pharmaceutical compound required to inhibit half of the enzyme activity in the specified biological process. Km is the Michaelis constant, which measures the affinity between the enzyme and the substrate, the smaller the Km, the lower the substrate concentration required for the enzyme to react, or in other words, the greater the affinity between the enzyme and the substrate. Vmax is the maximum reaction rate. Ki is the inhibition constant (kinetic inhibition rate constant), which reflects the binding affinity of the inhibitor to the enzyme, and the smaller the value, the stronger the inhibition. The Ki / Km value reflects the competition between the enzyme substrate and the inhibitor, and the larger the value, the worse the ability of the inhibitor to compete for the enzyme active site, and the less the enzyme is affected by the inhibitor.

[0102] As can be seen from Table 1, the measurement results show that the IC50 value of GR79 is 6.8685±1.3785mM; the IC50 value of G79M3 is 14.975±2.820mM. The IC50 of G79M3 is about 2 times that of GR79, which indicates that the mutant G79M3 increases the glyphosate resistance of wild-type GR79.

[0103] The Km of GR79 is 39.341±3.21μM, and the Vmax is 9.285±0.223U / mg; the Km of G79M3 is 21.212±2.675μM, and the Vmax is 22.416±0.811U / mg. It can be seen that the Km of the mutant G79M3 is reduced, and the substrate affinity is increased.

[0104] The Ki value of GR79 synthase is 75.360±5.029μM, and the Ki value of G79M3 is 129.744±12.331μM, indicating that the binding ability of the mutant G79M3 to glyphosate is lower than that of the original enzyme GR79.

[0105] The Ki / Km ratio directly reflects the glyphosate resistance of the enzyme, and the Ki / Km of G79M3 is 6.117, and the Ki / Km of GR79 is 1.916, which is 3 times that of the wild-type GR79, indicating that the glyphosate resistance of G79M3 has been greatly improved. The expression of G79M3 in cells will endow the host with high glyphosate tolerance.

[0106] The preferred embodiments of the present disclosure are described in detail above with reference to the drawings, but the present disclosure is not limited to the specific details in the above-described embodiments. Various simple modifications can be made to the technical solutions of the present disclosure within the technical concept of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0107] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, various possible combinations are not described again in the present disclosure.

[0108] In addition, various different embodiments of the present disclosure can also be combined in any appropriate manner, as long as they do not deviate from the idea of the present disclosure, and they should also be considered as disclosed in the present disclosure.

Claims

1. A method for improving the activity of 5-enolpyruvate shikimic acid-3-phosphate synthase, the method comprising mutating three amino acid residues of wild-type 5-enolpyruvate shikimic acid-3-phosphate synthase, characterized in that, The amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase is shown as SEQ ID NO. 2, the amino acid residues to be mutated include a tyrosine residue at position 40, a phenylalanine residue at position 114 and a serine residue at position 358; and the mutation is substitution of the amino acid residues.

2. The method of claim 1, wherein, The mutation includes: mutation of the tyrosine residue at position 40 into an isoleucine residue, mutation of the phenylalanine residue at position 114 into a serine residue, and mutation of the serine residue at position 358 into a threonine residue.

3. A protein having 5-enolpyruvylshikimate-3-phosphate synthase activity, characterized in that, The protein is a protein obtained by mutation of three amino acid residues to be mutated in a wild-type 5-enolpyruvylshikimate-3-phosphate synthase, the amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase is shown as SEQ ID NO. 2, the amino acid residues to be mutated include a tyrosine residue at position 40, a phenylalanine residue at position 114 and a serine residue at position 358; and the mutation is substitution of the amino acid residues.

4. The protein of claim 3, wherein, The amino acid sequence of the protein is shown as SEQ ID NO.

1.

5. A gene encoding the protein of claim 4, characterized in that, The gene is a DNA molecule with a nucleotide sequence shown as SEQ ID NO.

3.

6. A recombinant vector, characterized in that, The recombinant vector is a recombinant expression vector, and the recombinant vector is inserted with the gene according to claim 5.

7. A transformant characterized in that, The host of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene according to claim 5, or the recombinant vector according to claim 6 is introduced into the transformant.

8. The transformant according to claim 7, wherein, The genetically engineered bacterium is selected from one of Escherichia coli, Bacillus subtilis, Rhizobium, Pseudomonas and Azotobacter vinelandii.

9. A method of preparing 5-enolpyruvylshikimate-3-phosphate synthase, characterized by, The method includes inoculating the transformant according to claim 7 or 8 into a culture medium for culture to obtain a cultured material.

10. Use of a 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate resistance, characterized in that, The 5-enolpyruvylshikimate-3-phosphate synthase contains the protein according to claim 3 or 4. The amino acid sequence of the wild-type 5-enolpyruvylshikimate-3-phosphate synthase is shown as SEQ ID NO. 2, the amino acid residues to be mutated include a tyrosine residue at position 40, a phenylalanine residue at position 114 and a serine residue at position 358; and the mutation is substitution of the amino acid residues.

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