CN116555220A - A sulfonylurea herbicide hydrolase TsmE mutant, its coding gene and application - Google Patents

Abstract

The invention discloses a sulfonylurea herbicide hydrolase TsmE mutant, its encoding gene and application. A sulfonylurea herbicide hydrolase TsmE mutant, the amino acid sequence of which is shown in SEQ ID NO.4. A gene encoding the mutant of the present invention. The application of the mutant of the present invention in degrading sulfonylurea herbicides. The present invention uses the wild-type TsmE as the starting template, and obtains the mutant M7 (S179A) with significantly improved enzyme activity through directed evolution technology. Compared with the wild type, the specific enzyme activity of mutant M7 to metsulfuron-methyl, metetsulfuron-methyl, tribenuron-methyl, bensulfuron-methyl, ethametsulfuron-methyl and chlorimsulfuron-methyl increased by 1.7, 1.6, 2.5, 1.3, 1.4 and 1.2 times. The mutant and its coding gene can be used to construct transgenic crops resistant to sulfonylurea herbicides, and can also be used to remove sulfonylurea herbicides in soil and water, and have very important theoretical and application values.

Description

A sulfonylurea herbicide hydrolase TsmE mutant, its coding gene and application

technical field

The invention belongs to the technical field of enzyme protein engineering, and relates to a sulfonylurea herbicide hydrolase TsmE mutant, its coding gene and its application.

Background technique

In recent years, with the gradual restriction and elimination of highly toxic and long-residue herbicides, the herbicide market has gradually entered the era of low-toxicity and high-efficiency products. Sulfonylurea herbicides have developed rapidly due to their advantages of high efficiency, broad spectrum and low toxicity, and have become the third largest herbicides after organophosphorus and acetamide herbicides. According to the Food and Agriculture Organization of the United Nations (FAO), the use of sulfonylurea herbicides was only 129 tons in 1992, but its use has increased 17 times by 2011, and its use has even increased in Europe and North America. more than 100 times. In my country, the application area of sulfonylurea herbicides has exceeded 2 million hectares per year, and is still expanding. At present, the global annual sales of sulfonylurea herbicides exceed 2 billion US dollars, accounting for about 10% of all herbicide sales.

Although sulfonylurea herbicides have low toxicity to mammals, some sulfonylurea herbicides, such as metsulfuron-methyl, sulfuron-methyl, chlorimsulfuron-methyl and ethametsulfuron-methyl, have relatively stable chemical properties, and the soil residual period is relatively stable. Very long, especially in alkaline soil, its residual period can be as long as 2-3 years, causing serious phytotoxicity to subsequent crops. Some sulfonylurea herbicides with short residual periods, such as bensulfuron-methyl, thifensulfuron-methyl and tribenuron-methyl, can be used as target herbicides for the construction of herbicide-resistant transgenic crops. Therefore, obtaining excellent sulfonylurea herbicide detoxifying enzymes and genetic resources has important application value for bioremediation of sulfonylurea herbicide residue pollution and herbicide-resistant transgenic engineering.

TsmE was identified from the bacterium Hansschlegeliazhihuaiae S113, which can catalyze the weed control of various sulfonylureas such as thifensulfuron-methyl, metsulfuron-methyl, sulfuron-methyl, bensulfuron-methyl, tribenuron-methyl, ethametsulfuron-methyl and chlorimsulfuron-methyl The agent hydrolyzes the esterase that deesterifies and detoxifies. However, TsmE has extremely high deesterification efficiency for thifensulfuron-methyl, but lower deesterification efficiency for other sulfonylurea herbicides. In this context, the present invention improves the catalytic activity of the enzyme through directed evolution technology, so that it can more efficiently catalyze the deesterification and detoxification of sulfonylurea herbicides, and enhance its practical application value.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a sulfonylurea herbicide hydrolase TsmE mutant with higher activity, its coding gene and its application.

The purpose of the present invention can be achieved through the following technical solutions:

A sulfonylurea herbicide hydrolase TsmE mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.4.

A gene encoding the mutant of the present invention.

As a preference of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.3.

The recombinant expression vector containing the sulfonylurea herbicide hydrolase mutant gene of the present invention.

As a preference of the present invention, the recombinant expression vector is obtained by inserting the sulfonylurea herbicide hydrolase mutant gene of the present invention between the NdeI and HindIII sites of pET-29a(+).

The genetically engineered bacteria containing the sulfonylurea herbicide hydrolase TsmE mutant gene of the present invention.

The application of the mutant of the present invention in degrading sulfonylurea herbicides.

The mutant of the present invention is used in removing sulfonylurea herbicide residues in soil and water.

The application of the gene described in the invention in degrading sulfonylurea herbicides and constructing transgenic crops resistant to sulfonylurea herbicides.

The application of the recombinant expression vector of the present invention or the genetically engineered bacterium of the present invention in degrading sulfonylurea herbicides; the sulfonylurea herbicides are tribenuron-methyl, sulfensulfuron-methyl, thifensulfuron-methyl, Bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron-methyl, or chlorimsulfuron-methyl.

The beneficial effects of the present invention are as follows:

In the present invention, the wild-type sulfonylurea herbicide hydrolase TsmE is used as a starting template, and the mutant M7 (S179A) with significantly improved enzyme activity is obtained through directed evolution technology. Compared with the wild type, the specific enzyme activities of mutant M7 to metsulfuron-methyl, metetsulfuron-methyl, tribenuron-methyl, bensulfuron-methyl, ethametsulfuron-methyl and chlorimsulfuron-methyl increased by 1.7, 1.6, 2.5, 1.3, 1.4 and 1.2 times. The mutants and their coding genes can be used to construct transgenic crops resistant to sulfonylurea herbicides, and can also be used to remove sulfonylurea herbicides in soil and water, and have very important theoretical and application values.

Description of drawings:

Figure 1 Construction diagram of mutant M7 recombinant expression vector.

Fig. 2 SDS-PAGE pattern of sulfonylurea herbicide hydrolase TsmE mutant. Among them, lane 1 is the protein Marker, and the other lanes are wild-type TsmE and mutant M7 in sequence.

Detailed ways

Example 1. Directed evolution screening of TsmE mutants with significantly improved enzyme activity

1.1 Synthesis of wild-type tsmE gene

The nucleotide sequence (1197bp) of the wild-type tsmE gene was synthesized, as shown in SEQ ID NO.1 in the sequence listing, which encodes TsmE protein (398 amino acids), as shown in SEQ ID NO.2 in the sequence listing. The synthetic tsmE was cloned into the pUC57 vector, and the recombinant vector was named pUC-tsmE, and then transformed into Escherichia coli DH5α.

1.2 Construction of tsmE gene mutation library

Using the plasmid pUC-tsmE as a template, error-prone PCR was carried out with primers 1 and 2, so that the tsmE gene was mutated due to random base mismatches. The error-prone PCR product was cloned into pMD19-T vector, and the enzyme-linked product was transformed into Escherichia coli DH10B (ilvG ⁺ ) sensitive to metsulfuron-methyl by heat shock. The random mutation library was spread on an LB plate supplemented with 100 mgL ^-1 Amp, 200 mgL ^-1 IPTG and 200 mgL ^-1 X-Gal, and cultured overnight at 37°C.

The primers and error-prone PCR reaction system are as follows:

Primer 1: ATGGAAACCGATAAAAAAACCG, as shown in SEQ ID NO.5 in the sequence listing;

Primer 2: TCAGCTTTCGTTCTGATCTAAG, as shown in SEQ ID NO.6 in the sequence listing;

The error-prone PCR amplification system is:

PCR amplification program:

a. Pre-denaturation at 95°C for 3 minutes;

b. Denaturation at 94°C for 0.5min, annealing at 52°C for 1.0min, extension at 72°C for 1.5min, and 30 cycles;

c. Extend at 72°C for 10 minutes and cool to room temperature.

1.3 Screening the tsmE gene mutation library

The transformation product in the above mutation library was inoculated on a basal salt medium containing metsulfuron-methyl at a concentration of 50 μM (adding 5 gL ^-1 glucose, 200 mgL ^-1 valine, 200 mgL ^-1 leucine and 100 mgL ^-1 ampicillin penicillin) for 3 days. In view of the fact that Escherichia coli DH10B (ilvG ⁺ ) is very sensitive to high concentrations of metsulfuron-methyl, the resistance gene can convert metsulfuron-methyl into an acid product that is non-toxic to bacteria, thereby releasing the inhibitory effect on the growth of Escherichia coli DH10B (ilvG ⁺ ), Therefore, this principle is used to perform high-throughput screening on the above-mentioned mutant library. Select colonies of recombinant strains that can grow well on the screening plate. The resistance and deesterification ability of the selected strains to sulfonylurea herbicides were determined, and the recombinant strains with significantly improved deesterification activity of sulfonylurea herbicides were screened, and the tsmE sequence mutations in the recombinant strains were analyzed.

The formula of _the above basal salt medium _is : 1.0gNH4Cl, 0.5gNaCl, _1.5gK2HPO4 , _0.5gKH2PO4 , _0.2gMgSO4 · _7H2O , add deionized water to make up to 1L. Add 2% agar powder to the solid medium.

1.4 Acquired mutated resistance genes

After several rounds of screening, two TsmE mutant resistance genes were obtained, named M7 gene. The 535th and 536th positions of the M7 nucleotide sequence are mutated from the original AG to GC, and the sequence is shown in SEQ ID NO.3; the 179th position of its amino acid sequence is mutated from the original serine to alanine, and the amino acid sequence is as follows Shown in SEQ ID NO.4.

High expression of embodiment 2 mutant M7 gene in BL21 (pET-29a (+))

2.1 Construction of bacterial expression vectors and acquisition of recombinant microorganisms

The nucleotide sequence (1212bp) of the M7 gene with NdeI/HindIII restriction sites was synthesized, as shown in SEQID NO.7 in the sequence listing. The synthesized M7 gene was cloned into the pUC57 vector to obtain the recombinant vector pUC-M7. Recombinant vector pUC-M7 and pET-29a(+) plasmids were digested with restriction endonucleases NdeI and HindⅢ respectively, and the digestion system was as follows:

10×MBuffer5μL

NdeI (10U·μL ^-1 )2.0μL

HindⅢ(10U·μL ^-1 )2.0μL

DNA (pUC-M7 or pET-29a(+) plasmid) 30μL

ddH ₂ O 11 μL

After enzyme digestion at 37°C for 12 hours, the excised M7 gene fragment was enzyme-ligated with the digested bacterial expression vector pET-29a(+), and the constructed recombinant expression vector was named pET29a-M7. The recombinant expression vector was transformed into the expression host strain BL21(DE3) to obtain the recombinant microorganism BL21(M7).

2.2 Expression and purification of TsmE mutants

Inoculate BL21 (M7) in LB medium, culture at 37°C on a 200rpm shaker until the OD _600nm is between 0.4-0.6, add IPTG to a concentration of 0.4mM, and induce culture at 16°C for 12 hours. 100ml of bacterial liquid was collected by centrifugation, washed twice with PBS (50mM, pH7.4), resuspended with 10ml of PBS buffer, and ultrasonically disrupted (AutoScience, UH-650 Ultrasonic processor, 30% intensity) for 5- After 10 minutes, centrifuge at 12,000 rpm for 30 minutes, collect the supernatant, and purify the TsmE mutant with a nickel ion affinity chromatography column, and perform protein electrophoresis on the purified enzyme, as shown in FIG. 2 .

Embodiment 3, TsmE mutant enzyme activity assay

Enzyme activity reaction system (1 mL): Add purified TsmE mutant enzyme and 100 μM sulfonylurea herbicide substrate in 50 mMPBS buffer (pH 7.4). Each reaction was started by adding the reaction enzyme, and after 10 minutes of reaction at 30°C, an equal volume of acetonitrile was quickly added to terminate the reaction. With no enzyme added as a control, high performance liquid chromatography (HPLC) was used to detect the reduction of each sulfonylurea substrate to determine the activity of the mutants. The HPLC conditions are: 3000TitaniumSystem high performance liquid chromatography; ThermoScientificSyncronisC18 chromatographic column (5μm, 250mm×4.6mm); mobile phase is acetonitrile/water (60/40, v/v), added 0.5% acetic acid in water; flow rate 1mL/min; injection volume 20 μL; the detection wavelengths are 230nm and 255nm; the column temperature is 40°C. One unit of enzyme activity is defined as: the amount of enzyme required to catalyze the hydrolysis of 1.0 μmol of substrate in 1 min at pH 7.4 and temperature 30°C is defined as one unit of enzyme activity (U). The experimental results are shown in Table 1.

The above experimental results show that: compared with wild-type TsmE, the specific enzyme activity of mutant M7 to metsulfuron-methyl, metsulfuron-methyl, tribenuron-methyl, bensulfuron-methyl, ethametsulfuron-methyl and chlorimsulfuron-methyl was increased respectively. 1.7, 1.6, 2.5, 1.3, 1.4 and 1.2 times.

Table 1 Enzyme activity of TsmE mutant M7 to various sulfonylurea herbicides.

The sources of microorganisms used in the above examples are as follows: Escherichia coli DH5α was purchased from Bao Biological Engineering (Dalian) Co., Ltd., Escherichia coli high expression vector pET-29a (+) was purchased from Novegen Company, and expression host bacteria Escherichia coli BL21 (DE3) was purchased from From Shanghai Yingjun Biotechnology Co., Ltd.

Claims (10)

1. A sulfonylurea herbicide hydrolase Tsm mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 4.

2. A gene encoding the mutant of claim 1.

3. The gene according to claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 3.

4. A recombinant expression vector comprising the sulfonylurea herbicide hydrolase mutant gene of claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein the recombinant expression vector is obtained by inserting the sulfonylurea herbicide hydrolase mutant gene according to claim 2 or 3 between the NdeI and HindIII sites of pET-29a (+).

6. A genetically engineered bacterium comprising the sulfonylurea herbicide hydrolase TsmE mutant gene of claim 2 or 3.

7. Use of the mutant according to claim 1 for degrading sulfonylurea herbicides.

8. Use of the mutant according to claim 1 for removing sulfonylurea herbicide residues in soil, water bodies.

9. Use of the gene according to claim 2 or 3 for degrading sulfonylurea herbicides and constructing sulfonylurea herbicide-resistant transgenic crops.

10. The recombinant expression vector of claim 4 or the application of the genetically engineered bacterium of claim 5 in degrading sulfonylurea herbicides; the sulfonylurea herbicide is tribenuron-methyl, sulfometuron-methyl, thifensulfuron-methyl, bensulfuron-methyl, sulfometuron-methyl, tribenuron-methyl or chlorimuron-methyl.