Construction Of Sulfonylurea Herbicide Degrading Genetically Engineered Microorganism

Sulfonylurea herbicides are widely used due to its high efficiency, broad-spectrum, low toxicity and high selectivity properties with a half-life for one year or more in soil. Acetolactate synthase (AHAS) is the key enzyme of the synthesis of isoleucine, leucine, valine in plants, while sulfonylurea herbicides can inhibit the synthesis of these amino acids by inhibiting the activity of AHAS so that to inhibit protein synthesis. Some sulfonylurea herbicides are easily hydrolyzed in an acidic environment; some are stable, refractory and have antibacterial effects in alkaline and neutral conditions. Sulfonylurea herbicide residues in the soil will not only decrease the yield of subsequent sensitive crops, but also cause serious damage for the ecological environment. There are three ways to clean up polluted environments by sulfonylurea herbicides:microbial degradation, photolysis and chemical hydrolysis. The photolysis usually occurs in the soil surface. In neutral and acidic soils, the degradation of sulfonylurea herbicides is mainly the results of chemical hydrolysis and microbial degradation. In alkaline soils, microbial degradation is the main factor of the degradation of sulfonylurea herbicide.When applied directly to the contaminated sites to carry out bioremediation, sulfonylurea pesticide-degrading bacteria will be influenced by the complex environment and indigenous microorganisms in the soil, so that it is difficult to achieve the purpose of bioremediation. The main objective is to connect promoter P1 suitable for playing a role under laboratory culture conditions or promoter P2 suitable for soil with sulfonylurea herbicides hydrolase gene sulE by overlap extension PCR, and then PCR fragments were ligated into the broad-host vector pBBR1MCS-5 and transformed into Pseudomonas putida KT2440 suitable for expression in plant rhizosphere soil to construct genetically engineered bacteria (named as KT-sulP1 and KT-sulP2, respectively). The degradation characteristics of the two genetically engineered bacteria (KT-sulP1 and KT-sulP2) and wild sulfonylurea pesticide-degrading strain Hansschlegelia zhihuaiae S113 for sulfonylurea herbicides was studied in the laboratory liquid fermentation experiments and soil experiments. In laboratory liquid fermentation experiment, the effects of temperature, pH and initial concentration of pesticides on thifensulfuron degradation were studied. The optimum temperature of engineered bacteria KT-sulP1 and KT-sulP2 are 30℃ and the optimum pH are 7.0. Strain KT-sulP1 has the best degradation effect on thifensulfuron. It can degrade 50 mg/L of thifensulfuron within 24 hours and degrade 200 mg/L of thifensulfuron completely within 48 h. Effects of initial inoculum of microorgnism and initial concentration of pesticides on degradation of thifensulfuron were studied in sterilized soil and in unsterilized soil. The results showed that strain KT-SUIP2 had the better degradation effect on thifensulfuron in soil experiments. The degradation rate of thifensulfuron in unsterilized soil cantaining 30 mg/kg thifensulfuron was 73.4% and the degradation rate of thifensulfuron in sterilized soil was 71.7%. There was a linear relationship between the degradation effect and inoculation amount. The seedling length, seedling weight, root length and root weight of maize were all inhibited when thifensulfuron at different final concentrations (0.5,0.8,1.0,2.0 mg/kg) was added into soil, and the restraining effect enhanced with the increase of thifensulfuron concentration. The inhibition effect was removed when strains S113, KT-sulP1 and KT-SUIP2 were inoculated into the soil. Strain KT-SUIP2 had the best remediation action on maize damage caused by thifensulfuron. After strain KT-SUIP2 inoculated into the soil for 10 days, the four parameters (seedling length, seedling weight, root length and root weight of maize) were similar with the control group without addition of thifensulfuron.Pseudomonas putida KT2440 was certified by the Recombinant DNA Advisory Committee (RAC) of the United States as the host strain of the first host-vector biosafety system for gene cloning in soil bacteria. The broad-host vector pBBRlMCS-5 contained many antibiotic resistance genes, which had a certain environmental risks when released to environment as the vector of genetically engineered microorganism. To avoid the risk, it is necessary to construct genetically engineered microorganisms by introducing sulE gene into the chromosome of Pseudomonas putida KT2440 without selection markers. Gene sulE with the promoter p1 or p2 by PCR was amplified from strain KT-sulP1 or KT-sulP1, ligated into the random integration vector pUTTnsKm, transformed into E. coli DH5axPir. Two genetically engineered strains of Pseudomonas putida KT-putsi and Pseudomonas putida KT-putS2 were constructed by triparental conjugation method and the continuous selects of antibiotic resistance and sucrose, where gene sulE was integrated into the chromosome of Pseudomonas putida KT2440 without exogenous resistant markers. Strains KT-puts2, KT-puts2, KT-sulP2 and KT-SUIP2 followed the similar growth patterns, but strains KT-sulP2 and KT-sulP2 had better degradation effect for thifensulfuron than strains KT-puts2 and KT-puts2, which might be associated with a lower number of gene copies in strains KT-puts2 and KT-puts2.