Polyphasic-taxonomic Classification Of Chlorimuron-ethyl-degrading Strain And Multi-omics Analysis Of Its Degradation Characteristics

In this study,five strains of efficient chlorimuron-ethyl degrading bacteria were isolated from the activated sludge of wastewater treatment of a sulfonylurea herbicide production plant with chlorimuron-ethyl as the sole nitrogen source.The 16S r RNA gene sequence comparison analysis results showed that the strain LAM7117 is a suspected new species,and the strain LAM2020 is a suspected new genus.Based on phylogenetic research,the strain LAM7117 is a new species of Arthrobacter,named Arthrobacter sulfonylureivorans sp.nov;Strain LAM2020 belongs to the first report to have chlorimuron-ethyl degradation function,which enriches chlorimuron-ethyl-degrading microbial resources,and strain LAM2020 has a degradation rate after 5 days at 30?,p H 7.0,and substrate concentration of 100 mg/L.Reaching 90.5%,it has a wide range of application potential.For this reason,the degradation characteristics and degradation mechanism of strain LAM2020 have been studied.Single factor experiments and response surface methods were used to optimize the degradation conditions of chlorimuron-ethyl by the degradation strain LAM2020.The results showed that strain LAM2020 had the best degradation effect under the conditions of 30°C,p H 6.5,and substrate concentration of100 mg/L.The detection results of the ESI source of degradation metabolites of strain LAM2020 by high performance liquid chromatography-mass spectrometry(LC-MS)showed that in the negative ion mode,ethyl phthalate,benzenesulfonyl isocyanate,and phthalic acid were detected.Imine,ethyl phthalate benzenesulfonamide,benzenesulfonamide phthalate,2-amino-4-chloro-6-methylpyrimidine.Based on the above-mentioned metabolites,the degradation pathway of chlorimuron-ethyl in strain LAM2020 was speculated:the ester bond in chlorimuron-ethyl was broken;the urea bridge in chlorimuron-ethyl was broken.The whole genome sequencing of the degrading strain LAM2020 can be obtained.The genome of LAM2020 contains 5024 genes with a total length of 4424553bp.Preliminary analysis predicts 1 cytochrome P450 gene,10 hydrolase genes,and 2carboxylesterase genes.10 glycosyltransferase genes.At the same time,the transcriptome sequencing of the degradation strain LAM2020 predicts that the esterase degradation gene H related to chlorimuron-ethyl degradation is significantly up-regulated.The gene is 777bp in length and has a protein size of 258Aa.The esterase has Gly-X-Ser-X-Gly Basic motif;through the Blast analysis of NCBI database,the highest similarity between the esterase and the existing gene is 82.56%.After gene H was cloned,expressed exogenously,and purified,it was found that the degradation rate of gene H could reach 99.5%after 1 day at 30?,p H 7.0,and substrate concentration of 50 mg/L.Through metabolomics analysis,combined with HPLC detection of short-chain organic acids(oxalic acid and fumaric acid)produced by the degradation strain LAM2020,it was confirmed that the hydrolysis of chlorimuron-ethyl led to the rupture of the urea bridge of chlorimuron-ethyl.The strain uses glucose as a carbon source to produce TCA and glyoxylic acid cycle in the cell to generate energy for the body.At the same time,it produces oxalic acid and fumaric acid,which are excreted from the body,thereby causing chlorimuron to break the urea bridge under acidic conditions.Hydroponic experiments of sensitive crop corn showed that the degrading bacteria LAM2020 has a certain detoxification function to chlorimuron-methyl.On the basis of5 mg/L chlorimuron-ethyl pesticide,adding LAM2020 degrading bacteria with OD₆₀₀=0.2 to the plant height and fresh weight of corn has an inhibition rate of 4.62%and 7.27%.