| Organophosphorus pesticides?OPs?are widely used to control household and agricultural pests.Due to their high toxicity to non-target organisms and potential delayed neurotoxicity,OPs have caused severe environmental pollution and ecological damage.In this study,a strain with highly efficient degradation of chlorpyrifos was isolated.This strain was identified by 16S rRNA and gyrB sequences,and physiological and biochemical characteristics.The degradation characteristics of OPs were studied,and the complete genetic information of this strain was obtained by whole genome sequcing.The mechanism of microbial degradation and transformation of phoxim was explored by using transcriptome sequencing technology.At the same time,the related degradation genes were obtained.The gene characteristics,degradation function,and enzymatic properties of degradation enzymes were studied.The zebrafish,an environmental model organism,was used to evaluate the toxicity of phoxim enzymolysis products.This study has a certain theoretical significance and potential application value.The main results as following:?1?Twenty laboratory-preserved Bacillus spp.which had both growth-promoting and phosphate-decomposing capacities were compared,and a high-efficient chlorpyrifos-degrading strain YP6 was selected.Based on its 16S rRNA and gyrB sequences,and physiological and biochemical characteristics,strain YP6 was identified as Bacillus amyloliquefaciens,and was deposited at the China Center for Type Culture Collection?CCTCC NO:M 2018875?.Bacillus amyloliquefaciens YP6 showed efficient degradation-capacity to a broad-spectrum OPs?chlorpyrifos,dichlorvos,dipterex,triazophos,and phoxim?,especially the highest degrading efficiency of phoxim.Strain YP6 could grow with phoxim as the sole phosphorus,but not grow with phoxim as the sole carbon source.Furthermore,it could grow rapidly and degrade phoxim efficiently in LB broth medium.Response surface methodology was performed to optimize the degradation conditions of phoxim by strain YP6 in LB broth medium.The optimum biodegradation conditions were 40?C,pH 7.20,and an inoculum size of 4.17%?v/v?.Under optimal conditions,strain YP6 could tolerate and degrade phoxim up to 700 mg·L-1.Metabolities of phoxim degraded by strain YP6 were confirmed by high performance liquid chromatography-mass spectrometry?HPLC-MS?,and a possible degradation pathway was proposed.?2?The genome of strain YP6 was sequenced by using the PacBio+Illumina Hiseq sequencing technology,and the access number of Gen Bank was CP032146.Strain YP6genome had a chromosome with no plasmid.The total size of the genome was 4009619 bp with a GC content of 45.9%.4322 genes were detected from the genome sequence,and 4210coding sequences?CDSs?were predicted and annotated to the public databases.Genome analysis revealed that strain YP6 was most closely related to Bacillus amylollquefaciences MT45.Compared with the genomes of 35 Bacillus amyloliquefaciens that had completed whole genome sequencing and assembly in NCBI database,strain YP6 had the most CDSs.While,the numbers of gene annotated to“Carbohydrate transport and metabolism?G?”,“Translation,ribosome structure and synthesis?J?”,“Transcription?K?”,“Cell wal/cell membrane/envelope synthesis?M?”,and“The signal transduction mechanism?T?”were the fewest.The genome of strain YP6 also contains genes coding for some biomass-promoting substances?such as tryptophan,siderophores,and phytase?,secondary metabolites with antibacterial activity?such as surfactin,fengycin,bacilysin,macrolactin,and bacilaene?,flagellum,bacterial chemotaxis,and some enzymes involved in xenobiotic?such as aminobenzoate,atrazine,benzoate,chloroalkane,and chlorobenzene?degradation and metabolism.?3?According to transcriptome sequencing results,689 genes were upregulated,and 851genes were downregulated in strain YP6 during the degradation of phoxim.Sugar ABC transporter,multidrug ABC transporter,and MFS transporter may be involved in the transportation of phoxim.Hydrolases,monooxygenases,NADPH-cytochrome P450reductases,and glycosyltransferases may be involved in phoxim hydrolysis,sulfoxidation,dealkylation,and glycosyl transfer process.Bacterial chemotaxis,two-component sensors,DNA uptake and recombination,and DNA repair may be involved in the processes which strain YP6 could immediately adjust and respond to phoxim exposure.In addition,the expression of some genes at the transcription level were verified by qRT-PCR,and the results were consistent with the transcriptome data.?4?Based on product analysis,genome and transcriptome data,three alkaline phosphatase genes?D2M30?2812,D2M30?0296,and D2M30?1007?and one NADPH-cytochrome P450 reductase gene?D2M30?0765?encoding AP1,AP2,AP3,and P450B-1,were predicted to be involved in the degradation of phoxim.Through bioinformatics analysis,the primary structure,secondary structure,and tertiary structure information of AP1,AP2,AP3,and P450B-1 were obtained,and the protein models of AP2,AP3 and P450B-1 were also successfully obtained.Recombinant AP1,AP2,AP3,and P450B-1 with biological activity were obtained by heterologous expression.The functional verification of the recombinant enzymes on the phoxim degradation showed that AP2,AP3,and P450B-1?except for AP1?all showed good degradation performances for phoxim.The optimal pH values of AP2,AP3,and P450B-1 were 5.5,10.0,and 5.0,respectively.Their optimal temperatures were,in order,30,40,and 40°C.The effect of different concentrations and different metal ions on the activity of recombinases were also studied.Additionly,using p-nitrophenyl phosphate?pNPP?as the substrate for AP2 and AP3,and 7-ethoxycoumarin as the substrate for P450B-1,the values of Km were,in order,260.4,12.2,and 1.6 mmol·L-1,the values of kcat were,in order,2.3×105,3.3×106,and 1.7×10-2 s-1,the values of kcat/Km were,in order,8.8×102,2.7×105,and 1.1×10-2 L·s-1·mmol-1.?5?Recombinant enzyme AP2,P450B-1,and the combination with AP2 and P450B-1could all degrade phoxim,and the degradation rates were 61.1,34.8,and 73.2%,respectively.According to the HPLC-MS analysis,AP2 mainly acted on the P-O bond of phoxim,and P450B-1 mainly participated in the deethylation reaction of the O-C2H5 bond.The zebrafish acute toxicity tests showed that the three enzymes have a certain degree of detoxification effect on phoxim.Thereinto,the combination of AP2 and P450B-1 degraded phoxim rapidly and completely,and the toxicity of the degraded product was significantly lower than that of phoxim. |
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