Characterization And Preliminary Mechanism Investigation Of Aflatoxin Degradation By Lysobacter Sp.CW239

Aflatoxins（AFT）a group of secondary metabolites produced by Aspergillus spp.;as a potent mycotoxin,aflatoxins are highly carcinogenic,teratogenic,and mutagenic effects on human and animals.Among the aflatoxins,aflatoxin B1（AFB1）is recognized as the most toxic,and widely contaminated in nuts and food crops.It poses a huge threat to humans and livestock,and also results in losses to the world’s agricultural economy.Therefore,it is great significant to obtain effective methods for AFB1 detoxification.Biological detoxification method has attracted much attention because of its high degradation efficiency and specificity.At present,many degrading microbial strains have been isolated and indentified,but few studies focused on the biodetoxify enzymes in these strains.Due to the complex chemical structure and degradation processes of AFB1,the AFB1degradation mechanism clarification is a very difficult work.Hereby,aflatoxins detoxification enzymes（or genes）screening and the biological detoxification mechanism investigation should be great significant to the development biological detoxification technology in agricultural production and food industry.Lysobacter sp.CW239 is a strain ofγ-Proteobacteria which was isolated and deposited in our laboratory;strain CW239 can degrade low concentration AFB1 efficiently.After inoculated strain CW239 into a nutrient broth that containing 20μg/L AFB1 and incubated the culture for 72 h,the AFB1 degradation ratio was 75%.Subsequently,the cell-free supernatant,bacterial cells and intracellular extract of CW239 were prepared and determined for AFB1 degradation,respectively.As a result,only cell-free supernatant showed the AFB1 degradation activity.The degradation tests indicated that,the neutral p H and relatively high optimal temperatures（40-90℃）were appropriate to AFB1 degradation,and the degradation activity was increased steady by the improvement of incubation temperatures.Aflatoxin degradation activity of cell-free supernatant can significantly inhibit by 0.01 mol/L Cu²⁺,Zn²⁺,Ca²⁺and Fe²⁺.After pre-treatment of cell-free supernatant by EDTA,EGTA,proteinase K or SDS（or SDS plus proteinase K）at 30℃for 6 h,the degradation activity was significantly reduced,but none of treatment can inhibit the activity intensively.The degradation results from cell-free supernatant indicated that the active degradation components were quite complex and may contained metalloproteinases and micromolecular（s）.Based on the genomic sequence of Lysobacter concretionis（type species of Lysobacter）and the information of detoxify enzymes in the previous studies,a gene hppd239（4-hydroxyphenylpyruvate dioxygenase）that mediates the AFB1 degradation in cell-free supernatant was successfully screened.The cell-free supernatant prepared from the gene expressed E.coli BL21 transformant（containing p GEX-4T-1/hppd239）can efficiently degrade low concentration of AFB1.After the gene hppd239 expression in E.coli BL21,the color of cell-free supernatant prepared from p GEX-4T-1/hppd239transformant became darker（i.e.,reddish-brown）,but the cell-free supernatant of p GEX-4T-1 transformant was transparent（light yellow）.The degradation result found that approximately 70%of AFB1 was degraded by cell-free supernatant from p GEX-4T-1/hppd239 transformant within 24 h.However,the recombinant protein r HPPD239 produced from p GEX-4T-1/hppd239 transformant showed almost no degradation activity on AFB1.The active component from the p GEX-4T-1/hppd239transformant supernatant was thermotolerant（thermodependent）which was consistent to the cell-free supernatant of strain CW239.To elucidating whether hppd239 is the vital gene responsible for AFB1 degradation in strain CW239,the gene hppd239 was knockout from the genome of CW239 using the suicide plasmid p K18mobsac B by homologous recombination.Comparing with wide-type CW239,the mutant CW239^Δhppd equally showed efficient degrading activity to AFB1,the mutant CW239^Δhppd only showed a significant lower AFB1 degradation ratio at 48 h.As the degradation time prolonged,the degradation ratios of the mutant CW239^Δhppd showed no significant differences to the wild-type strain CW239（P>0.05）.On the basis of mutant construction,the HPPD inhibitor sulcotrione was further tested on CW239 and E.coli BL21（p GEX-4T-1/hppd239）transformant.As a result,the degradation activity of E.coli BL21（p GEX-4T-1/hppd239）was inhibited significantly by sulcotrione,but the CW239showed no significant effect to inhibitor.That is,in addition to the gene hppd239,other genes（or other degradation agents）can mediate AFB1 degradation in strain CW239;the gene hppd239 mediated degradation should be only one of the multiple pathways.Hereafter,the degradation components in the cell-free supernatant prepared from p GEX-4T-1/hppd239 transformant were determined by high performance liquid chromatography tandem mass spectrometry（LC-MS/MS）,and the gene hppd239 mediated metabolic changes were analyzed.The 13 candidate active agents screened from LC-MS/MS for aflatoxin degradation was artificially synthesized and tested for the degradation activity.Combining the comprehensive analysis of metabolomics and the degradation active components screening,the gene hppd239 mediated metabolic changes in host strain E.coli BL21 were determined.Among the metabolic pathways,the coumarin antibiotic novobiocin biosynthesis pathway and TCA cycle pathway were the greatly interfered,followed by purine and tyrosine metabolism processes.In order to screen AFB1degradation agents from these candidate agents,the relationship between the metabolic processes changing and AFB1 degradation was analyzed,and the candidate degradation agents will be examined further for the following study.