Screening Of Vomiting Toxin Degrading Bacteria And Establishment Of CRISPR/Cas9 Gene Knockout Method

The scientific name of the vomiting toxin was Deoxynivalenol,DON,and the chemical name is 3α,7α,15-trihydroxy-12,13-epoxide monotelomerase 9-en-8-one,belong to B-type monotelomerase alkene group toxin,and it was a secondary metabolism produced by Fusarium spp..Pure vomiting toxin was a white fine needle shaped crystal,and the molecular formula was C₁₅H₂₀O₆,the relative molecular weight was 296.32.Vomiting toxin was widely found in grains,including cereal products.After ingestion of vomiting toxin,people and animals may experience vomiting,nausea,and even death,causing serious harm to human and animal health.In this study,53 strains were isolated and purified from natural environment,and 5strains with high efficiency to degrade vomiting toxin were screened.The colony morphology and 16S r DNA sequencing of 5 strains with high degradation efficiency showed that the phenotypes of the 5 strains were highly similar:their color were mainly milky white with a touch of yellowish inside,and their shape were nearly round.16S r DNA sequencing showed that strain WH-4 had high homology with Bacillus DPBS066,strain JZ-8 had high homology with Bacillus cereus B33,and strain XY-2-2 had high homology with Bacillus NQS29,strain TM-2-4 had high homology with Bacillus cereus PL316 and strain XY-3-3 had high homology with bacillus tropicalis 5TC-1.The characteristics of degradation of vomitin by strains were studied,including the process of dynamic degradation of vomitin by degrading bacteria,the optimal degradation temperature,and the effects of degrading bacteria and their metabolites on the growth and sporulation of Gibberella spp.The results showed that the best pH degradation condition for the strain was 6.5-7.5 neutral environment.The optimum degradation temperature was 37 C.Metabolites of degrading bacteria inhibited the growth of Gibberella,but the effect was not obvious.Metabolites of strains JZ-8,TM-2-4 and XY-2-2 had significant inhibitory effects on the sporulation of Gibberella spp.The most obvious inhibitory effect was strain TM-2-4,which reduced the spore production of Gibberella spp.by 47%compared with the blank control.After combining the whole genome sequencing data and the related literature reported detoxification genes,we screened all but 27 detoxification candidates in strain TM-2-4,and then performed protein characterization,signal peptide prediction and protein tertiary structure analysis to select the candidate genes（gene313,gene314,gene1024 and gene1026）for gene knockout validation.The CRISPR/cas9 system was used to knock out the target gene,and after obtaining the target strain,the original strain TM-2-4 was used as a control to investigate whether the degradation efficiency of vomitoxin was changed by the strain after gene knockout,and the results showed that the degradation efficiency of vomitoxin was not changed by the strain after gene knockout.