Isolation And Characterization Of Bacterial Strains And A Gene Encoding A Protein For Detoxifying Fusarium Toxins

Fusarium toxins are a group of toxic secondary metabolite produced by many species of the genus Fusarium during the infection of wheat,barley,maize and other small grain cereals.Among them,deoxynivalenol(DON)is the most frequent mycotoxins in cereal matrices worldwide.DON has serious toxic efects on human and farm animals.In addition,DON is also phytotoxic,damaging plant tissues and acting as a virulence factor that stimulates fungal infection.Therefore,isolation and characterization of agents capable of detoxifying Fusarium toxins is essential for food/feed safety and human and animal health.Detoxification of DON by microorganisms is considered a promising method because of its efficiency,specificity,and environmental soundness.However,so far,almost all of the bacteria with de-epoxydation activities toward trichothecenes were anaerobic,limiting their empirical use.In addition,the underlying metabolite pathways remain unknown and the detoxification genes have not yet been cloned in the strain acting the C3 hydroxyl of DON.In this study,one pure bacterial strain,Sphingomonas S3-4,displayed stable DON-degradation activity in the aerobic condition was isolated from wheat field.The strain S3-4 can completely elimilate DON in the medium and the toxin in the wheat infected with F.graminearum.The products of DON degradation were detected by High performance liquid chromatography(HPLC),gas-chromatograph/mass spectroscopy(GC/MS)and nuclear magnetic resonance(NMR)spectroscopy,the results strongly indicate that the strain S3-4 acted the C3 hydroxyl of DON,and DON was transformed into 3-oxo-DON and 3-epi-DON.The murine macrophages and wheat seedlings were used to evaluate cytotoxicity and phytotoxicity of metabolites.Compared with DON,3-oxo-DON and 3-epi-DON have obviously lower toxicity than DON.The gene responsible for oxidation of DON into 3-oxo-DON was isolated using the genome sequencing,function-based screening and comparative analysis.This gene belongs to a new AKR family,family 18,and A1,designated AKR18A1,as the first member of this family.The HIS fusion AKR18A1 protein expressed in E.coli BL21 can catalyze the reversible oxidation/reduction of DON to 3-oxo-DON.The recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal.Furthermore,the AKR18A1 gene was disrupted using the method of homologous recombination,and the mutant showed no degradation activity to DON.The DNA sequences of the AKR18A1 gene were codon-optimized,synthesized and transformed into Arabidopsis thaliana,and the transgenic plant contained AKR18A1 gene showed more tolerance to DON than the wild-type Arabidopsis.In addition,a bacterial consortium,PGC-3,with DON degradation activity under aerobic condition was isolated from soil samples using an in situ soil enrichment method.The consortium PGC-3 acted the C12/13 epoxide group of DON,and DON was transformed into de-epoxy DON(d E-DON),PGC-3 could also de-epoxydize NIV into de-epoxy NIV(d E-NIV),which indicated by HPLC and GC/MS profiles.The murine macrophages were used to evaluate cytotoxicity of metabolite,and d E-DON showed less toxicity to the cells than that of DON.PGC-3 exhibited stable de-epoxydation activity at a wide range of p H(5-10)and temperatures(20-37?)values under aerobic conditions.PCR-DGGE results showed that sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium.Analyses of the 16 S r DNA sequences indicated that PGC-3 comprised 10 bacterial genera.Among these,one species,Desulfitobacterium,was possibly a major contributor to the observed DON de-epoxydation activity.This study provides promising agents for the control of Fusarium pathogens and detoxifcation of mycotoxins in plants and in food/feed products.These results also establish the foundation to further dissect the complete pathway for degradation of Fusarium toxins and their regulation.