Screening Of Zearalenone-detoxification Strain And Constructing Of Fosmid Library

Zearalenone(ZEN)is a nonsteroidal estrogenic mycotoxin produced by various Fusarium species on cereal grains and causes hyper estrogenism and related toxicosis of animals and humans.Our study aimed to focus on screening strains which can transform ZEN to none estrogenic toxicity derivatives,understanding ZEN transformation characterization and mechanism,constructing metagenomics fosmid library on the soil samples,screening functional DNA fragment to removing ZEN,and providing a theoretical basis for the ZEN biodetoxification and application.The main conclusions are as follows:(1)Three bacteria capable of transforming ZEN were screened from 140 different soil samples where gibberellic disease occurred frequently through 96-well micro plate,which named Fu2-3,Fu41-3 and Fu44-1 and were identified as Sphingobium,Bacillus tequilensis and Bacillus amyloliquefaciens based on 16 S rDNA gene sequence.(2)The growth curve and ZEN degradation curve of two Bacillus strains Fu2-3 and Fu41-3 and Bacillus strains 101,246,373,KCH,R1 stored in fermentation technology group were measured.The results showed that strain KCH needed the shortest time to entered the stationary phase,the strain101 reached the highest biamass of this stage,and the strain 373 showed the fastest rate to removing ZEN(10 ?g/mL).(3)Hormonal effects of ZEN detoxification products of the strains were detected by high performance liquid chromatography(HPLC)and bioluminescence yeast estrogen screen(BLYES)assay which indicated that,the degradation products of strain KCH,R1,and 373 still contained considerable estrogenic activity,probably ZEN was transformed to a derivative with estrogenic toxicity.While strain 101 and Fu2-3 resulted in practically non-estrogenic activity,showing the best detoxification effect.(4)The profile of ZEN detoxification by the strain 101 was investigated,the results showed that the cell membrane could bind a small part of ZEN,but the main detoxification was by the biotransformation of ZEN structure via active substance which was proved to be present in the extracellular fraction.This active substance was deadly affected by temperature and EDTA,the ZEN elimnination activity could be greatly reduced in the condition of high temperature and high concentration of EDTA.(5)Using LC-MS technology to analyze ZEN biodetoxification products of the strain 101,the molecular weight of product was 480.0 g/mol,the chemical formula was primarily identified as C24H32O10.and the dexoxification product was zearalenone-14-?-D-glucoside and zearalenone-16-?-D-glucoside.(6)The metagenomic Fosmid library was constructed using the soils sampled from the fields with gibberellic disease.The library titer was 9.5×103 CFU,three fosmid clones with the ability to transform ZEN were successfully screened.