Characterization Of DON-degrading Bacteria And Exploration On Its Degradation Mechanism

Deoxynivalenol（DON）,also known as vomitol,is a secondary metabolite produced by several Fusarium species,which is potentially harmful to human and animals after entering the food chain.However,the effects of various physical and chemical methods to remove DON are not ideal,also at the expense of the nutritions.Therefore,the development of biological detoxification method is imperative because of its green safety and high efficiency.Biological detoxification of DON can be divided into two categories:the adsorption of DON by microorganisms,and the degradation of toxic groups in DON by enzymes from microorganisms.Toxicological studies have shown that most of the microbial degradation products of DON are less toxic than DON,and even close to non-toxic.Therefore,the screening of microorganisms capable of high efficiency in the degradation of DON and the mining of their potential enzyme-coding genes are of great significance for the development of degradation bacteria/enzyme preparations and its application in DON removal in the food and feed industry.In this research,with the combination of the high-throughput sequencing and laboratory enrichment culture method,Devosia strain A6-243,able to degrade DON,was screened out from the soil.The degradation mechanism of DON by the strain A6-243 was investigated and the results showed that the epimerization of DON by A6-243 was achieved by the two-step process,where DON is first converted into 3-keto-DON followed by the reduction into 3-epi-DON.Meanwhile,it was found that the degradation of DON was mainly catalyzed by intracellular enzymes of Devosia strain A6-243,and the expression of the degrading enzymes did not require the induction of DON.The results of this study are summarized as follows:Isolation and identification of DON-degrading bacteria.In this study,a bacterial consortium LG-6 with DON degradation ability was obtained from soil,and it was found that the DON degradation effect varied among the bacterial consortium with different gradient of dilution ratios.Then high-throughput sequencing was used to analyze DON degrading bacteria in a bacterial consortium LG-6.The bacterial consortium LG-6-7 with dilution of 10^-7 times could completely degrade DON,while the bacterial consortium LG-6-8 with dilution ratio of 10^-8 times could not degrade DON.The microbial diversity of the two bacterial consortium was analyzed,and it was found that the bacterial consortium LG-6-7 included Pseudochrobactrum,Pseudomonas,Delftia,Devosia,and Achromobacter,while the bacterial consortium LG-6-8 included Pseudochrobactrum and Achromobacte,indicating that Pseudomonas,Devosia and Delftia were essential for the degradation of DON.In addition,DON（50μg/m L）was completely degraded within 48 hours in the culture with a bacterial consortium of Pseudomonas and Devosia.Further,by studying the effects of different cofactors on the degradation of DON by Pseudomonas and Devosia,it was found that Devosia strain A6-243 could degrade DON with exogenous addition of PQQ.Devosia strain A6-243 can degrade DON（100μg/m L）completely within 48 hours and produce degradation product A,while Pseudomonas could not degrade DON.Therefore,the high-throughput screening method is effective in analysis of the difference of DON degradation effect and strain differences among different dilution ratios in bacterial consortium,which could provide guidance for the efficient screening of deoxynivalenol degrading bacteriaIdentification of degradation products of deoxynivalenol by Devosia strain A6-243.The DON degradation conditions by Devosia strain A6-243 was tested and it was found that the degradation rate of DON can achieve 100%by Devosia strain A6-243 in the condition with PQQ concentration above 15μM,16～37℃,and pH 6～9.In addition,whether in the inorganic salt medium with poor nutrition,or more nutritious MMT,MMY,LB,NB medium,the DON degradation rate can reach 100%by Devosia strain A6-243.In the HPLC chromatogram of the degradation process of DON by the cell fragments of Devosia strain A6-243,it was found that the absorption peak of DON gradually decreased with the increase of incubation time,and the absorption peak of degradation product A gradually increased,indicating that DON was degraded into product A.By preparative liquid phase separation and organic solvent extraction,5mg degradation product A was obtained.The liquid-mass chromatograph showed that the molecular weights of degradation product A and DON were both 297 g/mol,and the characteristic ion peaks 231,249,261,279 and 297 of degradation product A were identical to those of DON.At the same time,the retention time of degradation product A was shorter than that of DON,indicating that the polarity of degradation product A was larger than that of DON.Therefore,it was speculated that the degradation product A was the epimerization form of DON,and further analysis of NMR spectra confirmed that the degradation product A was 3-epi-DON.Mechanism of Deoxynivalenol Degradation by Devosia strain A6-243.Firstly,the relationship between the growth of A6-243 and DON degradation ability was studied.The results showed that at the beginning of the logarithmic phase of growth,Devosia strain A6-243 could efficiently degrade DON,where the enzymes capable of degrading DON was expressed.Besides,the expression of DON degrading enzyme in the A6-243 doesn’t need DON induction.Secondly,the location of the degrading enzyme in Devosia strain A6-243was investigated,and we found that the degrading enzyme exists in both cell lysis supernatant and cellular debris of Devosia strain A6-243,which can completely degrade DON within 48 h,while heat treatment group and Devosia strain A6-243 fermentation supernatant cannot degrade DON.These results suggested that Devosia strain A6-243degrade DON by the action of intracellular enzymes.Finally,the ability of degradation on DON and 3-keto-DON by Devosia strain A6-243 was compared,and the results showed that the degradation rate of both DON and 3-keto-DON can achieve 100%,indicating that in Devosia strain A6-243 the conversion of DON is also a two-step process,where DON is first oxidized to form 3-keto-DON followed by the reduction into 3-epi-DON.The3-epi-DON did not decrease over the culture time,indicating that 3-epi-DON is the final product of DON degradation and this process is irreversible.