Screening Identification And Heterologous Expression Of Zearalenone-degrading Enzymes

Mycotoxins are toxic metabolites or secondary metabolites produced by fungi,and their high toxicity and strong carcinogenicity seriously threaten animal performance and human health.According to the estimation of FAO,there are 25%of grains around the world suffered contamination by mycotoxins each year.Nowaday,Chinese contamination of mycotoxins in feeds and food has become an increasing serious problem,which caused huge losses to the food industry,feed industry and animal husbandry.Mycotoxin degradation methods mainly include physical,chemical and biological degradation,while biological degradation methods can degrade ZEN into non-toxic substances,with the advantages of high specificity,high conversion efficiency and safety environmental protection.However,there is no efficient and broad-spectrum mycotoxin degrading enzymes industrial application in China.Zearalenone(ZEN),a secondary metabolite of estrogen-like activity,is one of the most damaging mycotoxins.Microbial ZEN-degrading enzymes are widely available in the natural environment,and current research on ZEN-degrading enzymes has focused on the fungal-derived lactone hydrolase ZHD101 and its homologues.These enzymes can effectively convert ZEN into non-toxic products,but there are also bottlenecks that need to be solved,such as low degradation activity of the intermediate productα-zearalenol,poor thermal stability and low heterologous expression.To address these problems,it is of great theoretical and application value to explore the gene resources of ZEN degradation enzymes and achieve heterologous expression,focusing on ZEN degradation enzymes.In this study,a series of ZEN-degrading bacterial strains and ZEN-degrading enzymes were isolated and identified from the enrichment and domestication of ZEN-degrading bacterial populations in different environmental samples,and the heterologous expression of ZEN-degrading enzymes was carried out to construct different engineered strains,including combined expression of ZEN-degrading enzymes and hemoglobin.The main research results are as following.1.A total of 56 ZEN-degrading bacterial strains of different genera from different sources were screened by enrichment domestication method.Further rescreening with different concentrations of ZEN as the sole carbon source revealed that strain Z-33 had good ZEN degradation ability,and its ZEN degradation rate reached 84.98%when cultured at 30℃and ZEN concentration of 40μg/m L for 144 h.In addition,16S r DNA sequencing,physiological,biochemical and chemical assays indicated that Z-25was a novel strain with ZEN-degrading ability.Z-25 was named Shinella oryzae Z-25(=GDMCC1.2424~T=KCTC 82660~T).2.Using ZEN as the only carbon source,four bacterial communities Z1,Z2,Z3 and Z4 were obtained from deep-sea sediment,rhizosphere soil,moldy corn and moldy food by enrichment and domestication method.The results of HPLC showed that four bacterial consortia had abilities to degrade ZEN efficiently,The ZEN degradation rates reached 80.73%,93.59%,93.25%and 93.58%,respectively,after incubation at 30℃for 180 h at a ZEN concentration of 40μg/m L.3.To mining more genetic resources of ZEN-degrading enzymes,three ZEN-degrading bacterial consortia with ZEN degradation rates above 90%were selected for metagenomic analysis.8 potential ZEN degrading enzyme gene sequences were mined from the metagenomes of 3 bacterial consortia and the genome of strain Z-33.By using protein sequence alignment and model-based alignment based on the ZEN degrading ability of the lactone hydrolase ZHD101 and the peroxidase A4-Prx,these genes were heterologously expressed in Escherichia coli.Theα/βhydrolase Cpo C,which originated from the genus Caulobacter,degraded ZEN with 100%efficiency after incubation at 37℃for 3 h at a ZEN concentration of 20μg/m L,with specific enzyme activity of 960.92±8.30 U/mg.Further analysis of the protein sequence and structure of Cpo C revealed that Cpo C has only 25.51%identity with the amino acid sequence of ZHD101,a lactone hydrolase reported in papers,and they had completely different catalytic site triad,indicating that Cpo C has a unique protein structure and catalytic mechanism,and it is a novel zearalenone degrading enzyme.Furthermore,a model of the complex of Cpo C with ZEN was constructed,and it was predicted that the key sites for substrate binding of this enzyme might be Gly173and Asp231.4.To improve oxygen utilization of recombinant strains and ZEN-degrading enzymes,recombinant expression vectors consisting of hydrolytic lactase coding gene zhd101,peroxidase coding gene a4-prx and leghemoglobin coding gene leg H were constructed and heterologously expressed in E.coli,respectively.To provide some theoretical basis for the method of improving heterologous protein expression by co-expression of hemoglobin.In conclusion,this study obtained four ZEN-degrading bacterial consortia through enrichment and domestication,a new ZEN-degrading bacterial strain and a novel ZEN-degrading enzyme Cpo C,and constructed an efficient expression system of ZEN-degrading enzyme combined with leghemoglobin in E.coli,which provided important theoretical and technical support for development and utilization of Zearalenone degrading enzymes.