Isolation And Identification Of Estradiol Degrading Bacteria And Screening Of Degrading Genes

In China,a total of 3069 t steroid estrogen is excreted by human and animal annually.They will enter farmland and water mainly via sewage sludge used in agriculture and thus cause serious environmental pollution.17?-estradiol(E2)is the most harmful among estrogen and is mainly manifested in its effects on the growth and development of humans and animals,as well as the reproductive system,nervous system and immune system.Microbial degradation of such pollutants is currently the greenest,cheapest and most efficient method.However,the resources of safe and efficient E2 degrading bacteria are relatively scarce,and the mechanism of microbial degradation of E2 is still unclear.For this reason,it is of far-reaching significance to screen E2 degrading bacteria and analyze its functional genes and degradation mechanisms.In the light of these limitations,a study is carried out to isolate an E2 degrading bacterium and its degradation characteristics are studied to optimize the degradation conditions.A further study is performed to analyze its genetic characteristics by detecting whole-genome and transcriptome sequencing in order to screen the key genes involved in the degradation of E2.Fluorescence quantitative PCR is used to verify the result.The main contents and results of research are as follows:1.First,the gradient pressure acclimation method is used to isolate and screen a strain of bacteria MZT7 that can degrade E2 from the activated sludge in the manure storage tank of the farm.Through identification,it is a bacterium belonging to the genus Microbacterium sp.2.By studying the degradation characteristics of the strain MZT7,it is found that it has good tolerance to temperature and p H.Additional carbon and nitrogen sources will promote the degradation of E2.The optimal degrading condition in the orthogonal test optimize is under 30?,with p H as high as 8 and the inoculation of 2%.The degradation rate of E2 can reach 96.63% after culturing for 5 days under these conditions.In addition,using GC-MS to detect the degradation products,it was found that E2 can be converted into E1 with low estrogenic activity,and then further decompose it into other small molecules.3.A genome,with a GC content of 71.26% and a length of 4,011,347 bp,is extracted by analyzing whole-genome sequencing of strain MZT7.3785 coding genes contained in the whole genome are annotated by using the NR,COG,GO and KEGG databases,and two of which are annotated to the steroid degradation pathway,including HIP---Co A ligase and 3-ketosteroid 9?-monooxygenase.In addition,combined with the detection and identification results of intermediate products and related literature reports,several dehydrogenases,monooxygenases,dioxygenases and cytochrome P450 s that may be involved in the degradation of E2 are screened.Also,it is found that the strain MZT7 possesses genes for degrading many harmful substances such as benzene,naphthalene,polyphenols and polycyclic aromatic hydrocarbons,indicating that this strain has great application potential and research value.4.Taking the whole-genome sequencing result of strain MZT7 as a reference,E2 is used as a substrate to induce strain MZT7 and transcriptome sequencing analysis is performed to compare the gene transcription expression level of strain MZT7 under E2 stress conditions.The genes involved in the degradation of E2 are screened by enrichment analysis and verified by fluorescent quantitative PCR technology based on the results of genome sequencing.The sequencing results showes that there are 1109 differentially expressed genes,among which 773 are up-regulated and 336 downregulated.Differentially expressed genes are mainly enriched in functions such as transport,transmembrane transport and redox activity,indicating that the degradation of E2 by strains is a complex process of multi-gene regulation.In addition,14 upregulated genes are screened through dual-omics joint analysis,including 3-ketosteroid9?-monooxygenase in the steroid degradation pathway.RT-q PCR results shows that the result of these 14 gene expression trends and transcriptome sequencing are consistent,all of which are up-regulated expression.It has confirmed that the 3-ketosteroid 9-?-hydroxylase,NAD(P)-dependent alcohol dehydrogenase,flavin-dependent oxidoreductase,and ?-hydroxy acid dehydrogenase expressed by strain MZT7 Enzymes,acyl-Co A dehydrogenase and cytochrome P450 are the key genes for the degradation of E2.