| In recent years,endocrine disruptors such as estrogens have caused serious threats to human health and ecological safety,gradually attracting widespread attention from scholars at home and abroad,and their pollution levels,enrichment levels,migration patterns,and transformation processes in the natural environment.Bioremediation,that is the use of microorganisms to degrade environmental estrogen is one of the effective methods to solve estrogen contamination.The elucidation of the genomic structure and characteristics of estrogen degrading microorganisms is the basis and prerequisite for the use of microbial metabolic capacity for environmental estrogen degradation.In this paper,17?-estradiol(E2)was selected as the target contaminant.One strain of Rhodococcus with the ability of efficient degradation of E2 was screened,and its genomic structure,distribution and expression characteristics of the degradative enzyme genes were studied.The comparative analysis of the genome laid the foundation for the future construction of estrogen degrading engineering bacteria.1.Screening and identification of estrogen-degrading bacteriaAn estradiol-degrading strain was screened with estradiol as the substrate.The growth curve and the remaining amount of estradiol were determined using a microplate reader and high performance liquid chromatography.The strain was found to grow well and was observed.Using estradiol,the degradation rate of estradiol reached a maximum of 97% on the third day and reached 0.155 on the fifth day.Morphological observation,physiological and biochemical analysis and 16 S rDNA sequence analysis and identification were performed on the strains.The results showed that the strains were Actinomyces,actinomycetes,and Rhodococcus erythropolis.It is named Rhodococcus.DSSKP-R-001(abbreviated as RD1).Deposited in China General Microbiological Culture Collection Center.2.Genomic sequencing and functional gene annotation of Rhodococcus DSSKP-R-001Full-genome sequencing and functional gene annotation of Rhodococcus.DSSKP-R-001 using second and third generation sequencing technologies.The genome sketch sequence included a genome size of approximately 5.4 Mbp,a G+C content of 68.72%,and 5180 predicted protein-coding genes.The strain Rhodococcus.DSSKP-R-001 genome consists of three replicons,one chromosome and two plasmids.The chromosome size was 5.2 Mbp and the two plasmids were 0.09 Mbp and 0.09 Mbp,respectively.3736 genes,3472 genes,2590 genes,and 1932 genes were annotated using the COG,GO,KEGG,and Swiss-Prot databases,respectively.Through functional gene annotation,the results showed that a total of 10 genes related to estrogen-degrading enzymes were obtained,and the various enzyme genes have extensive distribution and unequal recurrence characteristics throughout the sequence.3.Verification and expression of estrogen-degrading enzymesThe presence and expression of estradiol degrading enzymes were verified by polymerase chain reaction(PCR)and reverse transcription polymerase chain reaction(RT-PCR).Ten enzymes that directly involved in steroid degradation,namely 3?-,were found.Hydroxysteroid dehydrogenase,3-ketosteroid-?1-dehydrogenase,acetaldehyde dehydrogenase,cholesterol oxidase,3-keto steroid-9? hydroxylase,dioxygenase,3,4-DHSA dioxygenation Enzymes,monooxygenase,acetyl-CoA acetyltransferase,3-ketoacyl-CoA thiolase,and four enzymes among them: 3-Ketosteroid-9? hydroxylase,monooxygenase,dioxygenase Acetaldehyde dehydrogenase was used to express mRNA.4.Genome comparative analysisComparative genomics was used to compare Rhodococcus.DSSKP-R-001 with Rhodococcus.sp.P14,Rhodococcus jostii.RHA1,and Rhodococcus opacus B4 by two-dimensional collinearity,parallel collinearity,and Venn diagrams.It was found that Rhodococcus.DSSKP-R-001 has the highest similarity with Rhodococcus.sp.P14.There are 2070 genes shared with Rhodococcus.sp.P14,Rhodococcus jostii.RHA1,Rhodococcus opacus B4,and Rhodococcus equi 103 S.Of these,1159 genes are involved in the metabolism of materials,showing the same evolution source. |
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