Preliminary Study On Transcriptional Regulation Mechanism Of Estrogen Degradation Gene Cyp123A9 By TetR In Rhodococcus Sp. P14

Exogenous estrogen is one of the most serious environmental Endocrine disruptors(EDCs),and its metabolism is closely concerned by researchers.However,the pathways of estrogen degradation in Gram-positive bacteria are not clear,and the background of transcriptional regulation of degraded genes is also obscure.Rhodococcus plays an important role in estrogen degradation.In the laboratory,we isolated a highly estrogen-degrading strain of Rhodococcus sp.P14,in which under17β-estradiol stress,an upregulation of estrogen-degrading gene cluster were found in transcriptomic data,including regulatory protein TetR and cytochrome P450(CYP123A9).The regulation gene and P450 were studied to explore the function and regulation of CYP123A9,a functional gene in the process of estrogen metabolism in Rhodococcus sp.P14.The main results of this experiment are as follows:(1)Cloning and heterologous expression of P450 and TetR.It was confirmed that CYP123A9 could transform estrone and testosterone into 16-hydroxyestrone and 6-hydroxytestosterone,respectively.Also,Thr242 of CYP123A9 was verified as the key binding site affecting the degradation of estrone and testosterone.(2)RT-qPCR verified that tetR and cyp123A9 could be co-transcribed.P14 in vivo verification of tetR overexpression significantly upregulated CYP123A9 expression.The promoter region was predicted and verified in Escherichia coli system,and the optimal promoter was screened by continuous truncation of the promoter and detection of the expression intensity of the reporter gene.Among the 14 promoters,promoter P12,as the optimal promoter of CYP123A9,was induced by tetR and activated downstream gene expression.(3)To verify the specific site of TetR binding to promoter P12,a reverse palindrome sequence ACGG-N14-CCGT was subjected to single point mutation and symmetric point mutation,and fluorescence intensity was detected.It was found that singlepoint base mutation strongly affected the e GFP transcription level,and symmetric point mutation could restore the fluorescence intensity to the original level or even higher without destroying the reverse palindrome structure.It is speculated that the reverse palindrome sequence is the TetR binding region and does not require strict conservation of bases.(4)In order to further study the binding ability of total P14 protein to regulatory protein TetR under different estrogen stimulation,TetR was recombinant expressed and purified with different pollutant induced P14 proteins.In the Pull down experiment,TetR binding protein under estrone,17β-estradiol and testosterone were compared.It was found that 17β-estradiol induced most TetR binding proteins,followed by E1 and almost no binding proteins with T.TetR binding proteins induced by 17β-estradiol included rpo C-encoded RNA polymerase subunit β ’.We hypothesized that RNA polymerase subunits were generated under E2 stress,and after complete assembly,they clustered near the estrogen degradation gene cluster,recognized and bound to the promoter region,and completed the transcription of downstream gene cyp123A9.This study has accumulated resources in preliminatively exploring the transcriptional regulation mechanism of TetR-mediated estrogen degradation in P14,adding a new case of positive regulation of TetR transcriptional regulation family and providing a new idea to solve estrogen pollution at the transcriptional level.