Degradation Of Steroid Hormones By Rhodococcus R-001 17?-HSD

With the expansion of aquaculture,more and more attention has been paid to the pollution caused by steroid hormones to the natural environment.Steroid hormones,typically represented by estrogens and androgens,interfere with reproductive development,metabolic balance and immune function of animals and humans in the environment.Androgenic steroids have been listed as class 2A carcinogens by the World Health Organization,so it is urgent to degrade the steroid hormones in the environment.Steroid hormones are an important carrier of carbon sources.It was found that microorganism could cleavage different carbon sites and modify different types of steroid rings by its own enzymatic reaction to reduce steroid hormone activity.17?-hydroxy steroid dehydrogenase(17?-HSD),as a highly specific enzyme for C-17 dehydrogenation of steroid hormones,catalyses the interconversion of some steroid ketones,which plays an important role in the degradation of steroid hormones.However,17?-HSD has been relatively poorly studied in microorganisms capable of degrading steroid hormones,and the range of steroid substrates it degrades requires further investigation.Therefore,Rhodococcus equi DSSKP-R-001(R-001),a strain with strong substrate adaptability to various steroid hormones,was selected in this paper.A gene encoding17?-HSD(C7H75?RS25245)in its genome was named as Rhodococcus R-001 17?-HSD.The homology between the gene and other 17?-HSD was confirmed by phylogenetic tree construction.Transcriptional analysis and high performance liquid chromatography(HPLC)showed that the gene played a major role in the degradation of steroid hormones by Rhodococcus R-001.By means of homologous recombination technique and SDS-PAGE analysis,R-001 17?-hsd was constructed into E.coli BL21(DE3)/p ET28 a for heterologous expression,and the induction conditions of the recombinant protein were optimized.The effect of 17?-HSD on the degradation of steroid hormones by recombinant bacteria was studied by HPLC and RT-PCR.At the same time,the genetic stability of recombinant Escherichia coli was briefly analyzed by using genetic engineering techniques.The purpose of this paper is to provide a theoretical basis for the exploration of biodegradable steroid hormones and to provide a scientific basis for the restoration of national ecological environment.The results are as follows:(1)The highest homology of 17?-HSD was 98% with the 17?-HSD annotated in other known strains,confirming that this DNA fragment in R-001 was a 17?-HSD coding gene.The degradation efficiency of testosterone and estradiol at 12 h was 98.8% and 89.5%,respectively,in the inorganic salt environment with testosterone or estradiol as the only carbon source.During the degradation of testosterone or estradiol,17?-HSD gene was significantly expressed at the m RNA level,suggesting that 17?-hsd gene played a major role in the degradation of steroid hormones.(2)The 17?-hsd gene was successfully constructed into the plasmid p ET28 a,and the molecular weight of the protein was 29.8 k Da after heterologous expression in E.coli BL21(DE3).After optimizing the inducing conditions of recombinant protein expression,it was found that 0.025 m M IPTG induced the best expression at 20?.At 37?,0.05 m M IPTG induced the best expression.(3)The recombinant strain BL21(DE3)/p ET28a-17?-hsd had good genetic stability.The growth rate of the 100 generation recombinant bacteria was slightly slower than that of the original recombinant bacteria at the early stage of culture.Under kanamycin pressure,the plasmids of Escherichia coli with less than 40 generations had good stability,and the plasmid stability of Escherichia coli with 100 generations was 79%.The plasmid concentration did not decrease significantly in the passage process of less than 100 generations,the gene in the plasmid remained stable,and the protein expression was stable.(4)The recombinant strain BL21(DE3)/p ET28a-17?-hsd has the function of degrading steroid hormones and the degradation efficiency of testosterone is higher.The degradation rate of estradiol was 55% and that of testosterone was 98% at 24 h.Escherichia coli BL21(DE3)p ET28 a without 17?-hsd gene had no obvious ability to degrade the substrate.The reverse transcription results showed that 17?-hsd gene was induced expression and played a leading role in the degradation of the recombinant strain.In conclusion,this paper mainly studied the function of degrading steroid hormones of Rhodococcus R-001 17?-HSD through sequence alignment technology and genetic engineering technology,and constructed a recombinant E.coli BL21(DE3)/ PET28a-17?-hsd with the function of degrading steroids and good genetic stability.From the perspective of exploiting gene function,it provides a new idea for the future development of obligate pollutant degradation.