| Microbial degradation of steroids is a hot topic in the study of removing endocrine disruptors.The key to the degradation of steroid hormones lies in the expression and regulation of protease related genes in microbial genome.Among them,steroid dehydrogenase has been proved to be the key enzyme of steroid metabolism in most organisms,especially in humans and animals.However,there are few reports on steroid dehydrogenase gene and its expression regulation.In order to further elucidate the gene of steroid dehydrogenase and its mechanism in the degradation of steroid hormones,In this paper,Rhodococcus equi DSSKP-R-001(R.D-001 for short)was used as an experimental strain to compare the genomic distribution characteristics and metabolic pathways of steroid dehydrogenase gene in R.D-001 and other steroid degrading strains;by PCR and RT-PCR,the expression level and metabolic ability of three kinds of steroid dehydrogenase genes induced by different steroid substrates were compared;through the method of bioinformatics,the structure and function of three kinds of sterol dehydrogenase were compared to reveal their biochemical characteristics and physiological functions.It will lay a foundation for the cloning,expression and application of steroid dehydrogenase gene in the future.The results of this study are as follows:1.Comparative study of R.D-001 steroid dehydrogenase genomeThe genome of R.D-001 is relatively stable and contains a large number of related genes encoding steroid ring degrading enzymes.66 kinds of dehydrogenase genes were found in the KEGG database,accounting for 1.29% of the total number of coding genes.There are 19 kinds of dehydrogenase genes related to steroid degradation,which are widely distributed and relatively conserved in the R.D-001 genome.Among them,three key dehydrogenase genes involved in steroid catabolism are 3-ketosterol-1-dehydrogenase(KstD),3-oxo-5?-steroid 4-dehydrogenase-1(SRD5A1)and 17?-hydroxysteroid dehydrogenase(17?-HSD).There are a large number of conserved and homologous steroid degradation genes near the three kinds of steroid dehydrogenase genes.The R.D-001 genome contains 5 KstD ORFs(3KstD1,1 KstD2,and 1 KstD3),where the KstD3 ORF is located in a highly conserved genomic region encoding steroid catabolism.Three kinds of sterol dehydrogenase are involved in the metabolic pathway of steroid hormone synthesis and degradation,which further explains their important role in steroid metabolism.2.Comparative study on the degradation of steroid by R.D-001 steroid dehydrogenaseR.D-001 can grow in the medium with estradiol(E2),estriol(E3),testosterone(TTR)and progesterone(PGT)as the sole carbon source and energy source,and has strong metabolic capacity for all four kinds of sterol hormones.The degradation rate of PGT and TTR was over 98% at 12 h,while the degradation rate of E2 and E3 was about 80% at 96 h,the degradation ability of PGT and TTR was higher than that of E2 and E3.Under the induction of four kinds of steroids,all three kinds of dehydrogenase genes were expressed,but under the induction of different steroids,the three kinds of steroids dehydrogenase genes were affected by the substrate specificity,and their relative expression levels were different,and they could show different preference when degrading different steroids.3.Comparative study on the structure and function of three kinds of sterol dehydrogenase of R.D-001R.D-001 three kinds of sterol dehydrogenase have strong amino acid sequence identity.The 3-ketosteroid-?1-dehydrogenase(KstD)encoded by the 001952 gene contains the FAD binding domain,and it is speculated that the amino acid residues in the catalytically active site may be Tyr104 and Tyr121;the 17?-hydroxysteroid dehydrogenase(17?-HSD)encoded by the 005108 gene contains the NADH binding region and active site,and it is speculated that the catalytic residue sites may be Tyr149 and Ser150;the active site of 3-oxo-5?-steroid 4-dehydrogenase 1(SRD5A1)encoded by 000996 gene is temporarily unpredictable.KstD and 17?-HSD have no signal peptide and transmembrane domain,and are important intracellular oxidoreductases,while SRD5A1 has a signal peptide and 6 transmembrane domains,which are transmembrane proteins.The three enzymes have four phosphorylation sites for protein kinases.Amino acid residues and phosphorylation site residues in the active site may affect the activity of the enzyme,which in turn affects the enzyme's preference for different steroid substrates.In conclusion,through the in-depth analysis of the sequencing results of R.D-001 genome,the gene characteristics,expression products and physiological functions of steroid dehydrogenase were fully explained by means of comparative genomics.The results will be helpful to promote the study of the molecular mechanism and functional regulation mechanism of steroid dehydrogenase,and provide a theoretical basis for the construction of high-efficiency environmental estrogen degradation engineering strains. |
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