Heterologous Expression Of Kstd From Rhodococcus R.D-001 And Its Degradation Of Steroid Hormones

Steroids are a kind of endocrine disruptors,which can lead to some adverse effects such as sex imbalance,decreased reproductive ability and cancer at very low levels.Therefore,the pollution of steroids in the environment has attracted wide attention of researchers.At present,scholars at home and abroad have done a lot of work on the use of microorganisms to degrade steroids,including the isolation and identification of steroid-degrading bacteria,the optimization of degradation conditions,degradation kinetics and so on.However,the growth rate of traditional steroid-transforming bacteria is slow and the culture environment is high,which is not conducive to large-scale industrial culture,which limits the application of environmental remediation.Genetically engineered strains can effectively avoid these shortcomings.At present,the use of genetically engineered bacteria to degrade steroids has become a hot issue for people to solve environmental pollution.In this study,based on the method of genome sequencing,five key steroid degradation enzymes Kst D,were annotated from Rhodococcus equi DSSKP-R-001(R.D-001)to construct genetically engineered bacteria by gene cloning.The ability of genetically engineered bacteria to degrade steroids was detected by high performance liquid chromatography,and the physicochemical properties,structure prediction and conservative domain analysis of the protein were carried out by bioinformatics method.The basic properties of the enzyme were further explained at the molecular level.The main results of this paper are as follows:1.Five Kst D enzyme genes of 1539 bp,1713 bp,1539 bp,1692 bp and 1689bp from Rhodococcus R.D-001 were cloned and constructed.The recombinant plasmids were heterogeneously expressed in E.coli BL21(DE3)to construct recombinant bacteria containing K1,K2,K3,K4 and K5 genes.The target proteins of 61 k Da,60 k Da,54 k Da,60 k Da and 60 k Da were successfully obtained by inducer(IPTG).2.All the five genetically engineered bacteria could use 4-androstenedione(AD),progesterone(PGT),testosterone(TTR)and 17?-estradiol(E2)as the only carbon source for growth,and the degradation rates of AD and TTR were more than 90%,and the degradation rates of E2 and PGT were more than 30%.Among them,strains K1 and K4 had the best effect on the degradation of AD,K2,K3 and K5 had the best effect on the degradation of TTR,and the degradation efficiency of PGT was the lowest.Among the 5 recombinant bacteria,strain K2 had the best degradation effect and had broad-spectrum adaptability.In the degradation experiment of 72 hours,the degradation rate of AD,TTR,PGT and E2 reached 95.66%,99.02%,46.54%and 50.22%,respectively.RT-PCR experiments showed that the expression of kstd2 gene was induced,and the relative expression level of m RNA was different under the induction of different steroid substrates,in which the relative expression level of m RNA induced by AD was the highest,followed by the expression of Kst D2 induced by E2.At the same time,strain K2 showed a preference for AD and testosterone,followed by estradiol and progesterone,which further indicated that the genetically engineered strain showed a selective preference in the degradation of different steroids.3.The total number of Kst D2 atoms is 8468 and the molecular formula is C₂₆₇₉H₄₁₉₀N₇₆₄O₈₁₇S₁₈.The gene sequence encoding Kst D2 is 1713 bp and can encode 568amino acids.The starting codon is ATG,and the stop codon is TGA.The enzyme has no signal peptide and transmembrane region,is a hydrophilic soluble protein and belongs to an important intracellular oxidoreductase.It is predicted that the theoretical isoelectric point(PI)is 5.74 and the theoretical molecular weight is 60.75 k Da.20 amino acid sequences with steroid degradation function were selected to construct the phylogenetic tree,and the closely related proteins were selected for multi-sequence alignment.It was found that the protein belonged to 3-keto steroid-?1-dehydrogenase.The predicted secondary structure showed that the main structure of the enzyme was?-helix and random crimp.The 4c3y.1A crystal structure of strain R.erythropolis SQ1 was used for homologous modeling,and the similarity between Kst D and template 4c3y.1A was 35.90%.It was speculated that the tertiary structure of Kst D may be homologous tetramer.These results provide a theoretical basis for the further study of the specific site residues of the mutant substrate binding sites.The above results show that the genetically engineered strain K2 has a strong ability to degrade steroids in a short time and has a strong application potential to degrade steroids.This study will help to promote the study of the molecular mechanism of steroid degradation in Rhodococcus R.D-001 and provide a basis for the improvement of steroid metabolic pathway.It also provides some theoretical guidance for the successful application of using genetically engineered bacteria to degrade steroids in the environment.