Identification And Function Study Of Key Genes For Estradiol Degradation In Pseudomonas Putida SJTE-1 And The Preliminary Analysis Of Its Regulation Mechanism

Microbial degradation is one of the main means for the removal of environmental estrogens pollution.Many estrogen-degrading strains have been isolated,but its degradation gene and regulation mechanism is not very clear,limiting its remediation application.Estrogen-degrading bacteria can convert estradiol?Estrodial,E2?into estrone?Estrone,E1?before their subsequent transformation.The oxidative conversion of estradiol to estrone is considered as limited step of the whole estrogen metabolism pathway,and the enzyme catalyzing this step is the key enzyme for estradiol degradation;the change on its expression level will directly affect the strains'degradation efficiency.Until now this key enzyme has not been identified yet and its function is not clear;the mechanism of transcriptional regulation to this enzyme is still unknown.This thesis tried to determine the key enzyme for estradiol transformation in Pseudomonas putida SJTE-1 and its potential regulation factors.Using gene cloning and in vitro expression,the potential estrogen degradation genes were purified and their enzymatic properties were analyzed to determine the key enzyme for the first transformation step of estradiol.On this basis,the putative transcription factors involved in the regulation of this enzyme were expressed and purified.Electrophoretic mobility shift assay?EMSA?and DNase I footprinting were used to screen the transcription factors with specific binding activities,determine their binding region properties and dissociation effectors.Twenty two potential degradation genes and six transcription factor encoding genes of P.putida SJTE-1 were selected as targets.After in vitro gene cloning,hetergous expression and affinity purification,sixteen recombinant enzymes and four transcription factor protein were obtained.Seven recombinant proteins encoded by SJTE-1₀062/1052/3864/4227/1172/1008/3029 genes can oxidize E2;and the product of gene SJTE-1₀062?named 3,17?-hsd?was with the highest catalytic activity,which encodes 3,17?-hydroxysteroid dehydrogenase?3,17?-HSD?.This enzyme uses NAD⁺as cofactor;its Km is 0.0802 mM and its Vmax is 0.9706 mM/s.Two valent metal ions can affect its enzyme activity with concentration dependence;Mg²⁺,Ca²⁺,Mn²⁺,Ni²⁺can improve its activity,while Cu²⁺,Fe²⁺,Zn²⁺have inhibitory effect.The optimum catalytic temperature of this enzyme is 42;but it is very sensitive to temperature.30-minute treatment at 42 can make it almost complete loss of its activity.Alkaline environment is conducive to the enzyme acitivity;pH 9.0 is its optimum pH,its activity can maintain for 6 h.Substrate spectrum analysis found that besides E2,the enzyme can also oxidize the side chain hydroxyl of testosterone?TES?and phenanthrene,but not estrone.HPLC detection showed that E1 was the main product of E2 oxidation,and the conversion efficiency of E2 was 96.5%in 5 minutes.This indicated that this enzyme can efficiently catalyze the oxidation of 17-carbon at the D ring of steroids and plays an important role of degradation of E2.On this basis,we screened the potential transcription factors involved in the regulation of 3,17?-HSD.In vitro expressed four transcriptional factor proteins was purified and used to detect their binding activities to the upstream region of3,17?-hsd gene.EMSA results showed that the AraC family regulator encoded by SJTE-1₀063 gene and the LysR family regulator encoded by SJTE-1₁173gene could bind this DNA fragement obviously,and binding effect can increase with the protein concentration up.Steroids can effeciently dissociate this binding product,and the dissociation of testosterone was the strongest in the same concentrations.E2 can dissolve the binding at low protein/DNA molar ratio,and E1 can dissociate the binding under the high molar ratio condition.Further DNase I footprinting experiments confirmed that AraC and LysR proteins both have two binding regions in the upstream fragment of 3,17?-hsd gene;one is near its start codon and another one is in the upstream of about 100 bp.These results showed that transcription factor AraC and LysR may participate in the direct regulation of 3,17?-HSD.This study will promote the molecular mechanism study of estrogen degradation and transcriptional regulation in Pseudomonas putida SJTE-1,and support its further genetic engineering and efficient application.