红球菌P14降解雌二醇途径关键酶17β-羟基类固醇脱氢酶的研究

As an active environmental endocrine disrupting chemical (EDC), steroid hormones are well known to be difficult for degrading and harmful for human health. The pollution of steroid hormones has drawn widespread public attentions. Microbial bioremediation is considered to be a major and efficient strategy to remove steroids in different environments. In the our previous studies, we successfully isolated a marine bacterium strain Rhodococcus sp. P14, which showed a strong ability of estrogen degradation And it's biodegrading pathway and the whole genome were detected. We found that strain P14 could synthetize 17β-hydroxysteroid dehydrogenase (17β-HSD) which catalyzed estradiol into estrone. It was the first 17β-HSD from bacteria being able to catalyze estradiol oxidization. In the upstream of 17β-HSD gene cluster, two TetR family regulatory proteins (TetR) were detected. So we speculated that the expression of this gene might be regulated by the TetR. By the use of bioinformatics, biochemistry and molecular biology technique, this project intends to investigate the substrate binding site and the catalytic reaction mechanism of the functional enzyme 17β-HSD. The relationship between the enzyme structure and function will also be studied. Furthermore, the mechanisms of regulatory proteins (TetR) and sRNA for regulating 17β-HSD gene expression and translation will also be investigated. The results will reveal the molecular mechanism degrading estradiol by the bacterium strain P14 and will accelerate the application of hydroxysteroid dehydrogenase in the conrtol of steroid hormones pollution.

环境内分泌干扰物中，类固醇激素干扰活性强、难以降解可长期存在于环境，对人类健康造成极大危害，因而受到广泛关注；利用微生物修复是去除环境类固醇激素的非常有效方法。申请者前期获得了具强降解雌激素能力的海洋红球菌P14，并研究了其降解途径及基因组信息，发现菌株P14中具有一个能催化雌二醇的羟基类固醇脱氢酶17β-HSD，且是细菌中第一个报道的、能催化雌二醇形成雌酮的羟基类固醇脱氢酶；其基因上游存在两个TetR家族调控蛋白TetR，推测该基因的表达可能受TetR的调控。本项目拟利用生物信息学、生化与分子生物学等技术，研究17β-HSD底物范围、底物结合位点、催化反应机制、酶结构与功能的关系，探索调控蛋白对17β-HSD基因转录水平和sRNA对17β-HSD基因转录后水平的调控机制。预期结果为揭示红球菌P14降解雌二醇的分子机制、促进17β-羟基类固醇脱氢酶在环境甾体类污染物降解中应用奠定基础。

目前，随着环境中雌激素污染的日益加剧，对微生物降解雌激素研究已成为国际上环境领域的热点内容。本项目以具有高效雌激素降解能力的海洋红球菌P14为研究对象，对雌二醇降解关键酶17β-羟基类固醇脱氢酶（17β-HSD）的功能与其表达调控机制进行研究。通过在大肠杆菌中异源表达三个17β-HSD同工酶基因（包括17β-HSD、17β-HSDx和17β-HSDs），发现17β-HSD与17β-HSDs都具有高效转化雌二醇生成雌酮的能力。通过定点突变手段，发现Lev148、Ser142、Tyr150、Gly249、Tyr155是17β-HSD催化活性功能域关键氨基酸。同工酶17β-HSDx则具有催化多种类固醇激素的能力，其可分别催化雌二醇、睾丸酮和雌三醇生成雌酮、雄烯二酮和16-羟基雌酮。这些结果表明17β-HSD在海洋红球菌P14降解雌激素中具有重要功能。最后通过转录组学分析，发现多种调控蛋白具有影响17β-HSD表达的潜能；并通过系列分子生物学手段，证实TetR家族调控蛋白具有与17β-HSD启动子结合提高其表达的能力，在雌激素降解调控中具有重要作用。本项目阐明了海洋红球菌P14中雌激素降解关键酶17β-HSD功能与其表达的调控机制，为海洋红球菌在雌激素污染生物修复中的应用提供理论依据。