Lux通路蛋白调控Pseudoalteromonas sp. CSN423胞外金属蛋白酶E423表达的分子机制

Proteases from marine bacteria possess unparalleled advantages in catalytic properties and functions compared with that of terrestrial proteases. However, the industrial development of marine proteases was severely restricted due to their low yields and lack of systematic studies on their expression regulatory. Pseudoalteromonas sp. CSN423 is a protease-secreting bacterium isolated from the intertidal zones of Bohai Sea. UV-irradiation strategy was performed to obtain mutant strain with 5.1-fold higher protease E423 yield. Comparative genomics and proteomics of wild and mutant strain revealed that the increasing expression of E423 was relevant to Lux pathway factors. Then the strains with regulatory protein deletion mutation and complementation were constructed respectively to prove the regulatory effect and interaction during high-yield process of E423. The key sites and regulatory functions of regulatory proteins will be studied through heterologous expression, site directed mutagenesis, electrophoretic mobility shift assay(EMSA) and isothermal titration calorimetry (ITC), which will reveal the molecular regulatory mechanism of the expression of E423. Finally, whether this regulatory approach towards proteases expression is universal or not will be analyzed among genus Pseudoalteromonas. Our results in this project will provide important basis for the construction of engineering strains and application of marine sourced proteases.

海洋蛋白酶较陆地蛋白酶在催化特性和功能上具有柔性强，最适酶活温度低，耐盐性高，可耐受多种去污剂等优越性，成为工业酶开发利用领域的热点。但由于其产量较低，缺乏对蛋白表达调控的系统研究，造成干预手段缺乏且有很大的盲目性，严重制约了产业化开发。我们自渤海潮间带分离到一株高产蛋白酶菌株Pseudoalteromonas. CSN423，通过紫外诱变将其胞外金属蛋白酶E423产量提高了5.1倍。通过对比野生株及突变株基因组和蛋白组，推测E423表达量升高与Lux通路蛋白有关。本课题将构建野生株Lux通路蛋白的缺失突变株和回补株，验证其在E423高产过程中的调控作用，然后对调控蛋白进行异源表达、定点突变，结合凝胶阻滞和ITC分析，阐明调控蛋白的关键位点和调控方式，揭示Lux通路蛋白对E423表达调控的分子机制，最后探讨该类金属蛋白酶的调控方式在假交替单胞菌属中的普适性，为海洋蛋白酶的开发应用奠定基础。

海洋蛋白酶较陆地蛋白酶在催化特性和功能上具有无可比拟的优越性，成为工业酶开发利用领域的热点。但由于其产量较低，缺乏对蛋白表达调控的系统研究，造成干预手段缺乏且有很大的盲目性，严重制约了产业化开发。我们自渤海潮间带分离到一株高产蛋白酶菌株Ps. sp. CSN423，通过紫外诱变将其胞外金属蛋白酶E423产量提高了5.1倍。本项目探讨了调控Ps. sp. CSN423胞外金属蛋白酶E423表达的分子机制。首先，从不同组学层面对野生型菌株Ps. sp. CSN423和突变型菌株Ps. sp. CSN423-M进行了比较，筛选出导致金属蛋白酶E423酶量上升的主要调控因子；对比了野生型菌株和突变型菌株生长性状，发现群体感应信号分子AHLs可以影响E423的表达，证实E423的表达与群体感应相关。同时还发现菌株的生长状态还与ArcA/ArcB双组分系统有关，突变株Ps. sp. CSN423-M中arcB发生移码突变，当处于低氧、寡营养环境时，突变株生存能力严重受损。其次，利用同源重组技术构建了luxR3、luxO及arcB基因缺失株，利用pEV质粒构建了相应的基因回补株，证实E423的表达与Lux通路蛋白及ArcA/ArcB双组分系统有关。LuxR3作为重要的群体感应调控元件，对蛋白酶E423的表达有正调控作用，而luxO、arcB基因缺失会上调E423的表达，对E423的表达有负调控作用；构建基因敲除菌株Δhfq、ΔrpoN、ΔrpoS，发现Hfq通过增加luxR3 mRNA的稳定性从而上调luxR3的表达，LuxO及RpoN（σ54）的缺失，也会上调LuxR3的表达；凝胶阻滞分析结果显示，LuxR3可以通过与E423调控区结合从而上调E423的表达，而ArcA通过与LuxR3竞争结合E423的调控区影响E423的表达。在此基础上，我们进一步探索影响金属蛋白酶E423分泌的因素，发现金属蛋白酶E423的C末端PPC结构域可以促进E423的分泌，为后续构建新型高产蛋白酶海洋菌株提供了一定的理论依据。此外，我们利用金属蛋白酶E423酶解低值蛋白资源牡丹籽粕蛋白，获取了一系列具有较高抗氧化活性的功能肽，在金属蛋白酶E423的应用方面进行了初步探索。

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