基于蔗糖效应的RcsF激活蛋白调控荚膜异多糖酸合成机制研究

RcsF, as the signal sensing protein of the Rcs phosphorylation system, is the key regulatory factor of colanic acid (CA) biosynthesis and other environmental factors to respond. In our previous study, Kosakonia cowanii LT-1 has been screened and found to be able to produce CA with sucrose sensing. However, the specific mechanism of sucrose activating the RcsF is not clear. In order to analyse this mechanism, this project will start with the phenomenon of sucrose sensing, and the methods of fusion expression, co-immunoprecipitation, and protein mass spectrometry will be used to screen the activator proteins interacting with the RcsF. Further, the activator proteins will be verified by transcriptomic analysis and real-time polymerase chain reaction at the transcriptional level. On this basis, the activator proteins will be further determined in vivo through gene knock-out and gene retro-complementation. Then, the interaction way and the key interaction sites between the activator protein and RcsF will be determined by using isothermal titration calorimetry, surface plasmon resonance, molecular dynamics simulation, domain deletion, and site-specific mutagenesis. Finally, the specific mechanism of the activator protein of RcsF regulating the synthesis of CA based on sucrose sensing will be clarified. The results are expected to provide theoretical foundation for developing the strategy of high CA production and studying the regulation mechanism of microbial polysaccharides synthesized by similar microorganism. Hence, this work has an important academic significance and practical value.

RcsF作为Rcs级联磷酸化信号传导系统的信号感应蛋白，是启动荚膜异多糖酸合成及响应其它环境因素的关键调控因子。申请人在研究中获得一株具有独特蔗糖效应的荚膜异多糖酸高产菌株Kosakonia cowanii LT-1。目前，蔗糖效应如何激活RcsF的机制尚不明晰。为解析这一机制，本项目以蔗糖效应为基础，综合融合表达、免疫共沉淀及蛋白二级质谱技术挖掘RcsF激活蛋白，并利用转录组学和RT-PCR技术在转录水平层面确证激活蛋白。在此基础上，通过基因敲除与回补对激活蛋白进行体内验证，并通过等温滴定量热法、表面等离子体谐振、动力学模拟、结构域删除及定点突变等方法确定激活蛋白与RcsF的关键作用位点及互作方式，最终阐明基于蔗糖效应的RcsF激活蛋白调控荚膜异多糖酸生物合成的机制。研究结果有望为进一步开发荚膜异多糖酸高产策略及同类微生物多糖合成调控机制解析提供理论基础，具有重要的学术意义和实用价值。

荚膜异多糖酸是一种具有延长寿命和抗肿瘤功能的天然微生物多糖，但目前对Kosakonia cowanii LT-1依赖蔗糖合成荚膜异多糖酸的调控机制认知不清晰，阻碍了研究者利用代谢工程手段构建荚膜异多糖酸高产突变株的相关研究。在本项目中，申请人以Rcs级联磷酸化系统信号感应蛋白RcsF激活蛋白的挖掘和RcsF激活蛋白与RcsF互作方式为中心任务，以考氏科萨克氏菌K. cowanii LT-1为研究对象，通过基因组挖掘与比较、转录组学分析、基因敲除与回补、异源表达、分子对接与动力学模拟等技术手段解析了K. cowanii LT-1中基于蔗糖效应的RcsF激活蛋白调控荚膜异多糖酸合成机制，尤其是RcsF激活蛋白与信号感应蛋白RcsF的相互作用方式。具体地，对K. cowanii LT-1进行基因组测序，并基于基因组数据构建了菌株中荚膜异多糖酸的合成网络、分析了其合成基因簇；对Rcs级联磷酸化系统信号感应蛋白RcsF的激活蛋白进行挖掘，发现RcsF激活蛋白是一种位于周质空间的非依赖ATP的蛋白重折叠伴侣Spy蛋白，该蛋白编码基因的缺失可严重削弱K. cowanii LT-1合成荚膜异多糖酸的能力。对Spy蛋白与信号感应蛋白RcsF的互作方式进行分析，发现Spy与RcsF之间主要由氢键和盐桥作用，结合自由能△G为-37.8 kcal/mol。总体地，申请人在项目的资助下完成了基于蔗糖效应的RcsF激活蛋白调控荚膜异多糖酸合成机制解析工作，促进了人们对K. cowanii LT-1依赖蔗糖合成荚膜异多糖酸的理解，也为代谢工程改造获得荚膜异多糖酸高产菌株奠定了理论基础。