改造调控蛋白LacI建立二糖分子高通量筛选方法

The regulatory proteins which are regulated by small molecules can not only be used to regulate the expression of target genes, but also be used as reporters of their small-molecule inducers. Such reporters can then be used as high-throughput screening tools for the small-molecule inducers. Engineering the regulatory proteins' induction specificities will produce different regulatory protein mutants which can be induced by different kinds of small molecules, resulting in customized reporters for these different kinds of small molecules. The customized reporters can then be used as high-throughput screening tools for these small molecules. In this project the regulatory protein LacI from E. coli will be engineered to be induced by gentiobiose and lactulose,respectively. The resulted LacI mutants can thus be used as reporters of gentiobiose and lactulose, respectively. As an example, the reporter of lactulose will be used as a high-throughput screening tool of lactulose to engineer the β-glycosidase CelB from Pyrococcus furiosus for higher efficiency of lactulose biosynthesis by directed evolution strategy, which will prove the functionality of the reporter developed by regulatory protein engineering. The reporters of gentiobiose and lactulose will find applications in the studies of biosynthesis of these two disaccharides. Taking gentibiose and lactulose as examples, a general method for development of reporters of different kinds of disaccharides by engineering the regulatory protein LacI will be developed in this study, which will provide high-throughput screening tools for different kinds of disaccharides.In addition, the relationship between the structure and function of LacI will be investigated by the analysis of the mutations in the LacI mutants obtained in this study.

受小分子化合物诱导的调控蛋白不仅可用于调控目标基因的表达，还可作为其小分子诱导物的信号分子使用，实现对该小分子诱导物的高通量筛选。改造调控蛋白可改变其原有的诱导特异性，获得分别对各种不同小分子化合物产生诱导效应的调控蛋白。用该方法可为不同小分子化合物定制信号分子，实现它们的胞内高通量筛选。本项目将改造大肠杆菌调控蛋白LacI，使之分别对功能糖乳果糖或龙胆二糖产生诱导效应，以这两种二糖为例定制它们的信号分子。乳果糖信号分子作为其高通量筛选工具将被用于定向进化改造β-糖苷酶CelB提高乳果糖合成效率，以验证信号分子的功能。二糖的高通量筛选工具将在其生物合成研究中发挥重要作用。本项目拟建立改造LacI诱导特异性的方法，使之可推广应用于定制不同二糖分子乃至结构类似的非糖分子的信号分子，以实现这类分子的高通量筛选。本项目亦将对所获得的不同LacI突变体进行分析，深入认识LacI蛋白结构与功能的关系。

本研究成功构建了调控蛋白LacI的饱和突变体文库和Plac-荧光蛋白信号系统，用流式细胞分选法（FACS）进行筛选，成功获得了乳果糖的信号分子，证明了改造调控蛋白LacI的诱导特异性是有效可行的。乳果糖信号分子被证明能够正确报告细胞中乳果糖的浓度变化，从而成功作为其高通量筛选工具应用于定向进化改造来自Caldicellulosiruptor saccharolyticus的纤维二糖异构酶(cellobiose 2-epimerase，C2E)，C2E催化从乳糖合成乳果糖。我们成功获得了表达量和比活力均提升的突变酶。通过本研究，我们建立了一套改造LacI诱导特异性的筛选方法，可推广应用于定制其它结构类似的二糖分子甚至结构类似的非糖分子的信号分子。另外，我们着力解决乳糖操纵子诱导表达系统渗漏表达高的缺点，利用LacI突变体文库筛选到了渗漏表达降低约95%的LacI突变体。该突变体被证明与启动子DNA序列的结合亲和力更强。该突变体在IPTG存在条件下，诱导强度与野生型LacI相似，因此该突变体的诱导倍数比同等条件下野生型LacI的诱导倍数高20倍，更加适用于基因表达水平的精细调控。