基于核酶分子开关激活GFP报告基因的色氨酸高产关键酶突变文库的高通量筛选方法的研究

项目名称： 基于核酶分子开关激活GFP报告基因的色氨酸高产关键酶突变文库的高通量筛选方法的研究

项目编号： No.31300096

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 孙艳

作者单位： 中国科学院苏州纳米技术与纳米仿生研究所

项目金额： 24万元

中文摘要： 目前定向进化手段常用于分子改造代谢中的关键酶，构建具有代谢物高生产能力的工程菌。但是细胞内代谢物产量的鉴定一般只能通过培养后发酵检测，导致酶改造过程中不能直观筛选获得提高代谢物产量的酶的正向突变子。针对"如何高通量筛选单细胞内代谢物的关键酶突变子"这一核心问题，本研究拟构建一种基于核酶分子开关的细胞内非标记检测色氨酸的GFP基因编码的荧光传感器。将色氨酸响应的核酶分子开关与GFP相融合，在细胞中准确地响应色氨酸浓度变化来调控GFP表达,实现单细胞内色氨酸浓度的荧光可视化。运用荧光激活细胞分选技术，发展全新的高通量筛选色氨酸生产关键酶突变文库的方法。本研究所探讨出细胞内色氨酸响应的核酶分子开关可视化荧光生物传感器成功构建的一般规律和内在机理具有普适性，可用于其它细胞内代谢物的研究。将为高通量筛选代谢物产量提高的关键酶正向突变子奠定坚实的基础和提供新的思路，具有重要的科学意义和研究价值。

中文关键词： 核酶分子开关；高通量筛选；L-色氨酸；GFP荧光传感器；合成生物学

英文摘要： Recent advances in metabolic engineering have involved the construction of multi-step enzymatic pathways to optimize pathway productivity and yield. Typically, each of these optimization steps involves the construction of many pathway variants followed by the identification of the best resulting pathway. Directed evolution techniques have been used to improve the activity of a wide range of enzymes to obtaining metabolite-overproducing microbes. Therefore, optimization requires the ability to measure the production of the desired metabolite at high throughput using an appropriate screen or selection. When measuring the productivity of a small molecule, an ideal screening system would allow high-throughput analysis, enabling the characterization of large libraries of variants and discriminate between structurally similar compounds。While precise analytical techniques, such as liquid or gas chromatography coupled to mass spectrometry, are generally available to measure any desired small molecule, their slow speed limits the throughput of any resulting screen.Advances in synthetic biology have led to the design of modular, programmable, RNA-based control elements, or ribozyme molecular switches. Ribozyme molecular switches generally link an RNA aptamer to an RNA gene-regulatory component, resulting in a control elem

英文关键词： Ribozyme molecular switch；high-throughput screen；L-tryptophan；GFP fluorescent sensor；synthetic biology