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

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 element that regulates gene expression in response to binding of a ligand, such as a protein or small molecule. In order to use ribozyme switches as a platform for screening enzyme libraries, the switches must accurately and precisely discriminate between small differences in the concentrations of metabolites. In this study we will develop a generalizable in vivo screening strategy for product accumulation using engineered RNA switches as the key biosensor components. These novel biosensors link the concentration of a product metabolite to GFP expression levels in living cells. We use an RNA-based biosensor to quantitatively screen large enzyme libraries in high throughput based on fluorescence, either in clonal culture by flow cytometry or in single cells by fluorescence activated cell sorting (FACS). We demonstrate that the RNA-based biosensor has sufficient precision to distinguish small changes in fluorescence and therefore identify relatively small improvements in activity. Additionally, the biosensor can be coupled to FACS to allow screening of large enzyme libraries.As aptamer selection strategies allow ribozyme switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways. Using L-tryptophan producing Escherichia coli as a model system, we demonstrated that this RNA device could enrich pathway-optimized strains. This work highlights the power of novel single?cell screening technologies and their applications to genetics, evolution and the biotechnological uses of bacteria.

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。

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，本研究构建了基于核酶分子开关激活的响应细胞内色氨酸浓度变化的GFP基因编码的荧光传感器，利用该传感器高通量筛选色氨酸生产关键酶突变文库。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。