基于核糖开关的维生素B12生产菌的高通量筛选方法的研究

Vitamin B12 is the only vitamin compounds that containing metal elements, and has been widely used for the treatment of pernicious anemia and peripheral neuritis. There are bottlenecks in genetic operation of industrial strains as they often had been treated by several rounds of mutation, and the production is difficult to increase by the traditional methods. In our previous study, we obtained a vitamin B12-on riboswitch by screening several different B12 riboswitches based on the vitamin B12 industrial hyper-producing strain. In this project, we will try to improving the sensitivity and dynamic range of the riboswitch-based sensor by random saturation mutation of its aptamer domain, and the secondary structure of the optimal riboswitch will be analyzed. Moreover, to minimize the false positive rate, we substituted GFP with the tetracycline/H+ antiporter (encoded by tetA) as a selection marker for high-throughput screening. We will develop a B12-on riboswitch-based high throughput screening (HTS) method by combines atmospheric and roomtemperature plasma and vitamin B12 high-throughput assay method. And the HTS method will be used to screen hyper-producing strains from a random mutagenesis library.

维生素 B12是唯一含金属元素的维生素类化合物，主要用来治疗恶性贫血和末梢神经炎。工业生产菌多经过多轮诱变筛选，其遗传操作困难，用传统方法提高产量空间有限。本课题组在前期的研究中从维生素B12工业高产菌出发，通过对不同的维生素B12 riboswitch进行筛选，获得了与维生素B12浓度呈正相关关系的riboswitch。本项目中，我们将通过对riboswitch适体区进行随机饱和突变，来提高riboswitch元件的灵敏度和响应范围，并对效果好的riboswitch进行结构分析。进一步，我们用四环素/H+逆向转运蛋白替换GFP来优化筛选元件进而降低假阳性率。然后结合常压室温等离子体（ARTP）诱变系统及维生素B12高通量检测方法，建立维生素B12生产菌株的高通量筛选方法，并进行多轮筛选获得高产菌株。

维生素 B12是唯一含金属元素的维生素类化合物，主要用来治疗恶性贫血和末梢神经炎。工业生产菌多经过多轮诱变筛选，其遗传操作困难，用传统方法提高产量空间有限。本项目中，我们通过对与维生素B12浓度呈正相关关系的riboswitch进行随机饱和突变，提高了riboswitch元件的灵敏度和响应范围，并对效果好的riboswitch进行了二级结构分析。进一步，我们用四环素/H+逆向转运蛋白替换GFP来优化了筛选元件进而降低了假阳性率。随后，我们确定了诱变时间和高通量检测方法，结合常压室温等离子体（ARTP）诱变系统，建立了维生素B12生产菌株的高通量筛选方法，进行多轮筛选后获得了高产菌株。

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