谷氨酸棒杆菌内源表达元件的筛选

Selection of endogenous expression elements from Corynebactrium glutamicum

【目的】构建谷氨酸棒杆菌表达元件探测载体,筛选能够启动蛋白表达的序列片段。【方法】基于谷氨酸棒杆菌表达载体p XMJ19,利用Golden Gate新型克隆方法构建表达元件插入位点,使筛选的片段能够与报告基因快速无缝衔接,同时避免残留额外的序列对表达元件效果测试产生可能存在的干扰。对本课题组前期的谷氨酸棒杆菌BZH001高、中、低溶氧条件下的发酵样品转录组数据进行分析,筛选出稳定于高转录水平的6个基因,通过软件预测每个基因的启动子区域和5′UTR区域,两者构成能够启动基因表达的功能性元件,并将其从基因组中克隆出来。以增强型绿色荧光蛋白基因egfp作为报告基因,快速测量出表达元件的效果。【结果】获得5个不同效果的内源性表达元件,最好的元件插入探测载体后在谷氨酸棒杆菌中表达的荧光强度大于3 500 RFU/OD600。【结论】通过结合转录组数据,探测载体能够快速有效筛选表达元件,为将来人们对谷氨酸棒杆菌基因工程改造和生物系统的构建提供更多基础材料。

[Objective] We constructed a Corynebacterium glutamicum expression-element probe vector and selected sequences that can activate expression of protein. [Methods] Based on the classic plasmid pXMJ19, we set the inserted position with the new method of Golden Gate cloning strategy that can connect material sequence to the report gene seamlessly, avoiding remaining unwanted sequence that may interfere the measurement of expression elements effect. By analyzing thetranscriptomic data of C. glutamicum BZH001 cultivated under different dissolved oxygen level in our previous study, we screened six genes stabilizing in high transcriptional level, analyzed their promoter regions and 5＇UTR regions by the promoter software prediction, and then amplified the promoter sequences associated with 5＇UTR regions of the six genes. The activities of these expression elements were measured using the reporter gene egfp in the probe vector. [Results] Five expression elements with different performances were achieved, and the best one could reach over 3 500 RFU/OD600 of fluorescent intensity. [Conclusion] Associated with the transcriptomic data, we can screen effective expression elements using this probe vector, which can provide more genetic parts for the future genetic engineering and construction of biological system in Corynebacterium glutamicum.