大肠杆菌胞内半胱氨酸响应型启动子的筛选表征

Screening and characterization of cysteine responsive promoters from Escherichia coli

【目的】半胱氨酸是一种重要的含硫氨基酸,广泛应用于化妆品、药品、食品等行业,微生物发酵法合成半胱氨酸已成为当前研究的热点。基于比较转录组学分析等技术,筛选并表征大肠杆菌(Escherichia coli)胞内对半胱氨酸浓度变化显著响应的启动子。【方法】在Escherichia coli W3110培养基中外源添加不同终浓度的半胱氨酸,通过比较转录组学分析筛选转录水平显著响应半胱氨酸浓度变化的基因,融合目标基因的启动子片段与荧光报告基因egfp构建启动子文库,进一步测定不同半胱氨酸浓度条件下,含有不同启动子重组菌的绿色荧光蛋白(green fluorescent protein,GFP)荧光强度。【结果】筛选并挖掘了随着半胱氨酸浓度提高而转录水平显著提升的27个基因,并将基因的潜在启动子片段与荧光报告基因egfp融合构建启动子文库,筛选获得对半胱氨酸变化具备特异性响应的启动子PE2。最后,对PE2启动子−35区间隔区域AAAT进行随机突变,最终获得在1−7 g/L半胱氨酸浓度范围内特异性响应性能显著提高的启动子PE2-33。【结论】本研究筛选表征的启动子PE2-33是半胱氨酸特异性响应型启动子,为构建半胱氨酸特异性生物传感器并用于半胱氨酸高产菌的高通量筛选奠定了基础。

[Objective] Cysteine is an important sulfur-containing amino acid. Biosynthesis of cysteine has recently attracted increasing attention owing to its widespread application in cosmetics, medicine, food and other industries. Development of an efficient biosensor is indispensable for the screening of increased cysteine producers among a mutation library. In this study, on the basis of comparative transcriptome analysis, we screened and characterized some promoters from Escherichia coli that show significant response to changes in the cysteine concentration. [Methods] E. coli W3110 was cultured in LB medium with different concentrations of cysteine, and genes which showed significant improvement at the transcription level were screened. Then, the promoter fragment was amplified and fused with the fluorescent reporter gene egfp to construct a promoter library. Furthermore, the fluorescence intensity of recombinant bacteria with different promoters under different cysteine concentration was determined with a multi-functional microplate analyzer. [Results] A total of 27 genes, whose transcription levels increased significantly with the rise of cysteine concentration, were screened out and identified. The promoter PE2with specific response to changes in cysteine concentration was singled out. Subsequently, random mutation of AAAT was carried out in the PE2promoter-35 spacer region and we found that mutant promoter PE2-33had a higher specific response to the cysteine concentration range of 1-7 g/L. [Conclusion]The obtained promoter PE2-33in this study is a cysteine-specific response promoter, which lays a foundation for the construction of cysteine-specific biosensor and the high-throughput screening of cysteine-producing bacteria.