Screening and characterization of cysteine responsive promoters from Escherichia coli

Abstract:

[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 P_E2 with specific response to changes in cysteine concentration was singled out. Subsequently, random mutation of AAAT was carried out in the P_E2 promoter -35 spacer region and we found that mutant promoter P_E2-33 had a higher specific response to the cysteine concentration range of 1-7 g/L. [Conclusion] The obtained promoter P_E2-33 in 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.

Key words:Escherichia coli;cysteine;promoter;response