Abstract
Promoters serve as the switch of gene transcription, playing an important role in regulating gene expression and metabolites production. However, the approach to screening strong constitutive promoters in microorganisms is still limited. In this study, a novel method was designed to identify strong constitutive promoters in E. coli and S. marcescens based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of E. coli, Bacillus subtilis, and Corynebacterium glutamicum were randomly interrupted and inserted into the upstream of reporter gene gfp to construct three promoter libraries, and a potential strong constitutive promoter (P(BS)) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (P(BS76)) of the screened promoter was identified by sequence truncation. Third, a promoter library of P(BS76) was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter P(BS76-100) was 59.56 times higher than that of the promoter P(BBa_J23118). Subsequently, promoters P(BBa)_(J23118), P(BS76), P(BS76-50), P(BS76-75), P(BS76-85), and P(BS76-100) with different strengths were applied to enhance the metabolic flux of L-valine synthesis, and the L-valine yield was significantly improved. Finally, a strong constitutive promoter suitable for S. marcescens was screened by a similar method and applied to enhance prodigiosin production by 34.81%. Taken together, the construction of a promoter library based on random genomic interruption was effective to screen the strong constitutive promoters for fine-tuning gene expression and reprogramming metabolic flux in various microorganisms.