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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 <i>E. coli</i> and <i>S. marcescens</i> based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of <i>E. coli</i>, <i>Bacillus subtilis</i>, and <i>Corynebacterium glutamicum</i> were randomly interrupted and inserted into the upstream of reporter gene <i>gfp</i> to construct three promoter libraries, and a potential strong constitutive promoter (P_BS) suitable for <i>E. coli</i> 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 <i>S. marcescens</i> 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.