A Novel Method to Screen Strong Constitutive Promoters in Escherichia coli and Serratia marcescens for Industrial Applications

SIMPLE SUMMARY: With the advancement of synthetic biology and metabolic engineering, regulatory elements applied for the accurate expression of target genes have become more important. Among them, due to their important role in regulating gene expression at the transcription level, a number of homologous or heterologous promoters have been used to improve the yield of target metabolites in different microorganisms. However, the method to isolate strong constitutive promoters in different microorganisms is still limited. Our work describes a novel approach to identify strong constitutive promoters in Escherichia coli and Serratia marcescens. The identified promoters were further used for fine-tuning gene expression and reprogramming the metabolic flux of L-valine and prodigiosin in E. coli and S. marcescens, respectively, and finally, the higher-level L-valine synthesis strain and prodigiosin production strain were isolated. The method shown in our study can also be a useful strategy to identify strong constitutive promoters in other bacteria and isolate other effective genetic regulatory elements, such as ribosome binding sites, terminators, and N-terminal coding sequences (NCS), for tuning gene expression in different microorganisms. 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.