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Simple Summary: The synthesis of natural or non-natural products in microorganisms frequently involves multiple genes or pathways. Enhancing and balancing the expression of these genes is essential to prevent growth inhibition and product synthesis blockage resulting from the accumulation of intermediate products, thereby effectively increasing the yield of target products. However, there is a deficiency in robust tools for balancing gene expression in Bacillus subtilis. Herein, we identified a 5′ UTR sequence that enhances gene expression levels. The library constructed from this sequence enables a broad spectrum of gene variations. Utilizing this library to construct a synthesized rib operon markedly boosts riboflavin production. Therefore, a novel 5′ UTR sequence was screened and characterized, supplementing the synthetic biology toolkit for B. subtilis. The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5′ UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5′ UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5′ UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5′ UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5′ UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5′ UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products. [ABSTRACT FROM AUTHOR]

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. 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 (PBS) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (PBS76) of the screened promoter was identified by sequence truncation. Third, a promoter library of PBS76 was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter PBS76-100 was 59.56 times higher than that of the promoter PBBa_J23118. Subsequently, promoters PBBa_J23118, PBS76, PBS76-50, PBS76-75, PBS76-85, and PBS76-100 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. [ABSTRACT FROM AUTHOR]