Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis.

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]