A Simple, Rapid Assembly Method for Integrating Different Gene Order into Synthetic Operons

Although operons are a fundamental feature of prokaryotic genomes, their organization is non-random. The specific influence of operon architecture on gene expression, however, remains poorly characterized. In this study, we systematically analyzed the effects of operon length and gene position on expression levels in Escherichia coli and Bacillus subtilis. We found that promoter-proximal (5′ end) genes were expressed at higher levels and that expression of a given gene could be enhanced by increasing the overall length of the operon. To leverage these principles for metabolic engineering, we developed a Head-to-Tail PCR (HTPCR) method for the rapid assembly of synthetic operons with permuted gene orders. Application of this method enabled the construction of a synthetic rib operon that increased riboflavin yield by 35.38%. Collectively, these findings provide a theoretical framework and a practical methodology for designing efficient synthetic operons to enhance the production of target compounds.