Reconfiguring antisense RNA-mediated replication enables modular and autonomous plasmid copy number control

Dynamic plasmid copy number (PCN) control is a powerful tool for precise gene dosage regulation and synthetic biosystem optimization. However, its implementation in non-model bacteria such as Corynebacterium glutamicum remains challenging due to fundamental constraints in the regulation mechanisms of rolling circle replication plasmids. Here, we develop the first PCN control platform for rolling circle replication plasmids in C. glutamicum by engineering resorcinol- and vanillic acid-inducible artificial antisense RNAs (asRNAs) that overcome the inherent repA-asRNA structural coupling. This mechanism-driven strategy enables precise, dose-dependent PCN reduction (1–150 copies), validated via single-cell tracking. The implementation across synthetic gene circuits and metabolic engineering yielded substantial performance enhancements: biosensor's dynamic ranges were expanded by 25.9-fold, and production of 5-aminolevulinic acid and lycopene was markedly improved through precise, orthogonal and modular pathway fluxes balancing. Furthermore, the vanillic acid-responsive dynamic PCN system autonomously boosted protocatechuic acid production from lignin-derived substrates by 3.5-fold. This study establishes a universal framework for dynamic PCN control of rolling-circle replication plasmids, thereby unlocking precision gene dosage regulation in Gram-positive industrial chassis.