Dual-promoter systems drive plasmid loss via non-translational burden in geraniol-producing E. coli.

Plasmid-driven metabolic burden in Escherichia coli poses a critical challenge for industrial terpenoid production, yet the molecular origins of transcriptional stress remain unclear. Here, we engineered geraniol-producing E. coli strains with isogenic plasmid DNAs differing only in promoter architecture (Trc vs. T7, single vs. dual promoter) to dissect transcriptional versus translational contributions to plasmid instability. Dual promoter-driven strains exhibited acute production failure (92.1-94.8% reduction in peak geraniol titers) compared to the single Trc promoter control, accompanied by acute growth inhibition (34.6-37.9% lower specific growth rates). Remarkably, > 53% of this metabolic burden persisted after RBS/MCS deletion (calculated as [growth inhibition in S528]/[inhibition in S493] × 100%), directly linking instability to transcription initiation rather than protein synthesis. Promoter strength inversely correlated with plasmid retention: dual-T7 constructs reduced plasmid stability to 45.0 ± 9.5% within 12 h post-induction, while Trc systems maintained 80.0 ± 4.8% retention. Partial host adaptation restored cell growth (47% recovery by 12 h), but geraniol titer remained severely suppressed (21.7% of control), revealing irreversible metabolic flux imbalances. These findings reveal that strong promoters induce plasmid instability primarily through transcription-initiated burden, distinct from translational costs. Our work provides a genetic uncoupling strategy to quantify this burden and guides promoter optimization for stable microbial terpenoid production.