A hybrid system enables plasmid copy number control in yeast

Synthetic biology requires plasmid systems that offer not only stable, fixed copy numbers but also tunable copy numbers to enable multi-level regulation of gene expression. While dynamic plasmid copy number (PCN) control has been engineered in prokaryotes such as E. coli , a parallel capability has been lacking for eukaryotic systems. Here, we bridge this gap by developing a programmable PCN platform for S. cerevisiae based on its endogenous 2µ plasmid. First, we engineered a p2µ- Cir ⁰ system that exhibits a superior combination of high copy number (up to 20 per cell), enhanced population homogeneity, and improved segregation stability compared to conventional yeast episomal plasmids (YEps). This system supports protein expression levels up to 60-fold higher than a single chromosomal integrant. Introduction of a CEN element into p2µ enabled the construction of programmable YTp-C and YTp-I systems, which allow temporal PCN control with enhanced stability. These switchable vectors enable efficient PCN transition from 1 to 38 through time-dependent induction. Further incorporation of Leu2d-mediated metabolic selection in the YTp-CL and YTp-IL elevated the PCN to nearly 70 copies and boosted expression capacity to approximately 110-fold relative to chromosomal integration. We demonstrated the versatility of this platform through diverse applications, demonstrating that PCN elevation facilitated phenotyping of tRNA overexpression, enhancing the production of several compounds, including the therapeutic peptide GLP-1 precursor, 2-phenylethanol, β-carotenoid, and ergothioneine. These results establish the first quantitative and multi-dimensional PCN regulation toolkit for yeast, addressing the long-standing issue of instability arising from multiple copies and providing critical insights for synthetic biology that integrates gene dosage control across DNA, RNA, and protein levels.