A hybrid system enables plasmid copy number control in yeast.

Synthetic biology demands plasmid with tunable copy numbers. While dynamic plasmid copy number (PCN) regulation has been engineered in prokaryotes, a parallel capability remains unavailable in eukaryotic systems. Here, we develop a programmable PCN platform for S. cerevisiae based on the endogenous 2μ plasmid. The p2μ-Cir0 system achieves up to 20 copies per cell with enhanced homogeneity and stability, supporting 60-fold higher expression than chromosomal integration. Addition of a CEN element enables switchable PCN transitions from 1 to 38 copies, and Leu2d-based selection further raises copy number to ~70 and expression to ~110-fold. We demonstrate broad utility in tRNA phenotyping, GLP-1 precursor, 2-phenylethanol, β-carotenoid and ergothioneine production, and long-term strain preservation. These results establish a well quantified PCN regulation toolkit for yeast, addressing instability from multiple copies and enabling gene dosage control across DNA, RNA, and protein levels.