A hybrid system enables plasmid copy number control in yeast

Abstract

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. Synthetic biology demands plasmid with tunable copy numbers, however this capability remains unavailable in eukaryotes. Here the authors develop a dynamic plasmid copy number system for S. cerevisiae and show bioproduction applications.