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<p>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 <i>S. cerevisiae</i> based on the endogenous 2μ plasmid. The p2μ-<i>Cir</i><sup><i>0</i></sup> 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 <i>Leu2d</i>-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.</p>

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A hybrid system enables plasmid copy number control in yeast

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月28日

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  • Anni Li,
  • Qingyang Zhao,
  • Zhunyi Yang,
  • Jiesheng Cheng,
  • Dong Xu,
  • Yueyao Zhang,
  • Ting He,
  • Bingzhao Zhuo,
  • Xing Zhao,
  • Xiaolin Rao,
  • Hui Wang,
  • Lizhu Chen,
  • Zhouqing Luo

摘要

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.