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The role of plasmid copy number and mutation rate in evolutionary outcomes

Nature Ecology & Evolution volume 9, pages 1694–1704 (2025)Cite this article

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Abstract

Multicopy plasmids are widespread in nature and compose a common strategy for spreading beneficial traits across microbes. However, the role of plasmids in supporting the evolution of encoded genes remains underexplored due to challenges in experimentally manipulating key parameters such as plasmid copy number and mutation rate. Here we developed a strategy for controlling copy number in the plasmid-based Saccharomyces cerevisiae continuous evolution system, OrthoRep, and used our resulting capabilities to investigate the evolution of a conditionally essential gene under varying copy number and mutation rate conditions. Our results show that low copy number facilitated the faster enrichment of beneficial alleles whereas high copy number promoted robustness through the maintenance of allelic diversity. High copy number also slowed the removal of deleterious mutations and increased the fraction of non-functional alleles that could hitchhike during evolution. This study highlights the nuanced relationships between plasmid copy number, mutation rate and evolutionary outcomes, providing insights into the adaptive dynamics of genes encoded on multicopy plasmids and nominating OrthoRep as a versatile tool for studying plasmid evolution.

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Fig. 1: p1 CN achieved by three OrthoRep DNAPs expressed from eight promoters.
Fig. 2: Influence of CN on expression and growth rate.
Fig. 3: AmdSYM evolution experiment and CN stability.
Fig. 4: Assessment of amdSYM variants produced by evolution under varying CN and error rate.
Fig. 5: Comparison of relative activity of active allele libraries in acetamide versus butyramide.

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Data availability

Raw sequencing data can be found on the Sequence Read Archive under accession number PRJNA1236365. Comprehensive mutational data can be found in Supplementary Table 3.

Code availability

Custom code is described as pseudocode in the methods and Python scripts can be found via Zenodo at https://doi.org/10.5281/zenodo.15604699 (ref. 41) and MAPLE nanopore analysis via GitHub at https://github.com/gordonrix/maple.

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Acknowledgements

This work was funded by NIH R35GM136297 to C.C.L. A.‘O.’P. is supported by a Paul and Daisy Soros Fellowship for New Americans.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA

    Alexander ‘Olek’ Pisera & Chang C. Liu

  2. Center for Synthetic Biology, University of California, Irvine, Irvine, CA, USA

    Alexander ‘Olek’ Pisera

  3. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    Chang C. Liu

  4. Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA

    Chang C. Liu

Authors
  1. Alexander ‘Olek’ Pisera
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  2. Chang C. Liu
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Contributions

A.‘O.’P. and C.C.L. conceived the project ideas. A.‘O.’P. designed and carried out all experiments, collected and processed all data and analysed all data with input from C.C.L. A.‘O.’P. and C.C.L. wrote the paper.

Corresponding author

Correspondence to Chang C. Liu.

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Competing interests

C.C.L. is a co-founder of K2 Therapeutics, which uses OrthoRep in antibody engineering and evolution. The other authors declare no competing interests.

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Nature Ecology & Evolution thanks Fabienne Benz, Alvaro San Millan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8.

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Supplementary Tables

Supplementary Tables 1–5.

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Pisera, A.‘., Liu, C.C. The role of plasmid copy number and mutation rate in evolutionary outcomes. Nat Ecol Evol 9, 1694–1704 (2025). https://doi.org/10.1038/s41559-025-02792-7

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