Binary vector copy number engineering improves Agrobacterium-mediated transformation
The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.
Authors
- Jay D. Keasling (ORCID: https://orcid.org/0000-0003-4170-6088)
- Joshua McCauley (ORCID: https://orcid.org/0000-0002-3766-560X)
- Lucas Waldburger (ORCID: https://orcid.org/0000-0002-4735-5285)
- Jacob O. Brunkard (ORCID: https://orcid.org/0000-0001-6407-9393)
- Alexander J. Kehl (ORCID: https://orcid.org/0000-0002-0993-5828)
- Rita Kuo (ORCID: https://orcid.org/0000-0002-4571-626X)
- Liam D. Kirkpatrick (ORCID: https://orcid.org/0000-0002-2744-9102)
- John M. Gladden (ORCID: https://orcid.org/0000-0002-6985-2485)
- Nathan J. Hillson (ORCID: https://orcid.org/0000-0002-9169-3978)
- Claudine Tahmin
- Patrick M. Shih (ORCID: https://orcid.org/0000-0002-2119-3345)
- Matthew J. Szarzanowicz (ORCID: https://orcid.org/0009-0003-4398-5196)
- Michael Busche (ORCID: https://orcid.org/0000-0001-8845-3300)
- Gina M. Geiselman (ORCID: https://orcid.org/0000-0002-7910-0615)
- Shuying Liu (ORCID: https://orcid.org/0000-0002-8411-0332)
- Mitchell G. Thompson (ORCID: https://orcid.org/0000-0002-1490-8074)
- H. Pannu
- Ruoming Cui
Institutions
- University of Wisconsin–Madison (US)
- Lawrence Berkeley National Laboratory (US)
- Sandia National Laboratories California (US)
- Innovative Genomics Institute (US)
- Danish Academy of Technical Sciences (DK)
- Joint BioEnergy Institute (US)
- University of California, Davis (US)
- University of California, Berkeley (US)
- Technical University of Denmark (DK)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2024-11-04
- DOI
- https://doi.org/10.1038/s41587-024-02462-2
- Citations
- 32
- Primary Topic
- Plant tissue culture and regeneration
- Type
- article
- Field-Weighted Citation Impact
- 7.48
Funders
- National Science Foundation
- U.S. Department of Energy
- U.S. Department of Agriculture
- Life Sciences Research Foundation
- National Institutes of Health
- National Institute of Food and Agriculture
- Office of Science
- Biological and Environmental Research
- Lawrence Berkeley National Laboratory