Binary vector copy number engineering improves Agrobacterium-mediated transformation

Matthew J Szarzanowicz; Lucas M Waldburger; Michael Busche; Gina M Geiselman; Liam D Kirkpatrick; Alexander J Kehl; Claudine Tahmin; Rita C Kuo; Joshua McCauley; Hamreet Pannu; Ruoming Cui; Shuying Liu; Nathan J Hillson; Jacob O Brunkard; Jay D Keasling; John M Gladden; Mitchell G Thompson; Patrick M Shih

doi:10.1038/s41587-024-02462-2

Binary vector copy number engineering improves Agrobacterium-mediated transformation

Nat Biotechnol. 2024 Nov 4. doi: 10.1038/s41587-024-02462-2. Online ahead of print.

Authors

Matthew J Szarzanowicz^{1

2

3}, Lucas M Waldburger^{1

2

4

5}, Michael Busche⁶, Gina M Geiselman⁷, Liam D Kirkpatrick^{1

2

3}, Alexander J Kehl⁸, Claudine Tahmin³, Rita C Kuo^{1

5}, Joshua McCauley^{1

5}, Hamreet Pannu^{1

2}, Ruoming Cui^{1

2}, Shuying Liu^{1

2}, Nathan J Hillson^{1

5}, Jacob O Brunkard⁶, Jay D Keasling^{1

4

5

9

10

11}, John M Gladden^{1

7}, Mitchell G Thompson^{12

13}, Patrick M Shih^{14

15

16

17}

Affiliations

¹ Joint BioEnergy Institute, Emeryville, CA, USA.
² Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
³ Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA.
⁴ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
⁵ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁶ Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA.
⁷ Sandia National Laboratories, Livermore, CA, USA.
⁸ Department of Chemistry, University of California, Davis, Davis, CA, USA.
⁹ Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA.
¹⁰ QB3, University of California, Berkeley, Berkeley, CA, USA.
¹¹ Center for Biosustainability, Danish Technical University, Kongens Lyngby, Denmark.
¹² Joint BioEnergy Institute, Emeryville, CA, USA. mthompson@lbl.gov.
¹³ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. mthompson@lbl.gov.
¹⁴ Joint BioEnergy Institute, Emeryville, CA, USA. pmshih@berkeley.edu.
¹⁵ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. pmshih@berkeley.edu.
¹⁶ Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA. pmshih@berkeley.edu.
¹⁷ Innovative Genomics Institute, Berkeley, CA, USA. pmshih@berkeley.edu.

PMID: 39496930
DOI: 10.1038/s41587-024-02462-2

Abstract

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.

Grants and funding

DE-AC02-05CH11231/U.S. Department of Energy (DOE)