2024-11-07 アメリカ合衆国・ローレンスバークレー国立研究所(LBNL)
<関連情報>
- https://newscenter.lbl.gov/2024/11/07/new-plant-engineering-method-promises-to-dramatically-improve-biofuel-and-bioproduct-development/
- https://www.nature.com/articles/s41587-024-02462-2
バイナリーベクターコピー数工学がアグロバクテリウム媒介形質転換を改善する 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
Nature Biotechnology Published:04 November 2024
DOI:https://doi.org/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.
