Szarzanowicz Matthew J, Waldburger Lucas M, Busche Michael, Geiselman Gina M, Kirkpatrick Liam D, Kehl Alexander J, Tahmin Claudine, Kuo Rita C, McCauley Joshua, Pannu Hamreet, Cui Ruoming, Liu Shuying, Hillson Nathan J, Brunkard Jacob O, Keasling Jay D, Gladden John M, Thompson Mitchell G, Shih Patrick M
Joint BioEnergy Institute, Emeryville, CA, USA.
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Nat Biotechnol. 2024 Nov 4. doi: 10.1038/s41587-024-02462-2.
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.
质粒的拷贝数与其功能相关，但很少有人尝试优化高拷贝数突变体，以便在不同宿主中跨多种复制起点使用。我们使用高通量生长偶联选择测定法和定向进化方法，快速鉴定影响拷贝数的复制起点突变，并筛选可提高农杆菌介导转化（AMT）效率的突变体。通过将这些突变引入用于AMT的质粒骨架中的二元载体，我们观察到在四个不同测试起点（pVS1、RK2、pSa和BBR1）中，本氏烟草的瞬时转化得到了改善。对于表现最佳的起点pVS1，我们分离出了高拷贝数变体，这些变体在拟南芥中使稳定转化效率提高了60%-100%，在产油酵母红冬孢酵母中提高了390%。我们的工作提供了一个易于部署的框架，用于生成质粒拷贝数变体，这除了提高AMT效率外，还将在原核生物基因工程中实现更高的精确性。
