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有关，但很少有人尝试优化高拷贝数突变体以用于不同宿主的不同复制起点。我们使用高通量生长偶联选择测定法和定向进化方法来快速识别影响拷贝数的复制突变的起点，并筛选提高农杆菌介导的转化 （AMT） 效率的突变体。通过将这些突变引入用于 AMT 的质粒骨架内的二元载体中，我们观察到本氏烟草在四种不同测试来源 （pVS1、RK2、pSa 和 BBR1） 中的瞬时转化得到改善。对于性能最佳的来源 pVS1，我们分离了更高拷贝数的变体，这些变体在拟南芥中将稳定转化效率提高了 60-100%，在油质酵母 Rhodosporidium toruloides 中提高了 390%。我们的工作提供了一个易于部署的框架来生成质粒拷贝数变体，除了提高 AMT 效率外，还将提高原核基因工程的精度。