美国加州联合生物能源研究所Patrick M. Shih研究团队取得一项新突破。他们的最新研究利用二值载体拷贝数工程改进了农杆菌介导的转化（AMT）。2024年11月4日，国际知名学术期刊《自然—生物技术》发表了这一成果。

研究人员表示，质粒的拷贝数与其功能有关，但很少有人尝试在不同宿主的不同复制起源中，优化高拷贝数突变体的主题。

研究人员采用高通量生长偶联选择试验和定向进化方法，来快速识别影响拷贝数的复制突变的起源，并筛选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到用于AMT的质粒骨架内的二元载体中，研究人员观察到Nicotiana benthamiana在不同测试来源（pVS1、RK2、pSa和BBR1）中的瞬时转化得到了改善。

对于表现最好的来源pVS1，小组分离出更高拷贝数的变异，在拟南芥中提高了60-100%的稳定转化效率，在产油酵母Rhodosporidium toruloides中提高了390%。

他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异，除了能提高AMT效率外，还将使原核遗传工程更加精确。

附：英文原文

Title: Binary vector copy number engineering improves Agrobacterium-mediated transformation

Author: 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.

Issue&Volume: 2024-11-04

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.

DOI: 10.1038/s41587-024-02462-2

Source: https://www.nature.com/articles/s41587-024-02462-2