Occhialini Alessandro, King Gabriella, Majdi Mohammad, Fuentes Quispe Ivette A, DeBruyn Jennifer M, Lenaghan Scott C
Department of Plant Sciences, University of Tennessee, 301 Agriculture & Natural Resources Building, 2431 Joe Johnson Drive, Knoxville, Tennessee 37996, United States.
Center for Agricultural Synthetic Biology (CASB), University of Tennessee, 2640 Morgan Circle Dr., Knoxville, Tennessee 37996, United States.
ACS Synth Biol. 2024 Dec 20;13(12):4245-4257. doi: 10.1021/acssynbio.4c00724. Epub 2024 Dec 10.
Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells. One of these novel technologies is the mini-synplastome, an episomal plasmid with a chloroplast-specific origin of replication () used to express transgenes in plastids. In order to improve episome sequence stability overtime by reducing the frequency of spurious recombination events, an optimized version of mini-synplastome (Gen3) was designed. The innovation in the Gen3 design was to substantially reduce the size of the plastomic sequence containing to include only domains involved in replication and to reduce the sequence homology of the whole episome with the endogenous plastome. In this work, we have demonstrated that Gen3 can be used to install a multigene pathway in (potato) chloroplasts, and the episome is stable in a full-length circular form at high copy number throughout all plant developmental stages to anthesis in plants with normal phenotypic parameters. It is anticipated that in the next decade the mini-synplastome will be a valuable tool for installing complex genetic circuits in plastids.
质体是植物基因工程中许多生物技术应用的理想目标，从用作生产有价值分子的生物反应器到安装用于改善植物性状的转基因。30多年来，质体转化的常规方法一直依赖于同源重组整合载体。然而，非整合型附加体质粒最近作为一种用于植物细胞质体基因工程的创新工具受到了更多关注。其中一项新技术是微型同型质体基因组，这是一种具有叶绿体特异性复制起点的附加体质粒，用于在质体中表达转基因。为了通过降低假重组事件的频率来提高附加体序列随时间的稳定性，设计了微型同型质体基因组的优化版本（Gen3）。Gen3设计的创新之处在于大幅减小了包含复制起点的质体基因组序列的大小，使其仅包含参与复制的结构域，并降低了整个附加体与内源性质体基因组的序列同源性。在这项工作中，我们证明了Gen3可用于在马铃薯叶绿体中安装多基因途径，并且在具有正常表型参数的植物中，整个植物发育阶段直至花期，附加体都以高拷贝数的全长环状形式稳定存在。预计在未来十年，微型同型质体基因组将成为在质体中安装复杂遗传电路的有价值工具。
