Department of Food Science, University of Tennessee, Knoxville, Tennessee 37996.
Center for Agricultural Synthetic Biology, Institute of Agriculture, University of Tennessee, Knoxville, Tennessee 37996.
Plant Physiol. 2019 Mar;179(3):943-957. doi: 10.1104/pp.18.01220. Epub 2019 Jan 24.
Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.
植物合成生物学是一个快速发展的领域，不断涌现出新的工具来推动创新。特别引人关注的是将合成生物学应用于叶绿体生物技术，以生成能够产生新代谢物、疫苗、生物燃料和高价值化学品的植物。在组装大型 DNA 分子和组成多个转录单元方面取得的进展，极大地提高了快速构建用于基因工程的新型载体的能力。特别是 Golden Gate 组装为将合成遗传元件标准化组装到更大的 DNA 构建体中提供了一种简便的分子工具。在这项工作中，开发并验证了一个完整的模块化叶绿体克隆系统 MoChlo，用于在植物中快速灵活地进行叶绿体工程。从文献中挖掘并修改了 128 个标准化的叶绿体特异部件（47 个启动子、38 个 5'非翻译区 [5'UTR]、9 个启动子:5'UTR 融合、10 个 3'UTR、14 个感兴趣的基因和 10 个叶绿体特异的目的载体），并用于 MoChlo 组装，以及叶绿体特异的目的载体。通过组装各种大小的合成操纵子并确定组装效率来验证该策略。该方法成功地用于生成包含多达七个转录单元的叶绿体转化载体（单个载体中约 10.6-kb 合成操纵子）。为了使资源有限的研究人员能够参与叶绿体生物技术，并加速该领域的进展，按照所述内容，整套工具都可以以最小的成本从 Addgene 获得。因此，MoChlo 工具包代表了用于快速灵活设计异源代谢途径进行质体代谢工程的有价值的工具。
