Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, Missori.
Present address: State Key Laboratory for Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Biotechnol Bioeng. 2018 Sep;115(9):2305-2314. doi: 10.1002/bit.26747. Epub 2018 Jun 29.
The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.
好氧光合细菌集胞藻 PCC 6803（S6803）是一种广泛用于基础研究和生物技术应用的模式蓝藻。由于其多倍性，现有的 S6803 基因组工程方法需要多轮筛选才能修饰所有基因组拷贝，这既耗时又低效。在这项研究中，我们对 Cas9 工具进行了工程改造，使其能够一步实现无分离的基因组工程。我们进一步利用我们的 Cas9 工具删除了七个 S6803 天然质粒中的三个。我们的结果表明，所有三个小尺寸的天然质粒，但不是大尺寸的天然质粒，都可以用这个工具删除。为了进一步促进 S6803 中的异源基因表达，我们基于天然质粒 pCC5.2 构建了一个穿梭载体。该穿梭载体可以一步引入含有 Cas9 的 S6803 中，而无需分离，并可以在没有抗生素压力的情况下稳定维持至少 30 天。此外，在没有选择压力的情况下，连续培养 30 天后，穿梭载体上编码的基因仍然保持功能。因此，这项研究提供了一组新的工具，用于快速修饰 S6803 基因组和稳定表达异源基因，可能促进使用 S6803 的基础研究和生物技术应用。
