DataSheet1.PDF

Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.

质体工程为填补微生物藻光合细胞工厂技术性能与巨大潜力之间的鸿沟提供了重要工具。然而，截至目前，关于工业微藻质体工程的报道寥寥无几。这主要归因于这些物种细小的细胞尺寸和复杂的细胞壁结构，使得它们难以通过当前的质体转化方法（例如生物粒子转化和聚乙二醇介导的转化）进行转化。在本研究中，以工业油脂微藻海洋小球藻（Nannochloropsis oceanica）为模型，建立了一种电穿孔介导的叶绿体转化方法。荧光显微镜和激光共聚焦扫描显微镜证实了由内源质体启动子和终止子驱动的绿色荧光蛋白的表达。利用 Zeocin 抗性筛选获得了同质化菌株，通过测序和DNA印迹分析揭示了叶绿体基因组内稳定且定位特定的重组。电穿孔介导的叶绿体转化演示为工业微藻的质体基因组编辑开辟了众多途径，特别是那些对化学和微颗粒轰击转化具有抵抗性的叶绿体。