Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, College of Life Science and Technology, Jinan University, Guangzhou, China.
Biotechnol Bioeng. 2019 Nov;116(11):3006-3015. doi: 10.1002/bit.27110. Epub 2019 Jul 21.
There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.
人们对利用微藻作为生产宿主生产各种增值化合物越来越感兴趣。然而，由于缺乏同时控制多个基因的遗传工具，微藻的遗传改良受到了阻碍。在这里，我们鉴定了两个新的强启动子，分别命名为 Pt202 和 Pt667，并阐明了它们在同时驱动三角褐指藻关键脂肪生成基因表达中的潜在作用。对鉴定出的启动子序列的计算机分析预测了存在必需的核心顺式元件，如 TATA 和 CAAT 盒。通过使用 GUS 报告基因初步评估了启动子的调控作用，结果显示 GUS 表达强烈。此后，通过 Pt202 和 Pt667 这两个启动子，分别过表达了两个关键的脂肪生成基因，即苹果酸酶（PtME）和 5-去饱和酶（PtD5b）。组合基因过表达并没有损害一般的生理性能，同时中性脂质含量显著增加了 2.4 倍。脂肪酸甲酯的 GC-MS 分析表明，二十碳五烯酸（EPA；C20:5）显著增加。这些发现为微藻遗传工具增添了一个重要的工具包，它可以促进各种启动子驱动的多基因表达，并促进微藻进行工业生物生产。
