寇氏隐甲藻二十碳五烯酸定向合成的分子调控机制

Eicosapentaenoic acid (EPA) has important physiological functions such as regulating lipid metabolism and preventing cardiovascular diseases. It has been widely used in food, medicine, health care and other fields. The market demand has increased year by year, and the traditional sources of fish oil have been difficult to meet the demand. As a potential alternative source, EPA-producing microalgae are difficult to industrialize because of their low biological yield, low EPA yield and high culture cost. The docosahexaenoic acid (DHA) in microalgae oil is further transformed from EPA through carbon chain elongation and desaturation. From this point of view, a scientific hypothesis is put forward that blocking the transformation of EPA to DHA in Crypthecodinium cohnii cells with high DHA production might significantly increase the content of EPA. Firstly, the pathway of polyunsaturated fatty acid synthesis in Crypthecodinium cohnii will be analyzed, and the key enzymes of EPA/DHA transformation will be found. Then, the related genes will be knocked out or re-edited to block the transformation of EPA to DHA and realize the directional synthesis of EPA. This project is helpful to reveal the mechanism of polyunsaturated fatty acid synthesis system of Crypthecodinium cohnii, enrich the theoretical basis of molecular breeding of oil-producing microalgae, break through the technical difficulties of EPA microalgae fermentation, and promote the industrialization of EPA from microalgae.

二十碳五烯酸（EPA）具有调节脂质代谢、防治心血管疾病等重要生理功能，已被广泛应用到食品、医药、保健等领域，市场需求量逐年攀升，传统鱼油来源已经难以满足需求。作为潜在的代替来源，常见产EPA微藻生物产量低、EPA产率低、培养成本高，难以进行产业化开发。微藻油脂中的二十二碳六烯酸（DHA）是由EPA再经过碳链延长和去饱和进一步转化生成的。假若在高产DHA的寇氏隐甲藻细胞内阻断EPA向DHA的转化可能会大幅度提高EPA的含量。基于以上设想，本项目将首先分析寇氏隐甲藻多不饱和脂肪酸合成通路，找到EPA/DHA转化的关键酶，再对相关基因进行定点敲除或重新编辑，从而阻断EPA向DHA的转化，实现EPA的定向合成。本项目有助于揭示寇氏隐甲藻多不饱和脂肪酸合成系统的作用机制，丰富产油微藻分子育种的理论基础，突破EPA发酵技术难题，推动微藻来源EPA的产业化开发。

二十碳五烯酸（EPA）具有重要的生理功能，在食品、医药、保健等领域有广泛的应用前景。本项目建立了寇氏隐甲藻在转录水平上的参考基因组数据库，揭示PUFA合成系统的作用机制，获得EPA/DHA转化关键酶Δ5 Elo和Δ4 Des。并在此基础上构建了高产EPA的寇氏隐甲藻工程菌。改造后的工程菌所产EPA从原始菌株不足0.5%提升到了7.276%，含量提升10倍以上。葡萄糖是工程菌产EPA的最佳碳源。当氮源为酵母浸粉和谷氨酸钠混合氮源时，生物量及油脂含量明显高于单一氮源。工程菌在16-34 ℃条件下皆能生长，其中最适生长温度为28℃。高溶氧有利于细胞生长，但不利于工程菌EPA的积累。本项目有助于突破EPA微藻发酵技术难题，为隐甲藻来源EPA的产业化开发奠定了基础。