基于合成生物学构建与优化花生四烯酸异源合成新途径的应用基础研究

Arachidonic acid (ARA), which is an essential polyunsaturated fatty acid for human, has got more and more attention in the application of food, medicine, and other fields. At present, the main source of ARA is derived from the fermentative production of ARA-rich lipids by the filamentous fungus Mortierella alpina. However, some hindering obstacles, i.e., growth cycle is long, and the fermentation process is difficult to control, are still existed in this process. This makes the cost and price of the final product very high. This study aims to construct a novel ARA biosynthetic metabolic pathway (Δ8 pathway) in Yarrowia lipolytica which is a GRAS strain and has rapid growth rate and high biomass accumulation ability using the method of synthetic biology. The synthetic metabolic pathways harboring promoters with different strength were first constructed quickly using the DNA assembler technology. This would be beneficial for regulating the enzyme expression level accurately, and thus fulfilling the expression of exogenous genes coordinately. The accurate regulation upon promoter combinations is supposed to optimize the adaptation between artificial regulatory elements and functional modules as well as that between functional modules and host cell. This would lay a theoretical foundation for fulfilling efficient and low cost production of high quality ARA-rich lipid, and provide guidelines for constructing the synthetic metabolic pathways to achieve high other polyunsaturated fatty acids accumulation.

花生四烯酸是一种人体必需的多不饱和脂肪酸，因在食品和医疗保健等领域有着重要应用而得到广泛关注；目前，普遍采用丝状真菌高山被孢霉发酵生产；但这种方法发酵周期长，过程控制较难，导致花生四烯酸产量低、成本高。本研究针对上述问题，基于合成生物学原理，采用DNA assembler（体内DNA装配）技术在不能够合成花生四烯酸，但生长快速且生物量高的GRAS菌株解脂耶氏酵母体内，快速组装花生四烯酸合成的新途径，通过启动子水平的优化，将人工构建的花生四烯酸代谢途径中每个酶的表达量加以限制并精确调控，实现最优的多基因联合协同表达，优化功能模块和宿主细胞之间的适配程度，提高花生四烯酸生物合成新路线效率的同时，剖析人工构建的花生四烯酸合成代谢途径和宿主细胞的适配机制，并为其它类似的多不饱和脂肪酸代谢途径的构建与组装提供借鉴。

解脂耶氏酵母是一种被FDA公认的安全微生物。由于其自身独特的遗传特性，解脂耶氏酵母已受到很多研究团队的关注。尤其，随着代谢工程与合成生物技术的快速发展，很多团队将解脂耶氏酵母作为底盘细胞用于合成重要精细化学品。花生四烯酸是一种功能性多不饱和脂肪酸，目前，花生四烯酸的来源主要有组织提取法、微生物发酵法与代谢工程改造生物体的异源合成法。然而，动物组织提取法存在获取量低，无法满足市场需求；选用微生物（如高山被孢霉）发酵过程中存在生产强度低、生产成本高等问题；通过代谢工程改造生物体（如拟南芥、烟草）异源合成花生四烯酸同样面临一些缺点，如植物体生长受限、合成产量低。针对此，本研究以解脂耶氏酵母异源高效合成花生四烯酸为目标，通过建立解脂耶氏酵母体内多基因代谢途径一步组装技术平台，在该酵母体内分别组装两条不同的花生四烯酸合成途径，在此基础上，最终实现了花生四烯酸在解脂耶氏酵母体内的高效合成。