食用微拟球藻Nannochloropsis光合生产二十碳五烯酸的调控机制研究

Eicosapentaenoic acid (EPA, C20:5ω-3), an important nutraceutical approved and recommended by the United States Food and Drug Administration, is edible and can be found in a broad range of foods like salmon and krill. It belongs to the ω-3 polyunsaturated fatty acid family and has long been demonstrated to provide significant benefits to human health, particularly in prevention and treatment of cardiac diseases. Currently, EPA is mainly produced from marine fish oil, but its supply is limited and lags far behind the ever increasing demand. Nannochloropsis, an EPA-rich marine microalga, has been proposed as a promising alternative to fish oil, in that it possesses high photosynthetic efficiency for fast growth, is easy to culture with high cell density, and requires no arable land for mass production. In Nannochloropsis, EPA is mainly stored in polar membrane lipids particularly glycolipids, while triacylglycerol (TAG) contains extremely low content of EPA (commonly below 2%), representing a great challenge for the utilization of Nannochloropsis for EPA-rich oil production. The present project aims to elucidate the molecular mechanisms for EPA biosynthesis and regulation via comparative transcriptomics and lipidomics analyses, and to manipulate Nannochloropsis by metabolic engineering for enhanced EPA-rich oil production. The key genes involved in EPA biosynthesis, such as desaturase and elongase genes, will be cloned and functionally characterized. The overexpression of these genes, driven by constitutive promoter, is capable of pushing the carbon flux to EPA biosynthesis even under stress conditions. Besides, we have characterized a novel algal DGAT with high efficiency of transferring EPA to TAG. The introduction of this DGAT gene will sequestrate the free EPA, either de novo synthesized or released from the degraded glycolipids, into TAG, providing protection of EPA from β-oxidation as well as a sort of pulling power for EPA synthesis. The metabolic collaboration of pushing and pulling has great potential to redirect the carbon flux to EPA and allow the accumulation of high level of EPA-rich oil in Nannochloropsis. Overall, the present study will help us understand the regulatory mechanism of EPA biosynthesis and lay a solid foundation for developing phototrophic Nannochloropsis as green cell factories for the sustainable production of EPA-rich algal oil and as an alternative to fish oil-sourced EPA.

二十碳五烯酸(EPA)是美国FDA认可并推荐的可食用营养组分，广泛存在于三文鱼等海洋食品，对促进人体健康有着重要作用。目前，EPA主要产自鱼油，但其产量远不能满足日益增长的市场需求。因为生长快、易培养、不需要耕地、富含EPA，微拟球藻被认为有潜力取代鱼油EPA。微拟球藻EPA主要储存在极性脂特别是糖脂中,甘油三脂（TAG）中EPA含量极低。本项目旨通过比较转录组及脂质组学阐明EPA合成与调控的机制，并通过代谢工程提高油脂EPA的含量。首先克隆鉴定EPA生物合成的关键酶基因，导入微拟球藻组成型表达，将碳代谢流推向EPA合成。其次将新型高效微藻DGAT基因过表达，整合EPA到TAG使其免被氧化降解并提供代谢拉力促进EPA合成。这种代谢推力和拉力的共同作用引导碳代谢流向EPA合成，从而积累高水平的藻油EPA。此研究将为开发微拟球藻作为光合绿色细胞工厂替代鱼油生产藻油EPA打下坚实的理论基础。

多不饱和脂肪酸尤其是二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)，是人们公认的高值营养成分，有着广泛的用途和市场前景。人体不能合成EPA和DHA，需从食物中摄取。因为鱼油来源的多不饱和脂肪酸不能满足日益增长的市场需求，需寻求生产它们的生物新资源。微藻是EPA和DHA的最初生产者，其中，微拟球藻被认为有潜力来生产EPA。但是该藻TAG中的EPA含量极低。本项目拟阐明微拟球藻EPA的合成机制，明晰限制TAG中EPA积累的因素，并通过代谢工程手段定向提高。首先通过生理、生化、组学分析微拟球藻在不同条件下EPA的合成、动态分布、和储存，发现EPA主要存储在膜脂中，在TAG中的含量通常不超过1.5%，可能的原因在于：1）EPA生物合成相关的去饱和酶基因在TAG积累条件下下调表达，导致EPA的合成减少，2）甘油二酯酰基因转移酶（DGAT）对EPA的活力很低，EPA不能被有效地整合到甘油三酯（TAG）中存储，从而进入到脂肪酸β-氧化途径中降解。接着，我们研究了来源于4种微藻超过30个DGAT的功能，筛选到了对EPA有高活力的DGAT。然后，将这个高活力的外源DGAT（CzDGAT1A）导入到微拟球藻中表达，它定位于叶绿体内质网膜（cER），可将TAG中EPA的比重提高5倍。而将CzDGAT1A和内源的FAD基因同时过表达，则可将TAG中EPA的比重提高8倍。结合来讲，我们的研究厘清了微拟球藻TAG中EPA积累的限制性因素，理性设计靶点通过代谢工程手段成功地提高TAG中EPA比重，解决了项目提出的关键科学问题，为进一步开发该藻作为绿色细胞工厂和鱼油EPA替代生产者提供了理论基础和可行的技术思路。

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