食用裸藻Euglena gracilis 异养产多糖的调控机制研究

Euglena gracilis is a genus of unicellular flagellate eukaryotes containing many nutritive components. The most valuable composition is paramylon which has important function in immunology. This algae have been certificated by FDA in U.S. and also in Japan, it has been listed as a new resource food in our country, to date only company in Japan has realized industrialization through photoautotrophic cultivation. But the content of paramylon is low for the competitive relationship between paramylon and lipids, moreover the accumulation of paramylon is affected by “wax ester fermentation” which is unique in Euglena gracilis. Heterotrophic cultivation is an alternative way. So this project aims at the flows of substance and energy under the controlled heterotrophic conditions and focus on the flow and distribution of carbon source. The task of this project is to regulate and lead the flow of the carbon to the synthetic route of paramylon in heterotrophic cultivation. Firstly, the process of heterotrophic cultivation and cellular component will be analyzed. Then the intermediate metabolites and key enzymes in the metabolic pathway will be studied to illuminate synthesis path of target product-paramylon in heterotrophic cultivation of Euglena gracilis, meanwhile the interrelation and dynamic transformation between paramylon and other carbon constituent will be analyzed and resolved for the effects of central carbon metabolism and its effects on products synthesis. Finally, based on the above findings, the regulatory method will be implemented and the effects will be verified through heterotrophic cultivation of Euglena gracilis with high productivity.

裸藻是单细胞真核生物，具有重要的营养价值。其中的裸藻多糖在免疫学等方面有重要作用。该藻已取得美国FDA和日本的认证，我国也将其列为新资源食品，但迄今仅日本可实现裸藻的光自养培养且产业化。光自养条件下裸藻多糖与脂类间存在竞争底物关系，同时存在“蜡酯发酵”现象影响裸藻多糖积累。异养是可能的解决途径，本项目旨在解决异养可控条件下的裸藻细胞内物质与能量的定向流动问题，集中体现为碳流分配及其调控。研究异养培养过程裸藻多糖合成路径并调控引导碳源流向裸藻多糖合成以实现高效积累。首先对异养培养过程及细胞组分进行分析，其次对两大类含碳产物组成进行分析，并对中间代谢物及关键酶进行研究，探明异养条件下裸藻多糖的合成路径，同时分析裸藻多糖与其他含碳产物之间的相互关系和动态转化，并以此解析中心碳代谢与裸藻多糖合成的关系。最后基于以上研究结果，实施关键调控手段并验证其对代谢通路的影响以指导高效异养培养。

裸藻是一类介于动物和植物之间的单细胞真核生物，在光合作用机制方面具有重要的研究价值。另一方面，裸藻具有重要的应用价值，它能够全面地补充人体所需的营养，缓解隐性饥饿，是高效的天然膳食营养补充剂。尤其是其独有的生物活性物质裸藻多糖及其修饰物能刺激免疫系统尤其是巨噬细胞，增强免疫活力，其抗癌、抗细菌活性和抗病毒（HIV）的活性极强。降低胆固醇和降血糖作用效果明显。此外，裸藻多糖在保护肝脏、缓和过敏性皮炎、抑制嘌呤吸收和预防改善痛风等方面有独特作用。因此具有极大的应用价值和前景，且通过了包括美国、日本及我国的认证，为其广泛的应用提供了基础。.但目前仅有日本的一家公司利用光自养培养实现产业化，但存在诸如生产效率低，裸藻多糖含量低等问题。因此本文开展了裸藻的异养培养研究，实现了异养培养工艺的优化与放大，同时为提高异养裸藻的多糖含量及解除盐度抑制这两方面开展了机理的研究。具体结论如下：（1）优化并放大了异养培养工艺。筛选了合适的异养培养基，优化了碳氮源及初始浓度；优化了裸藻异养培养的理化条件，如合适的培养温度、种龄、消泡剂等。异养培养工艺优化后，放大到50 L、500 L及10,000 L发酵罐，细胞密度均超过50 g L-1。（2）利用代谢组学的方法（LC-MS/MS）进行了异养与光自养、混养的对比研究，研究了异养条件下裸藻多糖含量高的机理机制，筛选并鉴定了差异代谢物，并对代谢通路变化予以解析。为下一步通过代谢调控手段实现裸藻多糖的高产率奠定了基础。（3）针对异养补料分批培养过程中出现的裸藻细胞受到盐度胁迫和抑制的现象开展了相关机制的研究，解析了裸藻应对盐胁迫的响应机制。为进一步解除盐度胁迫，实现裸藻的高效异养培养建立了基础。