无光异养培养食用微藻Nitzschia PKU产生岩藻黄素的机制和调控方法

As a natural carotenoid with special biological activities such as promoting body fat decomposition, fucoxanthin are widely used as functional food and food additive in recent years. However, fucoxanthin which is mainly derived from large algae such as kelp and wakame, are in short supply. In our previous study, we found that the fucoxanthin content of the edible microalga Nitzschia PKU is nearly 100 times that of large algae. Moreover, as a heterotrophic culture microorganism, Nitzschia PKU has the potential for large-scale production, and is a better alternative source of fucoxanthin. Our previous studies showed that the content of fucoxanthin in Nitzschia PKU in the absence of light culture is only half of that under light conditions. Therefore, increasing the fucoxanthin content in the absence of light is quite necessary for the industrial production of fucoxanthin by Nitzschia PKU. Fucoxanthin is an important component of microalgal light-harvesting antenna protein FCP. The structure and transcriptional regulation of FCP complex under light is the hot topics in recent years. However, the mechanism of de novo synthesis and transformation of fucoxanthin in the absence of light is still unclear. In this project, the influence of light density on the synthesis and transformation of FCP pigments, supplemented by pigments synthesis inhibitors, and attempt to explain the accumulation of fucoxanthin and other pigments based on the dynamic changes of intermediate metabolites and related gene expression. The intrinsic mechanism will establish a matt culture model based on the efficient production of fucoxanthin by inducing FCP pigments synthesis. In the present project, we compared the culture model with or without light on the transcription of key enzymes in FCP pigment synthesis. Based on the dynamic changes of intermediate metabolites, we tried to explain the synthesis mechanism of fucoxanthin and its relationship with FCP pigment biosynthesis during heterotrophic culture, and establish a culture model in which fucoxanthin content can be regulated. This project will provide a theoretical basis for the production of fucoxanthin by microalgae growing in the dark.

岩藻黄素是一种天然类胡萝卜素，具有促进脂肪分解等特殊生物活性，近年被广泛用作食品功能因子和食品添加剂。目前市场上岩藻黄素主要来源于海带和裙带菜等大藻，产品供不应求。申请人前期研究所获得的微藻Nitzschia PKU岩藻黄素含量是大藻的近百倍，是可异养培养微生物，具有规模化生产的潜力，是更好的新原料来源。无光培养时Nitzschia PKU岩藻黄素含量仅为光照条件下的一半，因此无光时细胞岩藻黄素的积累仍具有巨大的提升空间。岩藻黄素是微藻捕光天线蛋白FCP的重要组成，光照条件下FCP整体的转录调控是近年研究热点，但无光时岩藻黄素的从头合成和相互转化机制尚不清楚。本项目通过比较有光、无光模型对FCP色素合成关键酶转录的影响，并根据中间代谢物动态变化，尝试阐释无光异养时岩藻黄素的合成机制及与FCP色素生物合成间关系，建立调控岩藻黄素积累的培养模型。本项目将为无光培养微藻产生岩藻黄素提供理论基础。

平滑菱形藻是可异养培养的硅藻，具有规模化生产的潜力，是更好的岩藻黄素原料来源。然而，无光培养时平滑菱形藻岩藻黄素含量仅为光照条件下的一半。为提高无光条件下细胞中岩藻黄素含量，阐释岩藻黄素可能合成机制，本项目建立了硅藻中多种类胡萝卜素的高效检测方法；阐释了无光条件下营养元素对平滑菱形藻中类胡萝卜素和脂肪酸的合成调控机制，明确了其对细胞中碳代谢物的分配关系；阐释了硅藻中FCP色素合成间的关系。获得如下四项主要研究成果：（1）利用超高效液相色谱-三重四级杆质谱联用方法，建立了海洋硅藻中常见7种类胡萝卜素的高效检测手段；（2）发现高浓度硅元素可促进细胞中岩藻黄素的积累，利用硅元素调控，提高海洋微藻平滑菱形藻在异养条件下岩藻黄素积累的浓度和产率，并阐述其调控机制；（3）阐释异养发酵条件下，碳源葡萄糖对平滑菱形藻中岩藻黄素等类胡萝卜素的诱导合成机制，建立了平滑菱形藻的氨基酸和中心碳代谢途径，发现葡萄糖存在的情况下，丙酮酸（1.86倍）、α-酮戊二酸（2.47倍）以及缬氨酸（1.57倍）水平显著提高。（4）利用转录组学和代谢组学分析研究，提出了葡萄糖诱导的海洋微藻内营养物质转化的新机制，发现八氢番茄红素去饱和酶（PDS）和zeta-胡萝卜素异构酶（ZISO）可能是类胡萝卜素生物合成的限速酶。