液体发酵过程发菜多糖生物合成的调控机制研究

Traditional food intergradient Nostoc flagelliforme is designated as Category I terrestrial cyanobacteria by Chinese government, and its polysaccharide has gained much attention for the anti-viral, anti-tumor, and immune regulatory activities. During the process of artificial liquid culture, the monosaccharide composition of polysaccharides was influenced by culture parameters, which consequently led to the differences on pharmacological activity, but the regulatory mechanism was still unclear. To reveal the underlying mechanism, the effects of various culture parameters on monosaccharide composition will be analyzed to find out the conditions with significant influences. Then the effects of these culture parameters on the three important processes of polysaccharide biosynthesis including monosaccharide metabolism, activation of monosaccharide, and monosaccharide assembly will be analyzed. The variance of cellular components including RNA, protein, and metabolite will be investigated to reveal the effects of culture parameters on monosaccharide metabolism. The changes of sugar nucleotide biosynthetic pathway will be analyzed to obtain the key enzyme and metabolic node. The genes for glycosyltransferase will be sequenced and the gene expression patterns will be investigated to determine the regulatory sites in the process of assembly of nucleotide sugars. Then the regulating mechanism of monosaccharide composition of polysaccharide will be illustrated. Finally, the sugar chain biosynthesis key nodes will be engineered, combined with fermentation optimization, to establish liquid culture system with controllable monosaccharide composition of polysaccharide. The research would reveal the regulation mechanism underlying cyanobacterial polysaccharide biosynthesis and provide theoretical guidance for the high-efficient production of high value polysaccharides.

传统食材发菜为我国一级保护陆生蓝细菌，其多糖因具有抗病毒、抗肿瘤、免疫调节活性而日益受到关注。前期研究发现在人工液体培养过程中，多糖糖链组成易受外界条件影响发生变化继而导致生物活性产生差异，但其调控机制尚不清楚。针对发菜多糖糖链组成调控机制这一关键科学问题，首先研究不同作用因素对糖链组成的调控规律，筛选显著影响因素；然后解析这些因素对糖链合成三个重要过程糖基代谢、糖基活化和糖基组装的影响，通过比较细胞组分（RNA、蛋白和代谢物）差异分析对糖基代谢的调控规律，分析糖核苷酸合成途径变化规律获得糖基活化过程的关键酶及代谢节点，克隆糖基转移酶基因并分析其时空表达特性确定对糖基组装的关键调控点，进而阐明糖链组成的调控机制；最后人工改造糖链合成过程关键节点，结合发酵代谢调控，建立多糖糖链组成可控的定向液体发酵体系。本研究对揭示蓝细菌多糖生物合成调控机制和指导高价值活性多糖的高效定向生产具有重要意义。

项目按照计划，为了研究液体发酵过程发菜多糖生物合成的调控机制，首先研究了发酵条件对发菜多糖结构、性质的影响，通过调控营养因素和胁迫因素改变发菜细胞的发酵条件，分离纯化得到了6种多糖组分，对比了它们在结构、性质和抗氧化、抗辐射和降血脂活性上存在的差异。接下来对液体发酵过程对发菜多糖生物合成影响及其机理展开了研究。从酶活水平上确定了碳源、氮源和光质条件下PGI和FBPase可能为影响发菜多糖合成的关键酶；通过Illumina高通量测序和iTRAQ定量蛋白质组学技术对不同光质条件下的发菜细胞进行了分析，发现光质影响多糖合成的机理是改变胞内的碳分配、使碳代谢流流向糖核苷酸合成的方向和调控胞内ROS水平，并对糖基转移酶基因进行了鉴定，进而比较了不同光质条件下糖基转移酶基因的表达情况；从8类12种化学诱导剂中筛选出水杨酸和茉莉酸可以显著提高发菜多糖产量，且对多糖的结构、理化性质和抗氧化活性没有显著影响，并发现了NO在水杨酸和茉莉酸影响发菜多糖合成中的调控作用。本研究为高价值活性多糖的高效定向生产和蓝细菌多糖生物合成调控机制提供了新见解。