海洋真菌多糖生物合成途径及其关键合成酶性质研究

At the base of many preliminary research works of our research team, the project will adopt more advanced technologies and methods under the advanced research supporting conditions of our school. The fungus Phoma herbarum YS4108 was choosed as our object studied in this project, which could produce the novel antitumor polysaccharide YCP. Both the single cell-preparation method and the Tn5 transposase-assisted transformation, which can be incorporated into a kit, promising to be an insertion-mutation tool with broad applicability to studies on functional genomics in fungi. The candidate enzymes that possibly involved in the pathway could be quickly identified by the in vitro biosynthesis method established. The biosynthetic pathway was reconstructed in vitro by using dialysed crude enzymes free from substrates. Through a series of substrate and intermediate test, the active sugar donors of the pathway were ascertained to be UDP-glucose(UDPG) and UDP-glucose 6-dehydrogenase. The other key enzymes would be cloned, expressed in E. coli, and purified by affinity chromatograph and their characters and functions would be showed their roles in the biosynthetic pathway of the polysaccharide YCP. The biosynthetic pathway of the polysaccharide YCP would be clarified at last. The theory base would be supplied in reconstructing synthesis in vitro by using dialysed crude enzymes free from substrates. The output and activity of polysaccharide would be improved. New structure polysaccharide would be gained. The physical and chemical properties of the polysaccharide would be changed. The research could give not only scientific instruction for the biosynthetic pathway of the polysaccharide by other Phoma herbarum fungi from the other labored and close relative genus, but also layed the foundation of using resources of this speice fungi resource.

本项目拟在课题组较多相关科研积累的基础上，利用我校先进的科研支撑条件，以本课题组分到的新颖抗肿瘤多糖YCP产生菌草茎点霉Phoma herbarum YS4108为研究对象，将单细胞制备方法和转座酶辅助的遗传转化技术用于该丝状真菌的多糖生物合成功能基因组研究。利用新建的YCP体外酶法化学合成技术快捷高效地鉴定可能参与YCP生物合成途径的候选关键酶类，克隆表达与纯化上述关键酶，并在全基因组测序基础上利用预测基因，采用基因组学和转录组学结合基因敲除技术研究参与多糖合成各酶的酶学性质与功能，揭示上述各关键酶在多糖生物合成过程中充当的角色，最终阐明YCP多糖生物合成途径。为进一步的体外酶法组合化学研究，以及提高多糖的活性和产量，最终获得新结构多糖、改变天然多糖理化性质提供理论基础。该研究不仅可为其它来源的草茎点霉真菌及相关种属真菌多糖生物合成提供科学指导，而且为拓展该类真菌资源的利用奠定科学基础。

在真菌全基因组测序基础上在基因组中定位了糖代谢相关的所有基因：尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）、UDP-葡萄糖脱氢酶（UGDH）、糖原蛋白（Glycogenin）、糖原合酶（GS）、糖原分支酶（GBE）、蛋白激酶C（PKC）、α-1,4-葡萄糖基转移酶(α-1,4-glycosyltansferase)、α-1,6-葡萄糖醛酸转移酶（α-1,6-glucuronosyltransferase）、尿苷二磷酸葡萄糖醛酸基转移酶（UGT）等，继而利用已建立的遗传转化方法，通过免疫学方法证明UGPase和糖原合成酶基因GS参与胞壁多糖YCP的生物合成，利用基因敲除和单糖定量等方法鉴定了葡萄糖醛酸转移酶。基本阐述了完整的phoman生物合成途径，从而为解决该多糖的来源问题铺平了道路。.同时对相同来源真菌CY018生物碱研究分得6个新生物碱，并对其抗病原细菌活性进行检测，发表论文1篇。并对蜱虫病毒的侵染机制进行了microRNA水平的检测和初步研究。利用新建立的抗流感病毒活性模型开展了多糖抗病毒活性筛选并对其抗病毒作用机制进行了初步研究。同位素示踪实验遇到困难，采用1-6位上各C原子均标记的葡萄糖饲喂进行生物标记，由于多糖分子量为240万，不能将全部信号加以标记，难以检测到。又进行了1,4位、1,3位及2,4位位上C原子均标记的葡萄糖饲喂实验探索及检测。为能顺利完成项目还进行了该真菌胞外多糖发酵条件优化及生物合成途径研究。.对内生真菌Myrothecium roridum IFB-E091固体发酵产物进行分离得到(S)-(−)-N-[2-(3-hydroxy-2-oxo-2, 3-dihydro-1H-indol-3-yl)-ethyl]-acetamide新化合物，其具有一定的抗肿瘤活性，发表在《药物学报》。从内生真菌土曲霉LQ中分离到一个新的生物碱Fumigaclavine I, 发表在Chinese Journal of Natural Medicines。并开展了多糖及其它天然产物的抗流感及蜱虫病毒活性筛选，相关抗病毒药物筛选模型已建立成功，已筛选药物353种，相关研究正在进行，并对病毒的侵染机制进行了microRNA水平的检测和初步研究，发表相关文章3篇，申请专利5项。授权专利1项(CN 104059994 B)。此外，为探明多糖合成酶作用（探究多糖合成的起