云芝新颖苔色酸木糖苷衍生物的糖基化机制研究

Glycosides include several important classes of small molecular metabolites as aglycon. Attachment of a monosaccharide or oligosaccharide moiety to the respective aglycon leads to a dramatic change in activity of parent molecules and enhances medicinal biodiversity. Recently we have isolated and identified a series of novel structure of xylosides in Trametes versicolor, whose silent genes were actively expressed during the co-cultivation with other basidiomycetes. Especially, an orsellinic acid-derived xyloside was synthesized and could significantly enhance the activity of Beas-2B cell line derived from the human respiratory tract. However, corresponding genes for xylosylation has so far not been reported in T. versicolor. In this project, we propose to identify the genes encoding UDP-xylose synthase and xylosyltransferase, and demonstrate their characteristics of enzymatic catalysis. To this end, the methodologies of bioinformatic genome mining, genetic manipulation through agrobacterium will be carried out to determine the UDP-xylose synthase and xylosyltransferase in T. versicolor, and to elucidate their expression profile under the co-culture. Furthermore, protein homology modeling and site-directed mutagenesis will be used to demonstrate 3D structure and active binding sites of xylosyltransferase, and to reveal the mechanism of xylosyltransferase catalyzing diverse substrates. Meantime, UDP-xylose synthase and xylosyltransferase will be overexpressed in T. versicolor and the change of metabolite profile of glycoside will be further analyzed. Overall, this proposed research will provide important insights into the mechanism of xylosylation in T. versicolor and other polyporaceae. This information can be further used to investigate the mechanism of regulation and activation of glycosylase in fungi.

糖苷化合物是以小分子次级代谢产物作为糖苷配基，以单糖或寡糖作为糖体的杂合天然产物，这种组合不但提高配基的生物活性，更加丰富其药用多样性。申请者前期利用担子菌共培养策略激活云芝（Trametes versicolor）沉默基因表达产生一系列新颖木糖苷结构，其中苔色酸木糖基衍生物具有显著促进Beas-2B细胞生长的先导药物价值，然而木糖苷糖基化机制却不清楚。本项目拟从鉴定木糖基合成酶和转移酶基因并研究其酶催化特性开展工作，整合生物信息学基因挖掘和农杆菌介导遗传操作策略从云芝基因组水平定位木糖合成酶和木糖基转移酶基因，解析其共培养激活表达的规律，继而蛋白质同源建模和定点突变阐释木糖基转移酶对底物适配多样性的蛋白质空间结构基础；同时在云芝中建立双酶组合过表达元件探究糖苷化合物代谢谱的变化特性。本项目对于阐释多孔菌科糖苷化合物的糖基化过程具有重要科学意义，为进一步探究糖基化酶的激活调控机制奠定基础。

担子菌多孔菌科来源的糖苷化合物具有丰富的结构多样性和显著的抗氧化、抗肿瘤、免疫等生物学活性，本项目在前期利用担子菌共培养策略从云芝和树舌灵芝共培养物种发现一例新颖木糖苷衍生物，以此探究木糖苷糖基化机制。在本项目中我们首先利用生物信息学预测分析云芝中潜在的木糖转移酶基因，通过构建系统进化树以及RT-PCR定位到关键的木糖苷转移酶基因GI:636605689；继而通过对该基因的克隆、异源表达和纯化，研究其基本的酶学活性；我们引用了C13标记动态分析技术追踪了共培养激活的83个特征产物的菌株来源问题，进一步证实云芝菌株独特的木糖苷衍生物形成机制；同时对共培养和单培养的云芝进行转录组测序分析，找到了上调/下调表达差异显著的基因，分析其对该木糖化合物形成的作用和影响；最后在云芝中建立UDP-木糖合成酶和木糖转移酶基因的组合过表达元件，通过底物添加和产物峰检测，初步探究该双元基因对糖苷化合物代谢谱的变化特性，为进一步探究糖基化酶的激活调控机制奠定基础。