出芽短梗霉中普鲁兰糖合成机理研究

Pullulan, similar with starch or glycogen, is one kind of α-D-glucan. Because of its special glycosidic bond (repeated by double α-1,4- glycosidic bonds and one α-1,6- glycosidic bond) distinguished from starch or glycogen, pullulan is water-soluble, high toughness, emulsibility, and alpha D-amylase resistance. Therefore, pullulan is widely used in food and medicine industry. .However, the mechanisms underlying biosynthesis of pullulan is unclear until now, which strongly restricts its production and application. How is the special glycosidic bond formed and which glucosyltransferases catalyze the reactions have not been discovered..The applicants have found that swollen cells of Aureobasidium pullulans are responsible for producing pullulan. Furthermore, no-pullulan and high-pullulan mutants from A.pullulans NG were selected successfully. We can obtain a large amount of purified swollen cells by controlling the cell differentiation of A.pullulans. At same time, we have established a new method utilizing Bromothymol Blue to analyze the activity of α-1,4-glucosyltransferase and α-1,6-glucosyltransferase accurately and efficiently. Pullulan productivities of the mutants were related with the activities of two glucosyltransferases. .Base on the previous works, we shall focus our researches on the function of glucosyltransferases in pullulan biosynthesis. After analyzing the functional sector of glucosyltransferases in swollen cell, which is responsible for producing pullulan, we tend to purify the special glucosyltransferases and analyze the N-terminal sequence of these enzymes. According to the protein sequence information, the genes coding the glucosyltransferases could be cloned and identified by reverse genetics strategy. Furthermore, the space-time express characteristics of such genes and its correlation with pullulan productivity will be detected to clarify the biosynthesis mechanisms of special glycosidic bond in pullulan. .It should provide a new approach to the researches in polysaccharide modification in molecular level and pullulan synthesis in vitro.

普鲁兰糖与常见的淀粉、糖原等同为大分子葡聚糖，却具有后两者不具备的多种特性，在食品医药等方面有广泛应用。但普鲁兰糖的合成机理还不清楚，其独特的糖苷键形成机制无人报道，催化其糖苷键形成的特异性葡萄糖基转移酶系还没被发现。申请人的前期研究结果表明普鲁兰糖是出芽短梗霉的膨大细胞合成的。我们诱变筛选到了出芽短梗霉的无糖和高糖突变株，建立了控制出芽短梗霉细胞分化的方法，得到大量纯化的膨大细胞，并且建立了可以准确、高效测定葡萄糖基转移酶活性的溴百里酚蓝法，测定结果表明突变株产糖量与葡萄糖基转移酶活性正相关。本项目希望在前期工作基础上，通过研究糖基转移酶在普鲁兰糖合成中的作用，分析其在细胞中的功能区域，分离纯化相应糖基转移酶并克隆其基因，分析相关基因的时空表达特性及其与普鲁兰糖产量的关系，分析普鲁兰糖糖苷键的形成机制，初步阐明普鲁兰糖合成机理，为体外合成普鲁兰糖和在分子水平上改良其他多糖提供新思路。

密度梯度离心法成功将膨大细胞和酵母状细胞分离，发现酵母状细胞乳白色，膨大细胞呈粉色。发酵液颜色可以作为细胞分化阶段的重要表型特征，对于监控普鲁兰糖发酵具有重要意义。差速离心与密度梯度离心结合成功分离亚细胞器。透射电镜结果显示，膨大细胞内富含一种囊状结构，可能与胞外物质合成与分泌有关。. 通过转录组差异表达基因分析，筛选到5个糖基转移酶基因，转录水平和普鲁兰糖含量正相关。基因comp6663含有GTA型糖基转移酶超家族保守结构域，属于GT-8，可能参与普鲁兰糖的从头合成有关。基因comp5913、comp8271、comp8503和comp11697都属于GTB型糖基转移酶家族，可能参与催化普鲁兰糖合成的延伸步骤。. 研究发现基因comp14856编码蛋白具有锌指结构和YEATS家族结构，二者均有具有转录激活功能，参与细胞分化和代谢调控。YEATS区域可能参与调控黑色素和多糖合成，W245是关键位点之一。锌指结构域参与出芽短梗霉多形性细胞分化，该区域S171L突变导致了分化膨大细胞的能力丧失，普鲁兰糖合成途径同时被关闭。糖基转移酶基因和发育调控基因将成为调控出芽短梗霉合成普鲁兰糖的重要分子靶点。