产黑色素短梗霉P16菌株普鲁蓝多糖合成途径的研究

Pullulan is an extracellular, regularly repeating, linear and biological macromolecule primarily consisting of maltotriose repeating units interconnected by alfa-1,4 glucosidic bond whereas consecutive maltotriose units are connected to each other by alfa-1,6 glycosidic linkages. Although this basic structure of pullulan was resolved in 1960 and the commercial production of pullulan began in 1976 by the Hayashibara Company in Japan, its biosynthesis pathway, relevant enzymes and genetic mechanisms behind pullulan production still remain completely unclear to date. In thie study, the genes related to the glucosyltrasferases and pullulan synthetases in Aureobasidium melanogenum P16 strain which is a high pullulan producing yeast will be continued to be knocked out and relationship between the disruptants and pullulan synthesis will be confirmed. All the genes encoding the glucosyltrasferases and pullulan synthetases will be heterologously overexpressed in Yarrowia lipolytica and Pichia pastoris or homologously overexpressed in A. melanogenum P16 and all the recombinant enzymes will be purified and characterized. Pullulan will be synthesized in vitro and the synthesized pullulan will be purified and the structure of the purified pullulan will be elucidated and the linkage between glucosyl residues in pullulan molecules will be identified. The whole biosynthesis pathway of pullulan in A. melanogenum P16 will be suggested，the related enzyme, their functions in pullulan biosynthesis and their genes will be identified. At the same time, interreaction between the different enzymes in vitro and in vivo will be studied and the possible complex of the enzymes will be suggested. In order to show that there are intereactions between different enzymes, two or three genes responsible for the pullulan biosynthesis in A. melanogenum P16 will be deleted and complemented and relationship between pullulan biosynthesis and deletion as well as complementation will be investigated. This study is of significant in elucidation of pullulan biosynthesis pathway in yeasts and further genetic modification for enhanced pullulan production.

普鲁蓝多糖是一种线性葡聚糖，由重复的麦芽三糖（由葡萄糖通过α-1,4糖苷键连接而成）单位经a-1,6糖苷键连接而成。 这一化学结构在上个世纪60年代就已经清楚，并在1976年就已经进行规模化商业生产。但是至今普鲁蓝多糖的生物合成途径、有关的酶及其基因还不清楚，尤其涉及的葡萄糖转移酶及其基因更是不清楚。 本申请项目就是继续敲除P16菌株中其他可能的葡萄糖转移酶和普鲁蓝多糖合成酶基因，研究敲除菌株与普鲁蓝多糖合成的关系；异源或同源表达所有有关的葡萄糖转移酶和普鲁蓝多糖合成酶基因，纯化葡萄糖转移酶和普鲁蓝多糖合成酶，在体外观察纯化的酶是否存在相互作用，利用纯化的酶和酶复合物在体外合成多糖，纯化体外合成的多糖，分析多糖的组成和化学结构;为了证实这些基因表达的酶在P16菌株中是否存在相互作用， 通过双基因、多基因敲除或超表达进行验证。该研究对于解析酵母菌胞外多糖合成途径具有重要的理论和实际意义。

发现来自红树林的类酵母Aureobasidium melanogenum P16菌株可以转化蔗糖产67g/l以上的普鲁蓝多糖，而从蜂蜜中分离的耐高渗类酵母菌TN3-1菌株可以转化葡萄糖产110g/l以上的普鲁蓝多糖合成。研究了P16菌株多个葡萄糖转移酶在普鲁兰多糖引物和前体合成中的作用，发现关键的多结构域合成酶AmAgs2在催化普鲁兰多糖前体物合成普鲁兰多糖的作用，并首次提出了完整的全新的普鲁兰多糖合成途径，发现多个信号通路如cAMP-PKA, HOG1, Snf1-Mig1, Nitrogen信号通路在普鲁兰多糖合成调控中的作用。发现α-淀粉酶、葡萄糖淀粉酶和异普鲁兰酶对普鲁蓝多糖分子量影响，敲除这些酶的基因可以制备高分子量普鲁兰多糖，便于制备药物胶囊和化妆品材料。为通过合成生物学方法提高胞外多糖合成量和研究胞外多糖的生物学功能提高重要的理论和技术基础。