糖转运蛋白在普鲁兰高效合成中的调控作用及生理机制

Pullulan, a kind of polysaccharide produced by Aureobasidium pullulans, has wide applications in the fields of food and medicine. The biosynthesis of pullulan usually needs a long cycle of cultivation during fermentation. The efficiency of pullulan biosynthesis can be improved by applying numerous approaches of process optimization and mechanism regulation, but the increase is limited. To solve this problem, we try to improve the rate of pullulan biosynthesis by manipulating the endogenous sugar transmembrane protein system for specific transformation of the precursors of pullulan (such as panose and isopanose). In this project, Aureobasidium pullulans CCTCC M 2012259, which can produce pullulan without melanin formation, is used as the starting strain. Based on the analysis of whole genome sequencing of the strain and the physiological character of panose/isopanose transporters involved in pullulan biosynthesis, mutants with significantly higher pullulan formation rate will be constructed by using genetic modification methods. The relationship between the manipulation of panose/isopanose transporters and physiological characteristics such as cell growth, cell membrane components, key enzyme activities, substance and energy metabolism within cells of mutants and the starting strain, will be illuminated through transcriptomics and proteomics approaches as well as bioinformatics analysis. The results of this project not only help to understand the role of sugar transporters in the regulation of efficient pullulan biosynthesis and its physiological mechanism, but also provide novel ideas for strain improvement and efficient production of other analogous microbial polysaccharides.

普鲁兰是一种在食品和医药等领域具有广阔应用前景的微生物胞外多糖。普鲁兰生物合成的周期通常较长，采用外源性的优化方法和调控手段可以提高普鲁兰的合成效率，但提升的幅度有限。为此，我们拟对普鲁兰合成前体物质（如潘糖和异潘糖）的内源特异性糖跨膜转运蛋白系统进行改造，以此提高普鲁兰的合成效率。本项目以一株不产黑色素的出芽短梗霉为研究对象，在全基因组测序结果分析的基础上，结合普鲁兰生物合成中潘糖/异潘糖特异性转运蛋白的生理特性，对出发菌株进行遗传改造，构建普鲁兰合成速率显著提高的突变株。借助于组学技术和生物信息学分析手段，解析潘糖/异潘糖特异性转运蛋白系统改造对细胞生长、细胞膜组分、关键酶活性、物质和能量代谢等方面的影响规律。研究结果不仅有助于理解糖转运蛋白在普鲁兰高效合成中的调控作用及生理机制，同时也为其他微生物多糖的高效合成及其菌株改良提供新的思路。

普鲁兰是一种由麦芽三糖重复单位聚合而成的直链高分子同型微生物胞外多糖，已广泛应用于食品加工、生物医药和化妆品等领域。微生物法合成普鲁兰的周期较长，导致普鲁兰合成效率不高。为此，本项目在对出芽短梗霉全基因组测序结果进行分析的基础上，预测得到与普鲁兰分泌有关的糖转运蛋白编码基因。采用基因工程技术，对出发菌株进行遗传改造，构建糖转运蛋白编码基因敲除/过表达的突变株。通过对普鲁兰分批发酵过程动力学、关键酶活性和表达水平、物质和能量代谢等参数进行比较分析，从细胞、分子和生理等多个维度解析了糖转运蛋白在普鲁兰高效合成中的调控作用及其促进普鲁兰分泌的生理机制。主要研究内容包括：（1）出芽短梗霉糖转运蛋白编码基因的预测、克隆及敲除；（2）糖转运蛋白基因mal31在普鲁兰生物合成中的作用及生理机制；（3）普鲁兰生物合成中底物的作用及其生理机制；（4）氯化钠影响普鲁兰生物合成的影响及机制；（5）硫酸锌在普鲁兰和β-葡聚糖生物合成中的作用及机制解析；（6）表面活性剂提高生物转化合成普鲁兰的效率；（7）Triton X-100提高普鲁兰合成效率及其作用机制；（8）基于廉价底物优化的普鲁兰生物合成。研究结果不仅有助于理解糖转运蛋白在普鲁兰高效合成中的调控作用，同时也为其他微生物多糖的高效合成及其菌株改良提供新的思路。