鞘氨醇单胞菌 NXdG微氧高效合成胞外多糖的机制及其应用研究

Microbial polysaccharides are a class of macromolecular polymers formed by the polymerization of monosaccharides or derivatives linked with glycosidic bonds. Sufficient oxygen is a prerequisite for the efficient synthesis of polysaccharides. However, as the viscosity of the fermentation broth increases, the concentration of dissolved oxygen rapidly decreases during the fermentation process, thereby a micro-oxygen environment was formed. This situation greatly reduces the activity of the strain and the production of the polysaccharide. Sphingomonas sanxanigenens NXdG is a strain that efficiently synthesizes exopolysaccharides under microaerobic conditions, providing a balance between low dissolved oxygen concentration and high energy consumption. In this project, the synthesis mechanism of NXdG under micro-oxygen condition was elucidated through the comparative analysis of genome and transcriptome and molecular genetic manipulation. In addition, an engineered strain producing xanthan was constructed that can utilize industrial waste under microaerobic conditions. This project is a theoretical study based on the current status of polysaccharide industry, and finally applies to the polysaccharide fermentation process, which will promote the development of microbial polysaccharide industry.

微生物多糖是一类由单糖或衍生物经过糖苷键聚合形成的高分子聚合物。充足的氧气是有效合成多糖并获得高产率的前提条件。然而，在多糖发酵过程中随着发酵液粘度的增加溶氧量迅速降低，由此形成的微氧环境极大的限制了菌株的活性和多糖的生产。Sphingomonas sanxanigenens NXdG是一株可在微氧条件下高效合成胞外多糖的菌株，为实现多糖发酵后期溶氧低和能量需求高之间的平衡提供了一个研究实例。本项目拟从基因组、转录组、分子遗传操作方面阐明菌株NXdG微氧高效合成多糖的机制，绘制微氧条件下菌株的能量代谢途径；并以NXdG为多糖合成底盘菌株，引入黄原胶合成基因模块，构建一株可在微氧条件下高值转化工业废弃物合成黄原胶的基因工程菌株。该项目是由多糖发酵现状激发的理论研究，并最终落实至多糖生产中，可为微生物多糖的低电能能耗、高产量发酵提供理论基础。

微生物胞外多糖是一类性能优越、应用广泛的生物技术产品。充足的氧气是多糖发酵的必备条件，但在好氧发酵过程中随着发酵液粘度增加，溶氧量降低，显著影响了菌株的活性和多糖产量。本项目以一株可在微氧条件下高效合成多糖的自发突变株Sphingomonas sanxanigenens NXdG为研究对象解析了其微氧产糖的机制，并以该菌株为底盘构建了产糖工程菌株。结果显示，NXdG合成多糖是原始多糖Ss的亚型多糖，命名为Hesan，其酰基含量较低，具有更高的弹性和更低的构象转变温度；微氧产糖机理与多糖输出蛋白Wzc的C末端关键ATP-binding Walker-A motif 结构域点突变有关，使多糖中单糖在聚合输出过程中不消耗或者低消耗ATP，极大的降低了菌株的产糖能耗，从而使其在微氧、即低能耗情况下合成胞外多糖；最后，基于NXdG菌株构建了一株可利用秸秆等农业废弃物合成黄原胶的工程菌株，并通过启动子工程进行了系统优化，最终将NXdPE(P916gum)的黄原胶产量提高至14.26 ± 0.80 g/kg。本项目为微生物多糖发酵提供了新的思路并从菌株水平上丰富了微生物多糖的种类；为Wzy依赖型多糖提供了一种新型的低能耗合成靶点，为多糖聚合输出蛋白复合体结构及功能的深入研究提供了实验基础；同时，本项目为农业资源变“废”为“宝”提供研究基础，有助于农作物秸秆的高值转化，同时有利于进一步推动我国多糖产业的发展。