透明质酸、肝素和硫酸软骨素作为主要的三类糖胺聚糖已广泛应用于医药等领域，然而，由于以上三类糖胺聚糖生物合成与分子量的调控机制尚不清楚，构建微生物细胞合成具有特定生物学功能的特定分子量的糖胺聚糖依然是一个挑战。本项目在前期研究的基础上，通过动态与理性调控三类糖胺聚糖的前体合成途径，结合糖基转移酶的功能域互换与分子改造，明确各糖基转移酶的功能域定位以及解析三类糖胺聚糖的合成分泌以及分子量调控机制；通过分泌表达与优化组装硫酸化修饰系统各组分，构建高效体外硫酸化修饰系统；通过快速进化水蛭透明质酸酶，获得水解肝素和硫酸软骨素的突变体，在此基础上，解析水蛭透明质酸酶和突变体晶体结构以及水解机制。整合以上研究，构建枯草芽孢杆菌微生物细胞工厂发酵葡萄糖等廉价碳源一步合成特定分子量的透明质酸、肝素和硫酸软骨素以及寡聚糖。

Hyaluronan, heparin and chondroitin sulfate as the main three types of glycosaminoglycans (GAG) have been widely used in medicine and other fields, however, due to the unclear regulation mechanisms of biosynthesis and molecular weight, it is still a big challenge to construct microbial cell factories to produce specific molecular weight GAG with desired biological functions. Based on our previous studies, we will identify specific functional domains of the glycosyl transferases and resolve the regulation mechanism of the biosynthesis, secretion and molecular weight through dynamic and rational regulation of the precursors biosynthesis pathways, function domain exchange and molecular engineering of the glycosyl transferases. Then an efficient in vitro sulfating modification system will be established by secretory expression, optimization and assembly of the sulfating modification system components. In addition, mutants that can hydrolyze heparin and chondroitin sulfate will be created by rapidly engineering the leech hyaluronidase (LHAase). On this basis, the crystal structure and hydrolysis mechanism of LHAase and its mutants will be resolved. Finally, we will construct the Bacillus subtilis microbial cell factories to directly produce hyaluronan, heparin, chondroitin sulfate, and its oligosaccharides with specific molecular weights.