氨基环醇类阿卡波糖是重要的2型糖尿病治疗药物。游动放线菌主要合成阿卡波糖，但也产生多种类似物和分支代谢物。由于缺乏遗传操作手段，以及其生物合成中间体多被磷酸化，使得阿卡波糖的生物合成机制研究仅停留在有限的几步。本项目将基于高效遗传操作体系、所定位的关键合成基因、功能基因组数据，并借鉴氨基环醇类井冈霉素生物合成体系，系统解析阿卡波糖的生物合成机制和相关的遗传调控机理。首先，通过体内基因敲除与产物检测、酶的异源表达与催化、完整催化系统的体外重构等，揭示生物合成酶的催化机制与顺序，阐明多种副产物的形成机制；其次，通过遗传操作和酶活性中心改造，一方面降低或消除副产物,提高阿卡波糖产量，另一方面积累活性优良的衍生物；最后，利用转录组、DNA亲和层析等，解析渗透压调控等阿卡波糖生物合成的调控机制。研究成果将推动氨基环醇类天然产物的生物合成机制研究，提高阿卡波糖的生产水平，并提供多个性能提升的衍生物。

The C7N aminocyclitol acarbose is very effective for the treatment of type 2 diabetes. While acarbose is the main product in its producer, Actinoplanes sp., there are many other by-products, including acarbose derivatives and shunt products. Due to the lack of efficient genetic manipulation and the presence of phosphorylated intermediates in the biosynthetic pathway, the study on the mechanism(s) of acarbose biosynthesis has been limited to just a few steps. In this proposal, the mechanisms of acarbose biosynthesis and the related genetic regulation will be systematically investigated, aided by a recently established efficient genetic manipulation system, a set of genetically confirmed biosynthetic genes, an accurate genome sequences, and enzymes/intermediates borrowed from aminocyclitol validamycin biosynthetic pathway. Based on gene inactivation, enzyme heterologous expression and catalysis and in vitro reconstitution of the whole pathway, the catalytic mechanism of each biosynthetic enzyme, the order of all reactions, and the routes to the accumulation of those by-products will be elucidated. Subsequent gene manipulation and rational enzymes engineering will be performed either to eliminate or reduce the accumulation of by-products, or to specifically accumulated derivatives with improved activities. In addition, the regulatory mechanism, esp. the osmosis related one, will be studied with transcriptome, DNA affinity capture of regulatory proteins, and so on. This project will strengthen the biosynthetic studies on the family of aminocyclitol natural products, exemplified by acarbose and validamycin, improve the industrial production of acarbose, and provide lead compounds with improved activities for the treatment of diabetes.