多杀菌素(I型聚酮)是由刺糖多孢菌产生的一种高效、低毒的“绿色”杀虫剂，具有极大的开发价值。经过多年对原始产生菌的诱变和遗传改造，虽然有很大幅度的提升但仍无法接近国外的水平，至今没有实现国产化。本项目拟首先建立刺糖多孢菌的BAC文库，利用BAC质粒初步实现多杀菌素在链霉菌中的异源合成；然后建立链霉菌代谢组学分析方法，通过定量代谢组学分析，辅之于蛋白质组分析，在代谢物和蛋白水平系统考察并确认制约多杀菌素合成的节点，通过相应的代谢工程改造提高多杀菌素的异源表达水平。而后针对多杀菌素各合成模块的特征，利用链霉菌启动子库实现各合成模块的优化和组装，促进多杀菌素的高产；这样的循环可以往复进行，从而循序渐进提高多杀菌素的产量。在此过程中运用发酵工程加以配合，充分对突变株的结果进行放大。本研究的顺利实施可为多杀菌素的异源高效合成奠定基础，并且相关的工具和研究策略可应用于其他微生物药物的高产。

Spinosad, produced by Saccharopolyspora spinosa, is an important type-I PKS-derived macrolide antibiotic with potent insecticidal properties. As a high efficiency, safety and environment-friendly pesticide, spinosad has been widely used in agriculture. However, due to the low productivity, the industrial production of spinosad is quite limited in China. In addition, although many previous works have been done for spinosad overproduction, the problem is still challenging. In this project, we firstly plan to construct a bacterial artificial chromosome (BAC) library of Saccharopolyspora spinosa. And the spinosad will be attempted to biosynthesize in Streptomyces host by using BAC plasmid that contains the whole spinosad biosynthetic gene cluster. Secondly, the metabolomics approach will be developed in Streptomyces spp. To reveal the rate-limiting steps involved in spinosad heterologous biosynthesis, the metabolomics approach (accompany with proteomics analysis) will be applied. And the metabolic engineering work can be carried out to promote the heterologous production of spinosad in Streptomyces. Lastly, the biosynthetic modules of spinosad will be optimized by in vitro reconstitution, and subsequently assembled by using a Streptomyces promoter library. These optimized biosynthetic modules will be introduced into the chromosome of Streptomyces host to further enhance the production of spinosad. Followed by several rounds of metabolomics-guided metabolic engineering, the production of spinosad will be gradually increased in Streptomyces. The successful of this project will lay the foundation for high-efficient biosynthesis of spinosad in Streptomyces, and the strategy and tools developed in this project may also be applied to dramatically improve the production of other antibiotics.