多杀菌素是由刺糖多孢菌发酵产生的新型杀虫剂，市场需求巨大。但因缺乏高水平发酵菌种，至今我国尚未掌握该产品的生产技术。构建高产菌株的最大障碍是该菌遗传操作困难，原因包括：甲基化限制修饰导致的DNA转化效率低以及同源重组能力弱。解决或规避以上问题是建立高效遗传操作体系的关键所在。本项目拟以DNA甲基化限制修饰缺陷的大肠杆菌作为研究平台，鉴定刺糖多孢菌的甲基化修饰系统，基于合成生物学理念，在接合转移供体菌中重构甲基化修饰系统，减弱或消除对外源DNA的限制修饰作用；以游离型质粒为载体，筛选低毒性Cas9，开发不依赖于同源重组修复的基因组编辑技术，包括基于NHEJ的CRISPR/Cas9基因组编辑技术，dCas9或nCas9介导的碱基编辑器，实现DNA的高效缺失或失活，为功能基因的筛选以及基因组改造提供高效的操作工具。预期成果将极大促进该菌功能基因组研究以及基于合成生物学策略的高产菌分子育种研究。

Spinosad is a novel insecticide produced by the fermentation of Saccharopolyspora spinosa, which has a huge market demand. However, due to the lack of high-level fermentation strains, China has not yet mastered the production technology of this product. The biggest obstacle for the construction of a high-yield strain is the difficulty in genetic manipulation of S. spinosa. The main reasons include the low efficiency of DNA transformation caused by methylation restriction-modification (R-M) systems and the weak ability of DNA homologous recombination. To solve or avoid the above problems is the key to establish an efficient genetic manipulation system. This project aims to identify the methylation modification system of S. spinosa by using E. coli with the defective of DNA restriction-modification system as research platform. Based on the concept of synthetic biology, the methylation modification system will be reconstructed in the donor E. coli strain (used in conjugal transfer) to weaken or eliminate the restriction modification of exogenous DNA. Using free plasmids as carriers, low toxicity Cas9 will be screened. Genome editing techniques independent of homologous recombination-based repair will be developed, including NHEJ-based CRISPR/Cas9 genome editing and dCas9 or nCas9-mediated base editors, and could achieve efficient DNA deletion or inactivation, which will provide efficient manipulation tools for functional gene screening as well genome modification. The expected results will greatly promote the functional genome research and the synthetic biology based-molecular breeding of high-yield strains.