S. avermitilis neau1069全基因组鉴定代谢杀虫剂多拉菌素调控基因及调控机理研究

发现的新微生物S. avermitilis neau1069代谢天然产物杀虫剂多拉菌素和9个新结构化合物，并具有特异的生物合成多拉菌素基因簇和机理。拟利用链霉菌高效转座诱变技术，从源头系统、全面的在全基因组水平上，鉴定出影响多拉菌素生物合成的相关调控基因。并进一步研究影响多拉菌素生物合成最大的2-3个调控基因，明确它们对多拉菌素合成基因簇中基因的转录丰度影响；以及以调控基因编码的蛋白为核心，以DNA-protein相互作用为基础，通过ChIP-seq技术发现调节蛋白结合的靶序列，并从全基因组中找到调节蛋白的靶基因集，在靶基因集中采用凝胶阻滞(EMSA)及DNA足印技术寻找结合的起始位点，明确调节蛋白的调控网络模块，揭示调控基因对多拉菌素生物合成的调控机理。研究为构建多拉菌素生物合成的复杂调控网络奠定基础和数据累积，也为构建多拉菌素高产工程菌，解决多拉菌素生产实际需求问题奠定坚实理论基础。

The novel strain Streptomyces avermitilis neau1069 not only generates natural insecticide doramectin together with nine new secondary metabolites, but exhibits distinctive biosynthetic gene cluster and biosynthetic mechanism associated with doramectin production. The genome-wide identification of regulatory genes involved in doramectin biosynthesis will be conducted by transposon mutagenesis technology. Then, the most important two or three regulatory genes will be selected as targets to investigate their influences on the transcript abundance of genes in the biosynthetic gene cluster of doramectin. On the basis of the inner interaction of DNA-protein, the potent DNA sequences in the genome, which bind to the regulatory proteins will be identified by ChIP-Seq assay. Subsequently, the EMSAs and DNase I footprinting assays will be employed to determine the start binding positions in the DNA targets, leading to clarify the regulatory network of regulatory proteins and elucidate the regulatory mechanism by the regulatory genes in the biosynthesis of doramectin. This research will pave the way for constructing complex regulatory network of doramectin biosynthesis and developing genetically engineered strain with high yield of doramectin to resolve the practical problems encountered in doramectin production.