全基因组鉴定Streptomyces microflavus neau3调控杀虫抗生素尼莫克丁生物合成相关基因及调控机理研究

以具有自主知识产权、产农用杀虫抗生素尼莫克丁S. microflavus neau3为研究对象。完成菌株全基因组测序；并采用链霉菌高效转座技术饱和诱变菌株，从突变菌株库中筛选影响尼莫克丁生物合成的表型突变体，测定突变体的突变基因序列，全基因组鉴定调控尼莫克丁生物合成相关基因；对发现影响尼莫克丁生物合成重要新基因，通过体内基因敲除与回补，明确重要基因的调节作用；然后以对调控尼莫克丁生物合成的重要基因编码的调节蛋白为研究核心，采取染色质免疫沉淀与新一代高通量测序联用技术，基于体内DNA-蛋白互作，钓取调节蛋白的DNA结合靶点，从全基因组范围找到它们的靶基因集，明确调节蛋白的调控网络模块。在基因互作水平上，揭示新的调控蛋白基因调控尼莫克丁生物合成的机理。为研究建立尼莫克丁生物合成的复杂调控网络奠定实验方法和积累实验数据，也为构建尼莫克丁高产、优良工程菌，解决尼莫克丁生产实际需求问题奠定重要基础。

Streptomyces microflavus neau3, nemadectin producer with independent intellectual property rights, will be used as the research object. First, the genomic sequence of this strain will be completed. Second, the mutant library of S. microflavus neau3 will constructed based on the transposon mutagenesis technology; and the phenotypic mutants with the impact on the biosynthesis of nemadectin will be screened and the mutant genes from these mutants will be sequenced, then compared with the wildtype to identify genes for regulating nemadectin biosynthesis. Third, the in vivo gene knockout and complementation technology will be employed to decipher the function of regulatory genes. After that the chromatin immunoprecipitation followed by high-throughput sequencing will be applied to study the regulatory proteins, to identify the binding sites and target gene collection at a genome scale and reveal the regulatory network based on protein-DNA interactions. Therefore, from the view of gene interactions, the mechanism of nemadectin biosynthesis directed by regulatory proteins will be demonstrated. This finding will lay important foundation for constructing excellent engineering stains with high yield nemadectin and solving practical problems in nemadectin production.