新型信号分子调控米尔贝霉素生物合成机制研究

冰城链霉菌是拥有自主知识产权，我国工业化生产环境友好杀虫剂米尔贝霉素和米尔贝肟的工业菌。前期证实一复杂结构黄色化合物为冰城链霉菌的信号分子，能够结合受体蛋白转录因子KelR4，影响米尔贝霉素的生物合成。为了认知黄色化合物作为信号分子调控米尔贝霉素生物合成机制，拟结晶或酶解、水解复杂结构黄色化合物，X-单晶衍射，核磁、质谱等解析黄色化合物的分子结构；免疫共沉淀、转录组测序、凝胶阻滞、足印及点突变寻找并确定KelR4调控的靶基因和它们结合的区域，并通过体内基因敲除与回补明确靶基因的功能；再经EMSA、点突变及微量热泳动明确KelR4与化合物结合的功能域，以及黄色化合物对KelR4结合不同的靶DNA的影响；体内组合敲除、过表达黄色化合物生物合成的关键基因和（或）kelR4基因和（或）靶基因对米尔贝霉素发酵单位的影响。理解新的信号分子调控机制，为针对新的调控机制进行高产优良工程菌的改造奠定基础。

Streptomyces bingchenggensis is an industrial strain with independent intellectual property rights, which produce the eco-friendly pesticides milbemycin and milbemycin oxime. Previous studies have confirmed that a yellow compound with complex and new structure is a signaling molecule in Streptomyces bingchenggensis, which can bind to transcription factor KelR4 and affect milbemycins biosynthesis. In order to reveal the mechanism of the yellow compound as a signaling molecule to regulate milbemycin biosynthesis, the yellow compound will be subjected to crystallization, enzymatic hydrolysis or hydrolysis, and its molecular structure will be determined by single crystal X-ray diffraction, nuclear magnetic resonance and mass spectrometry. Then the target genes and the binding motif of KelR4 will be found and determined by immunoprecipitation, transcriptome sequencing, electrophoretic mobility shift assay, footprinting and point mutation. Subsequently, the regulatory function of the target genes on the production of milbemycin will be studied by gene disruption and genetic complementation in Streptomyces bingchenggensis. Furthermore, the effects of the yellow compound on the DNA targets affinity of KelR4 and the interactions between KelR4 and the yellow compound for regulating milbemycin biosynthesis will be analyzed by electrophoretic mobility shift assay, point mutation and microscale thermophoresis. Finally, the effect on milbemycin fermentation units will be determined by deletion and overexpression of key genes for the biosynthesis of yellow compounds and/or kelR4 gene and/or the target genes of KelR4 in Streptomyces bingchenggensis. The project is aimed to understand the regulatory mechanism of a new signaling molecule and provide theoretical basis for engineering high-yield strains according to the regulatory mechanism.