重离子辐照诱变选育高产米卡芬净前体FR901379棘腔孢霉突变体及其分子机制研究

米卡芬净是一种重要的抗真菌药物，其前体FR901379由丝状真菌棘腔孢霉发酵生产，优质的FR901379生产菌株是米卡芬净生产工艺的核心竞争力。影响棘腔孢霉发酵的因素很多，前期工作显示菌株的理性改造缺少有理论支持的可靠靶点，诱变育种更具可操作性，而重离子辐照是一种非常有效的诱变方法。丝状真菌主要依靠非同源重组末端连接途径（NHEJ）修复DNA双链断裂损伤，其重离子辐照损伤修复会具有特殊性，研究较少。本项目将对产FR901379棘腔孢霉及其NHEJ缺失菌株分别进行重离子辐照，通过重测序分析NHEJ缺失对产生突变的影响，研究丝状真菌重离子损伤修复机制，评估增加NHEJ缺失菌株进行辐照对提高突变库多样性的作用。然后利用建立的高通量筛选方法对突变库进行筛选，选育高产FR901379的突变菌株，通过突变位点-生产性状关联性分析和棘腔孢霉体内验证，解析高产的分子机制，基于此进一步构建更高效的工程菌株。

Micafungin is an important antifungal drug. Its precursor FR901379 is industrial produced through the fermentation of filamentous fungus Coleophoma empetri. And excellent producing strain of FR901379 is the key to achieve high-efficiency production of micafungin. There are many factors that affect the fermentation of C. empetri. Our previous works showed that there no reliable target genes with theoretical supports for the rational genetic engineering of the C. empetri strain, and mutation breeding is more operable. And heavy ion irradiation is a very effective mutation breeding method. In this project, the wild-type and NHEJ-deficient strain of FR901379-producing C. empetri strain will be irradiated with heavy ions respectively. The effect of NHEJ deletion on mutations induced by irradiation will be statistically analyzed based on genome-resequencing of the mutants, which would provide more theoretical support for the application of heavy ion irradiation in filamentous fungi. And then, the mutants with excellent performance will be selected from the mutant libraries through the high-throughput screening method. In addition, we will exposit the mechanism of FR901379 biosynthesis by genome wide association study and in vivo verification, which would provide theoretical guidance for further construction of more efficient engineered strains.