冬虫夏草相关真菌生物合成基因簇的发掘及功能解析

Various fungal species have been reported to be associated with natural Ophiocordyceps sinensis microenvironment, which are the important resources of biological medicines, and are the real producer of active substances from Ophiocordyceps sinensis.Type I polyketide synthase and nonribosomal peptide synthetase ( NRPS ) are the key enzymes in the biosynthesis of natural products. In this study, we select the Cordyceps-colonizing fungi with important physiological and ecological effect as material, and obtain gene fragments of Type I polyketide synthase and nonribosomal peptide synthetase ( NRPS ) using degenerate PCR or genomic analysis. Fosmid library were constructed from the genomic DNA of the fungal species, and screening with homolog probe to obtain Type I polyketide synthase and nonribosomal peptide synthetase ( NRPS ) gene cluster. The sequence analysis, phylogenetic analyses, prediction of substrate specificity analysis of biosynthesis gene cluster were performed. To illuminate the functions of PKS and NRPS, we select Hirsutella sinensis and Paecilomyces hepiali as model fungus strain, by using gene deletion technology. These results would discover the biosynthesis gene cluster from special habitat,analysis the evolution and transfer of biosynthesis gene clusters in microenvironment, and provide new insight into the biosynthesis of secondary metabolism and mechanism of regulation in fungal species associated with Ophiocordyceps sinensis.

冬虫夏草微生物区系中存在许多种类的真菌，是一类非常重要的生物医药资源，也可能是冬虫夏草活性物质的真正生产者。Ⅰ型聚酮合酶（PKS）和非核糖体多肽合成酶（NRPS）是真菌次生代谢生物合成的关键酶。本项目以前期筛选的具重要生理与生态效应的冬虫夏草定殖真菌为材料，已完成全基因组测序的菌株，采用基因组挖掘技术获得生物合成基因簇，尚未进行全基因组测序菌株，通过简并引物PCR扩增，获得PKS和NRPS的部分基因序列；构建Fosmid文库，同源探针杂交获得PKS和NRPS的完整基因簇；进行结构域功能预测和系统进化分析；以中国被毛孢和蝙蝠蛾拟青霉为模式菌株，利用基因敲除技术研究PKS和NRPS功能。本项目实施，可发掘特殊生境真菌类群的生物合成基因资源，分析冬虫夏草微生物区系中生物合成基因簇的进化与转移，并初步解析其功能，为阐明冬虫夏草相关真菌次生代谢生物合成及调控机理提供理论依据。

冬虫夏草微生态系统中存在许多种类的真菌，是一类非常重要的生物医药资源，也可能是冬虫夏草活性物质的真正生产者。Ⅰ型聚酮合酶（PKS）和非核糖体多肽合成酶（NRPS）是真菌次生代谢生物合成的关键酶。本项目以高通量测序与培养组学技术系统比较分析了冬虫夏草菌膜和周围土壤的微生物多样性，高通量测序鉴定出真菌15个纲、41个目、79个科、112个属，培养组学方法，鉴定出真菌33个属，2种方法互补且有较大差异。结果表明，高通量测序和培养组学分离技术相结合可高效揭示冬虫夏草微生态系统中真菌多样性；特异扩增冬虫夏草微生态系统中可培养真菌PKS的KS区域和NRPS的A区域，系统分析PKS和NRPS的进化和转移规律，初步揭示了冬虫夏草相关真菌的次生代谢合成潜力；以转录组学和LC-MC分析中国被毛孢与蝙蝠蛾拟青霉在不同培养策略下，次生代谢生物合成基因表达情况及代谢物图谱的变化，以初步解析PKS和NRPS功能；共培养策略激活药用真菌沉默的生物合成基因簇，以发现新化合物；冬虫夏草相关真菌发酵中药材，形成复合菌剂，发现对2型糖尿病小鼠模型有很好降糖作用，能有效回复2型糖尿病小鼠失衡的肠道微生物多样性。