毛韧革菌中基于基因组信息的次生代谢产物挖掘

The metabolites of Stereum hirsutum FP-91666 possesses important drug development value. Genome sequencing suggested that there were a large number of potential secondary metabolites gene clusters in the genome. In previous works, some sample polyketides and sterols were obtained from S. hirsutum FP-9166 and then a new type hybrid of sesquiterpene and amino acid were achieved after optimization of culture conditions, but more complex natural products predicted by genomic analysis had not been found. In this project, we will mine new natural products based on the method of genome analysis: Inducing chemical molecules and optimizing culture conditions will be developed for mining secondary metabolites from S. hirsutum FP-9166; The mutant strains were constructed by disrupted and enhanced by the potential global regulatory (saraC) gene, and were isolated and purified the metabolites guiding by LC-MS; Based on the analysis of genome, 1-2 complete gene clusters were cloned and expressed, and isolated the metabolites. The systemic investigation in S. hirsutum FP-91666 would be discovered its secondary metabolites and more biosynthetic elements, which would provide the theoretical foundation and resource for the development of novel microbial drugs using synthetic biologic techniques.

毛韧革菌代谢产物具有重要的药物开发价值，目前已测序的菌株(Stereum hirsutum FP-91666)表明该菌含有丰富的次生代谢产物生物合成基因簇。与该菌发表的化合相比，还有大量的基因资源调控产生的化合物有待发掘，如申请人前期从该菌株中获得聚酮和甾醇类化合物，进一步优化发酵条件后，获得了结构新颖的倍半萜和氨基酸的杂合物菌。本项目拟基于基因组的策略，利用化学小分子诱导、培养基筛选等技术挖掘毛韧革菌的代谢产物；通过对潜在的全局调控子（saraC）基因的中断、增强表达，并以LC-MS导向，分离纯化其突变株新产生的化合物；基于信息生物学的分析，选择1-2个完整的基因簇，通过克隆、表达载体构建以及异源表达，来分离鉴定产生的化合物。通过系统的对毛韧革菌深入研究，挖掘天然产物及其生物合成基因簇资源，发现该菌的潜在的全局调控子，为利用合成生物学技术进行创新微生物先导化合物的发现提供理论基础和资源。

基于前期研究结果和基因组信息分析，对毛韧革菌培养条件筛选，发现在YMG和WGB液体中化合物更加丰富，从这两种培养条件下共分离鉴定52个化合物，其中16个新化合物，部分化合物具有一定的抗细菌活性。从毛韧革菌的基因组信息中筛选出一个具全局转录调控功能的基因SaraC，通过农杆菌介导的遗传操作体系，构建了两个过表达SaraC基因的转化子（SA4和SA6），与野生型对比，通过对全基因甲基化组、转录组和代谢组的数据分析，确定SaraC是一个具有DNA去甲基化功能的全局转录调控因子；对增强表达SaraC的SA6转化子放大发酵，获得两个结构新颖的杂合类化合物。选择PKS生物合成1.2基因簇为目标，构建完整生物合成基因簇的异源表达载体，导入构巢曲霉获得异源表达菌株，在液体YMG培养基的发酵产物中检测到了对照组中并未出现的三个新分子离子峰，表明PKS生物合成1.2基因簇成功的在构巢曲霉中表达。