基于HCSs基因探针的海绵放线菌中新颖AT-less聚酮的发现及活性评价

酰基转移酶缺失聚酮合酶（AT-less PKSs）来源的天然产物结构新颖、成药率高。申请人发现3-羟基-3-甲基戊二酰辅酶A合酶（HCSs）基因可作为AT-less PKSs高效挖掘的探针，并在菌种库和复杂环境微生物组中成功运用，进而从自主构建的海绵放线菌库中挖掘到三株含新颖AT-less PKSs基因簇的菌株；经发酵优化和液质联用分析，发现其中两株菌有望产生新结构AT-less聚酮。本项目拟利用核糖体工程、引入底物广谱性的PPTase或插入强启动子以激活目标菌的AT-less聚酮生合成途径；对其进行规模发酵和质谱示踪高效分离，获得结构新颖的聚酮；运用HR-MS、NMR、单晶X-衍射及化学衍生等方法确定化合物平面及立体结构；系统评价化合物的抗菌、抗肿瘤和抗炎活性；通过基因敲除确认重要活性新化合物的生物合成基因簇。有望发现结构新颖、活性显著的AT-less聚酮，为新药研发提供先导化合物。

Polyketides, biosynthesized by acyltransferase-less polyketide synthases (AT-less PKSs), belong to one of the highest drug-making rates families, which have various structures. In previous research, we designed a new 3-hydroxy-3-methylglutaryl-CoA synthases (HCSs)-based gene probe for efficient mining of AT-less PKSs. The novel HCSs-based gene probe has been successfully used in the bacterial bank and complex environmental microbiome. Based on the gene probe, three strains containing novel AT-less PKSs gene clusters were discovered from the self-constructed sponge-associated actinomycetes library. After fermentation optimization and HPLC-MS analysis, it was found that two strains are expected to produce novel AT-less polyketides. This study intends to activate the AT-less PKSs gene clusters by perform metabolic regulation on target strains, including ribosome engineering, introducing a broad-spectrum substrate PPTase, or inserting a strong promoter; to obtain novel structure AT-less polyketides through large-scale fermentation and mass spectrometry-directed high-efficiency separation; to determine the structures of the isolated compounds by HR-MS and NMR spectroscopic analysis, X-ray single crystal diffraction and chemical modification; to systematically evaluate the antibacterial, antitumor and anti-inflammatory activities of compounds; to identify the biosynthetic gene clusters for important active new compounds through gene knockout. By the study of this proposal, we may discover AT-less polyketides with novel structure and significant activity, which will provide lead compounds for the development of new drugs.