基于代谢组学挖掘黄色粘球菌亲缘识别过程中诱导的次生代谢产物

Myxobacteria are a fertile source of novel natural products discovery. Genome sequencing shows numerous gene clusters specifying secondary metabolites remain unexpressed under laboratory conditions. The bottlenecks in the myxobacterial natural products research are to efficiently activate the silent gene clusters, and to straightforward identify the secondary metabolites of interest. The myxobacterial “kin-discrimination” refers to that cells from the same species distinguish between close genetic kin and non-kin. During the investigation of the kin-discrimination of the model myxobacterium Myxococcus xanthus DK1622, we obtained an immune protein-null strain ΔMXAN_0049 that forms a colony-merger-incompatible boundary with the wild type strain DK1622. The following proteomics study confirmed that a number of silent gene clusters are activated within the DK1622-ΔMXAN_0049 boundary. In this study, we propose to use metabolomics tool to facilitate the identification of the elicited compounds in the boundary, followed by antibacterial test, cytotoxicity assay, and effect on the growth of M. xanthus by the isolated compounds, which will provide new insights into understanding the myxobacterial “kin-discrimination” from the perspective of small molecules. To the best of our knowledge, the methodology of “activating silent biosynthetic gene clusters by kin-discrimination” are hitherto unreported in the field of microbial natural products, which is expected to have profound impact on mining the biosynthetic potential of the largest collection of myxobacteria in China.

现代基因组测序表明粘细菌产新颖化合物的潜能巨大，而目前研究的主要难点是如何激活沉默基因簇和快速靶向鉴定代谢产物。粘细菌的“亲缘识别”是指同类细胞的社会性合作和非同类细胞的竞争抑制。前期对模式黄色粘球菌的亲缘识别现象的研究，发现野生株DK1622与其免疫蛋白突变株ΔMXAN_0049在共培养时可产生菌落融合不兼容的分界线。随后蛋白质组学工作表明交界线处有沉默基因簇被激活，但诱导的具体化学成分及生物学功能尚不清楚。本项目：（1）拟采用代谢组学方法快速鉴定在DK1622-ΔMXAN_0049菌落界线中诱导的化合物；（2）测试所诱导化合物的抗菌、细胞毒活性、及对黄色粘球菌自身的影响；从而以小分子代谢水平的视角加深理解黄色粘球菌“亲缘识别”这一生物学现象。本项目利用“亲缘识别诱导沉默基因簇表达”在微生物天然产物研究领域未见有报导，对今后深入挖掘我国最大的粘细菌资源库的新药潜力具有重要意义。

黏细菌是活性先导化合物的重要来源。本项目通过整合基因组学和代谢组学，高效挖掘大型黏细菌资源库中的新药潜力。具体来讲，利用多种手段，包括亲缘识别、更换培养基、强启动子置换等诱导黏细菌中沉默基因簇的表达，之后利用代谢组学工具靶向分离鉴定所诱导的新化合物，从而加速发现了以美沙达唑、原囊菌酯、原囊菌素等为代表的10余个新骨架化合物。美沙达唑在斑马鱼模型中具有良好的心血管系统活性，原囊菌酯具有很好的抗病毒活性。我们解析了部分新化合物的生物合成途径和关键酶学机制，其中，美沙达唑为首例由维生素B12代谢途径与PKS/NRPS装配线杂化发生成的天然产物，革新了人们对微生物生物合成的认知，为通过合成生物学手段“智造”化学多样性打开一扇新的大门。此外，我们构建了全球范围内第一个开源的黏细菌天然产物数据MyxoDB（https://www.myxonpdb.sdu.edu.cn），对提升代谢组学挖掘黏细菌天然产物“暗物质”的效率，具有十分重要的意义。

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