基于隐性基因簇激活策略定向发现深海放线菌中新型抗多重耐药菌先导化合物

The never-ending emergency of multi-drug-resistant (MDR) pathogens makes exploration of novel antibiotics even more urgent than ever. The large scale microbial genome sequencing has revealed the presence of lots of cryptic secondary metabolites biosynthetic gene clusters, providing brand new viewpoint and unprecedented opportunities for drug discovery. Deepsea microorganisms are endowed with unique genes and metabolic pathways during evolution in the complex and challenging living conditions, and thus have become the emerging strategic bioresource all over the world. In this project, we are going to explore novel cryptic PKS/NRPS biosynthetic gene clusters in the deepsea Actinomycete strains, whose genomes harbor many novel cryptic gene clusters. The expression of the targeted gene clusters will be activated by using various genome mining strategies; comparative metabolite profiling will be performed to rapidly lock the activated novel compounds; after structural identification by using a combination of 1- and 2-D NMR experiments, their anti-MDR bacteria activities will be extensively evaluated; thus the relationship of “gene cluster—compound—activity” will be established. In addition, rational genetic engineering will be carried out to obtain new analogues of the bioactive compounds. Furthermore, the compounds with novel structure and great in vitro anti-MDR bacteria activities will be subjected to in vivo test using mouse model. Implementation of this project will lead to discovery of novel anti-MDR lead compounds, which would have profound values for the development of innovative anti-MDR drugs with independent intellectual property; simultaneously, it will provide important theoretical guidance for genome mining of other Actinomycete strains.

多重耐药病原菌的层出不穷使寻找新型抗生素迫在眉睫。基因组测序揭示了微生物存在大量的隐性次级代谢产物合成基因簇，为新药研发提供了全新的视角与空前的机遇。深海放线菌因其极端生境而进化出新颖独特的生理代谢过程，成为世界新兴战略生物资源。本项目以具有突出新型次级代谢产物的合成潜力的深海放线菌为研究对象，采用多重基因组挖掘策略，靶向激活其中新颖的聚酮/聚肽隐性基因簇，通过比较代谢组分析快速排重，分离和鉴定抗多重耐药菌的新颖结构化合物，建立“基因簇—化合物—活性”关联性，进一步通过理性基因工程进行活性化合物的结构改造与优化，筛选出活性显著、结构新奇的化合物进行体内抗感染活性评价。本研究将对开发具有我国自主知识产权的抗耐药菌感染创新药物具有重要意义，另一方面将为其他放线菌的基因组采掘提供重要的技术参考和理论指导。

抗生素耐药性已成为全球性危害公共卫生安全的最大隐患之一，寻找新型抗生素迫在眉睫。基因组测序揭示了微生物存在大量编码未知结构的次级代谢产物合成基因簇，为新药发现提供了全新的视角。然而，许多基因簇常常处于隐性或低水平表达状态。如何有效激活基因簇的表达，实现基因到化合物的定向挖掘成为亟待解决的关键问题。本项目采用调控元件改造、代谢途径删除、信号分子诱导等多重基因组挖掘结合发酵条件优化策略，重点靶向聚酮/聚肽生物合成基因簇，从海洋放线菌中发现了聚酮类抗生素youssoufenes等，环肽类抗生素atrovimycin等53个新颖结构化合物，其中28个具有抗多重耐药菌活性，先导化合物3个，youssoufene A1被天然产物权威评论性期刊Natural Product Reports评为Hot off the Press；在此过程中发现了能够有效激活放线菌中隐性基因簇的潜在靶基因，建立了“2D-NMR代谢组-基因组-活性”复合策略。通过体内基因敲除、高表达，体外生化实验、定点突变等手段对活性化合物的生物合成过程进行研究，初步阐明了youssoufenes、atrovimycins等7条聚酮/聚肽生物合成途径，解析了新颖的酰基转移酶、糖基转移酶、甲基转移酶、II型BV单加氧酶等的功能。进一步，通过基因工程、代谢工程等策略获得了新颖的活性化合物结构类似物42个。在Angew. Chem. Int. Ed.、JACS、ACS Catal.、Org. Lett.、J. Nat. Prod.等期刊发表基金标注论文36篇（其中SCI收录论文35篇，5篇IF>10，1篇获得Faculty Opinions推荐点评，1篇获ESI高被引论文）；申请发明专利12项，其中美国PCT专利2项，授权国内发明专利5项；培养学生获山东省优秀博士学位论文、山东省研究生优秀成果奖一等奖、刘瑞玉海洋科学奖等奖项；项目负责人获德国Thieme Chemistry Journal Award 2020等奖项。项目取得的研究结果丰富了我国海洋生物药用基因资源和化合物资源，为我国自主知识产权的海洋新药提供了物质基础，为新颖活性化合物的靶向挖掘提供了可靠参考和重要手段，对我国海洋生物资源的高值化利用具有促进作用。