基于功能宏基因组学策略的抗菌天然产物挖掘研究

Natural products from microbe have been very important resources for antibiotic discovery. Antibiotics produced by microbe were generally obtained by fermentation of the native producer and the majority of microbe could not be cultured in laboratory, prohibiting the new natural products discovery. Function-based metagenomics, where environmental DNA (eDNA) derived biosynthetic gene clusters were expressed in the heterologus host and the antimicrobial active clones can be identified directly by functional screening, provides a culture-independed way and a new strategy for novel antimicrobial natural products discovery. We have constructed a 1.5 million-clones metagenomic library derived from Mount. E’Mei soil, and identified 5 antibacterial clones by functional screening of 200 thousands clones in Streptomyces host. A compound with rare iso-thiozole ring was isolated from the fermentation broth of a clone, and high biofilm inhibitory and cleavage activities were found from the fermentation broths of two other clones. In this project, we will focus on discovery and biosynthetic analysis of novel antimicrobial compounds, and our studies will set the stage for new antibiotic discovery.

微生物天然产物是抗生素发现的重要源头，但微生物来源的抗菌天然产物的发现切由于绝大多数微生物的不可培养性而受到极大限制。不依赖微生物培养的功能宏基因组学，通过环境微生物生物合成基因簇在异源宿主中的直接功能表达，获得新抗菌活性天然产物，突破了微生物的培养瓶颈，是极具潜力的新策略。利用功能宏基因组学策略，我们建立了含150万克隆的峨眉山土壤的功能宏基因组文库，通过在链霉菌宿主中初步功能筛选20万个克隆，获得了5个抗菌活性克隆。对其中1个阳性克隆发酵产物的分离纯化发现了新结构化合物和天然产物稀有的异噻唑结构，同时另外2个阳性克隆的发酵产物表现了很强的对金黄色葡萄球菌生物被膜的抑制生成和清除的活性。本课题我们将在此基础上，进一步发现新的抗菌活性化合物，同时研究特殊产物的合成机理，为抗菌药物开发提供依据。

环境中存在着大量的非培养微生物，它们是包括天然产物和酶等生物活性物质的重要生产者。功能宏基因组学技术，直接从环境中克隆并表达微生物中相关的生物合成基因，为这些活性物质的发现提供了新的途径。在本课题中，利用功能宏基因组学策略，我们克隆环境微生物基因组DNA构建宏基因组文库，在文库中筛选出生产生物活性物质的阳性克隆，并鉴定阳性克隆产物，为新物质的发现建立基础。通过课题的实施，我们成功构建了用于功能筛选的大型宏基因组文库，获得了多个阳性克隆。通过对阳性克隆的针对性研究，分离鉴定了一类异喹啉类化合物Mansouramycins和首个天然吡唑醌化合物Salumycin，并首次鉴定了喹啉类化合物Mansouramycins的生物合成基因簇。同时，对筛选获得的生物活性酶进行了表征和分子改造。通过课题的实施，确立了功能宏基因组学策略发现微生物天然产物和活性酶的方法。在课题的资助下，已发表SCI论文4篇，中文核心期刊论文5篇。