定向挖掘含AHBA结构单元的新活性天然产物

Ansamycins, represented by rifamycins and maytansine, is a family of macrolactam natural products, which are assembled by type I polyketide synthase using 3-amino-5-hydroxy-benzoic acid (AHBA) as the starter unit. The prominent bioactivities of known ansamycins and the unexplored potential of new scaffolds predicted from the biosynthetic viewpoint, encourage us to search for new members of this family. Previously, more than 60 AHBA synthase gene positive actinobacterial strains have been obtained. Bioinformatics analysis of the biosynthetic gene clusters with novel AHBA synthase genes in these strains reveals that most of them are involved in the biosynthesis of ansamycins with new scaffolds, and the rest are involved in the biosynthesis of other novel AHBA-containing natural products. However, the following attempts to isolate these predicted products by large scale fermentation and TLC-guided separation were failed, which indicates that these gene clusters are probably “silent/cryptic” under standard laboratory conditions. Based on the above analysis and results, in this proposal, we are planning to activate these silent/cryptic gene clusters by manipulation of the pathway specific and/or global/pleiotropic regulators, which could be identified by transposon mutagenesis and dual reporter genes-based selection, to obtain novel natural products. The outcomes of this project will not only provides new leads for drug discovery, but also will establish an efficient and simple method to access the new natural products encoded by cryptic biosynthetic gene clusters in microbes, which will accumulate valuable experience for mining of the biosynthetic potential of microbes, and expand the research area of microbial natural products.

以利福霉素和美登木素为代表的安莎霉素是通过I型聚酮合酶以3-氨基-5-羟基苯甲酸（AHBA）为特殊起始单元合成的大环内酰胺类抗生素，其较高的成药率和潜在的新结构空间，促使我们定向寻找这一家族的新成员。前期，我们通过筛选积累了60余株AHBA合酶基因阳性菌。对较新颖AHBA合酶基因及其所在基因簇的生物信息学分析表明，其中绝大多数编码安莎类新骨架，另一部分则编码含AHBA单元的非安莎类新骨架。然而，随后的化合物分离提示这些新颖基因簇几乎都处于“沉默”状态。基于此，本项目拟通过途径特异性调控和全基因组范围的全局/多效性调控激活这些含AHBA合酶基因的沉默基因簇，实现基于生物信息学的新天然产物定向发现。本项目的研究成果一方面可以为新药创制提供源头支持，另一方面还有望建立简便有效的微生物“沉默”次级代谢合成基因簇激活方法，为微生物次级代谢潜力的深度发掘积累经验，拓展微生物次级代谢的研究方法和领域。

新活性天然产物是小分子药物研发的重要源泉。本项目通过全基因组测序等手段，获得并完善了6条含AHBA合酶基因的新颖基因簇。通过正调控基因单表达和共表达、启动子替换等策略成功激活并发现了2类共16个新骨架五酮安莎霉素（Microansamycins和Aminoansamycins）和1类与安莎存在亲缘关系的I型-III型PKS杂合化合物Venemycins，并对激活产物进行了初步的活性评价。通过对前期激活的安莎三烯和Venemycins开展生物合成后修饰研究，阐明了安莎三烯独特的O-(N-环己甲酰)-D-丙氨酰基侧链加载机制，表征了首个丙氨酰化酶；从Venemycins合成基因簇中鉴定了链霉菌中首个不需要黄素还原酶参与的单组份卤代酶VemK，VemK同时也是首个催化吡喃酮卤代的卤代酶。此外，我们还基于黑色素报告基因初步建立了沉默基因簇定向激活方法。上述结果以及尚在开展的后续研究不仅为小分子药物研发提供了新化合物，同时也为进一步结构改造提供了催化元件和前期基础。