State Key Laboratory of Bioreactor Engineering and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Institute of Pharmaceutical Biotechnology and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China.
Nucleic Acids Res. 2022 Apr 8;50(6):3581-3592. doi: 10.1093/nar/gkac181.
Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.
直接从微生物基因组中克隆生物合成基因簇（BGCs）有助于基于天然产物的药物发现。在这里，我们通过结合 Cas12a 和细菌人工染色体文库构建的先进功能，开发了一种快速而有效的体外平台，用于直接捕获大型 BGCs，命名为 CAT-FISHING（CRISPR/Cas12a 介导的快速直接生物合成基因簇克隆）。作为演示，CAT-FISHING 有效地从不同放线菌基因组 DNA 样本中捕获了几个大型 BGC，其中最大的一个大小为 145 kb，GC 含量为 75%。此外，我们还在链霉菌底盘中异源表达了从 Micromonospora sp. 181 中直接克隆的、长达 110 kb 的、隐匿的聚酮化合物编码 BGC。结果表明，这是一种新的大环内酯化合物 marinolactam A，具有有希望的抗癌活性。我们的结果表明，CAT-FISHING 是一种用于复杂 BGC 克隆的强大方法，我们相信它将成为基于天然产物的药物发现整个社区的重要资产。
