Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA.
Nat Commun. 2022 Sep 6;13(1):5256. doi: 10.1038/s41467-022-32858-0.
Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.
细菌基因组包含大量生物合成基因簇（BGCs），这些基因簇被预测编码尚未开发的天然产物。对以前未研究的 BGCs 的异源表达应该有助于从细菌培养物中发现更多具有治疗意义的生物活性分子，但 BGCs 的大规模操作仍然很麻烦。在这里，我们描述了一种平行鉴定、动员和异源表达 BGCs 的方法。我们的解决方案通过在一个大插入文库中克隆菌株文库的基因组来同时捕获大量的 BGCs，并使用 CONKAT-seq（靶向序列共现网络分析）测序管道来有效地定位携带完整 BGCs 的克隆，这些克隆是异源表达的候选物。我们发现了几种天然产物，包括一种对多药耐药金黄色葡萄球菌有效的抗生素，证明了利用这种方法在经济规模上的优势来系统地研究菌株库中隐藏的 BGCs 的潜力。
