細菌ドラフトゲノムからのBGCの特定とキャプチャのための迅速かつ費用効果の高いパイプライン

The exploration of microbial genomes through next-generation sequencing (NGS) and genome mining has transformed the discovery of natural products, revealing an immense reservoir of previously untapped chemical diversity. Bacteria remain a prolific source of specialized metabolites with potential applications in medicine and biotechnology. Here, we present a protocol to access novel biosynthetic gene clusters (BGCs) that encode natural products from soil bacteria. The protocol uses a combination of Oxford Nanopore Technology (ONT) sequencing, de novo genome assembly, antiSMASH for BGC identification, and transformation-associated recombination (TAR) for cloning the BGCs. We used this protocol to allow the detection of large BGCs at a relatively fast and low-cost DNA sequencing. The protocol can be applied to diverse bacteria, provided that sufficient high-molecular-weight DNA can be obtained for long-read sequencing. Moreover, this protocol enables subsequent cloning of uncharacterized BGCs into a genome engineering-ready vector, illustrating the capabilities of this powerful and cost-effective strategy. Key features • This protocol enables bioprospection through cloning of a novel BGC identified in an ONT bacterial draft genome. • A combination of ONT sequencing, antiSMASH, and TAR cloning can be used to clone BGCs from bacteria into a vector. • Cost-effective strategy for the discovery of BGCs of diverse natural product classes, including nonribosomal peptides, polyketides, and RiPPs. • Overnight sequencing in-house using cheap and easy-to-use instruments such as MinION, which allows multiplexing.