A Rapid and Cost-Effective Pipeline to Identify and Capture BGCs From Bacterial Draft Genomes.

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.