Enabling Access to Novel Bacterial Biosynthetic Potential From ONT Draft Genomic Data

Abstract

Natural products comprise a wide diversity of compounds with a range of biological activities, including antibiotics, anti-inflammatory and anti-tumoral molecules. However, we can only access a small portion of these compounds due to various technical difficulties. We have herein developed a novel and efficient approach for accessing biosynthetic gene clusters (BGCs) that encode natural products from soil bacteria. The pipeline uses a combination of long-read sequencing, antiSMASH for BGC identification and Transformation-associated recombination (TAR) for cloning the BGCs. We hypothesized that a genome assembly using Oxford Nanopore Technology (ONT) sequencing could facilitate the detection of large BGCs at a relatively fast and low-cost DNA sequencing. Despite the relative low accuracy and sequence mistakes due to high GC content and sequence repetitions frequently found in BGC containing bacteria, we demonstrate that ONT long-read sequencing and antiSMASH are effective for identifying novel BGCs and enabling TAR cloning to isolate the BGC in a desired vector. We applied this pipeline on a previously non-sequenced myxobacteria Aetherobacter fasciculatus SBSr002. Our approach enabled us to clone a previously unknown BGC into a genome engineering-ready vector, illustrating the capabilities of this powerful and cost-effective strategy.